New features of triacylglycerol biosynthetic pathways of peanut seeds in early developmental stages.

PubMed

Yu, Mingli; Liu, Fengzhen; Zhu, Weiwei; Sun, Meihong; Liu, Jiang; Li, Xinzheng

2015-11-01

The peanut (Arachis hypogaea L.) is one of the three most important oil crops in the world due to its high average oil content (50 %). To reveal the biosynthetic pathways of seed oil in the early developmental stages of peanut pods with the goal of improving the oil quality, we presented a method combining deep sequencing analysis of the peanut pod transcriptome and quantitative real-time PCR (RT-PCR) verification of seed oil-related genes. From the sequencing data, approximately 1500 lipid metabolism-associated Unigenes were identified. The RT-PCR results quantified the different expression patterns of these triacylglycerol (TAG) synthesis-related genes in the early developmental stages of peanut pods. Based on these results and analysis, we proposed a novel construct of the metabolic pathways involved in the biosynthesis of TAG, including the Kennedy pathway, acyl-CoA-independent pathway and proposed monoacylglycerol pathway. It showed that the biosynthetic pathways of TAG in the early developmental stages of peanut pods were much more complicated than a simple, unidirectional, linear pathway.

Flavoenzymes: Versatile Catalysts in Biosynthetic Pathways

PubMed Central

Walsh, Christopher T.; Wencewicz, Timothy A.

2012-01-01

Riboflavin-based coenzymes, tightly bound to enzymes catalyzing substrate oxidations and reductions, enable an enormous range of chemical transformations in biosynthetic pathways. Flavoenzymes catalyze substrate oxidations involving amine and alcohol oxidations and desaturations to olefins, the latter setting up Diels-Alder cyclizations in lovastatin and solanapyrone biosyntheses. Both C4a and N5 of the flavin coenzymes are sites for covalent adduct formation. For example, the reactivity of dihydroflavins with molecular oxygen leads to flavin-4a-OOH adducts which then carry out a diverse range of oxygen transfers, including Baeyer-Villiger type ring expansions, olefin epoxidations, halogenations via transient HOCl generation, and an oxidative Favorskii rerrangement during enterocin assembly. PMID:23051833

A retro-biosynthetic approach to the prediction of biosynthetic pathways from position-specific isotope analysis as shown for tramadol

PubMed Central

Romek, Katarzyna M.; Nun, Pierrick; Remaud, Gérald S.; Silvestre, Virginie; Taïwe, Germain Sotoing; Lecerf-Schmidt, Florine; Boumendjel, Ahcène; De Waard, Michel; Robins, Richard J.

2015-01-01

Tramadol, previously only known as a synthetic analgesic, has now been found in the bark and wood of roots of the African medicinal tree Nauclea latifolia. At present, no direct evidence is available as to the biosynthetic pathway of its unusual skeleton. To provide guidance as to possible biosynthetic precursors, we have adopted a novel approach of retro-biosynthesis based on the position-specific distribution of isotopes in the extracted compound. Relatively recent developments in isotope ratio monitoring by 13C NMR spectrometry make possible the measurement of the nonstatistical position-specific natural abundance distribution of 13C (δ13Ci) within the molecule with better than 1‰ precision. Very substantial variation in the 13C positional distribution is found: between δ13Ci = −11 and −53‰. Distribution is not random and it is argued that the pattern observed can substantially be interpreted in relation to known causes of isotope fractionation in natural products. Thus, a plausible biosynthetic scheme based on sound biosynthetic principals of precursor–substrate relationships can be proposed. In addition, data obtained from the 18O/16O ratios in the oxygen atoms of the compound add support to the deductions made from the carbon isotope analysis. This paper shows how the use of 13C NMR at natural abundance can help with proposing a biosynthetic route to compounds newly found in nature or those difficult to tackle by conventional means. PMID:26106160

Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology.

PubMed

Robey, Matthew T; Ye, Rosa; Bok, Jin Woo; Clevenger, Kenneth D; Islam, Md Nurul; Chen, Cynthia; Gupta, Raveena; Swyers, Michael; Wu, Edward; Gao, Peng; Thomas, Paul M; Wu, Chengcang C; Keller, Nancy P; Kelleher, Neil L

2018-05-18

Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus. The acu-dioxomorpholine nonribosomal peptide synthetase features a new type of condensation domain (designated C R ) proposed to use a noncanonical arginine active site for ester bond formation. Using stable isotope labeling and MS, we determine that a phenyllactate monomer deriving from phenylalanine is incorporated into the diketomorpholine scaffold. Acu-dioxomorpholine is highly related to orphan inhibitors of P-glycoprotein targets in multidrug-resistant cancers, and identification of the biosynthetic pathway for this compound class enables genome mining for additional derivatives.

Characterization of Enzymes Catalyzing Transformations of Cysteine S-Conjugated Intermediates in the Lincosamide Biosynthetic Pathway.

PubMed

Ushimaru, Richiro; Lin, Chia-I; Sasaki, Eita; Liu, Hung-Wen

2016-09-02

Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the β-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evolution-guided optimization of biosynthetic pathways.

PubMed

Raman, Srivatsan; Rogers, Jameson K; Taylor, Noah D; Church, George M

2014-12-16

Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers. We convert the intracellular presence of the target chemical into a fitness advantage for the cell by using a sensor domain responsive to the chemical to control a reporter gene necessary for survival under selective conditions. Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. This scheme allows us to perform multiple rounds of evolution (addressing ∼10(9) cells per round) with minimal carryover of cheaters after each round. Based on candidate genes identified by flux balance analysis, we used targeted genome-wide mutagenesis to vary the expression of pathway genes involved in the production of naringenin and glucaric acid. Through up to four rounds of evolution, we increased production of naringenin and glucaric acid by 36- and 22-fold, respectively. Naringenin production (61 mg/L) from glucose was more than double the previous highest titer reported. Whole-genome sequencing of evolved strains revealed additional untargeted mutations that likely benefit production, suggesting new routes for optimization.

Enhancement of cordyceps polysaccharide production via biosynthetic pathway analysis in Hirsutella sinensis.

PubMed

Lin, Shan; Liu, Zhi-Qiang; Baker, Peter James; Yi, Ming; Wu, Hui; Xu, Feng; Teng, Yi; Zheng, Yu-Guo

2016-11-01

The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2mM of manganese sulfate on 0day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of a Direct Biosynthetic Pathway for UDP-N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

PubMed

Dadashipour, Mohammad; Iwamoto, Mariko; Hossain, Mohammad Murad; Akutsu, Jun-Ichi; Zhang, Zilian; Kawarabayasi, Yutaka

2018-05-15

Most organisms, from Bacteria to Eukarya , synthesize UDP- N -acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP- N -acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea , the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii , predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii , and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic

ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways

DOE PAGES

Le, Thuc M.; Poddar, Soumya; Capri, Joseph R.; ...

2017-08-14

It is known that leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the diseasemore » in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.« less

ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Le, Thuc M.; Poddar, Soumya; Capri, Joseph R.

It is known that leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the diseasemore » in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.« less

The aromatic amino acids biosynthetic pathway: A core platform for products

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lievense, J.C.; Frost, J.W.

The aromatic amino acids biosynthetic pathway is viewed conventionally and primarily as the source of the amino acids L-tyrosine, L-phenylalanine. The authors have recognized the expanded role of the pathway as the major source of aromatic raw materials on earth. With the development of metabolic engineering approaches, it is now possible to biosynthesize a wide variety of aromatic compounds from inexpensive, clean, abundant, renewable sugars using fermentation methods. Examples of already and soon-to-be commercialized biosynthesis of such compounds are described. The long-term prospects are also assessed.

Construction of a controllable β-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae.

PubMed

Xie, Wenping; Liu, Min; Lv, Xiaomei; Lu, Wenqiang; Gu, Jiali; Yu, Hongwei

2014-01-01

Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi-gene pathways by employing the GAL regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof-of-principle, a controllable β-carotene biosynthesis pathway (∼16 kb) was reconstructed and optimized by repeatedly using GAL10-GAL1 bidirectional promoters with high efficiency (80-100%). By controling the switch time of the pathway, production of 11 mg/g DCW of total carotenoids (72.57 mg/L) and 7.41 mg/g DCW of β-carotene was achieved in shake-flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture. The results demonstrated a controllable and genetically stable biosynthetic pathway capable of increasing the yield of target products. Furthermore, the strategy presented in this study could be extended to construct other pathways in S. cerevisisae. © 2013 Wiley Periodicals, Inc.

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

PubMed Central

2016-01-01

SUMMARY Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism. PMID:27074917

Biosynthetic Pathway for the Epipolythiodioxopiperazine Acetylaranotin in Aspergillus terreus Revealed by Genome-based Deletion Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Chun-Jun; Yeh, Hsu-Hua; Chiang, Yi Ming

2013-04-15

Abstract Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from cyclic peptides. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of 9 genes including one nonribosomal peptide synthase gene, ataP, that is required for acetylaranotin biosynthesis. Chemical analysis of the wild type and mutant strainsmore » enabled us to isolate seventeen natural products that are either intermediates in the normal biosynthetic pathway or shunt products that are produced when the pathway is interrupted through mutation. Nine of the compounds identified in this study are novel natural products. Our data allow us to propose a complete biosynthetic pathway for acetylaranotin and related natural products.« less

Sioxanthin, a novel glycosylated carotenoid, reveals an unusual subclustered biosynthetic pathway.

PubMed

Richter, Taylor K S; Hughes, Chambers C; Moore, Bradley S

2015-06-01

Members of the marine actinomycete genus Salinispora constitutively produce a characteristic orange pigment during vegetative growth. Contrary to the understanding of widespread carotenoid biosynthesis pathways in bacteria, Salinispora carotenoid biosynthesis genes are not confined to a single cluster. Instead, bioinformatic and genetic investigations confirm that four regions of the Salinispora tropica CNB-440 genome, consisting of two gene clusters and two independent genes, contribute to the in vivo production of a single carotenoid. This compound, namely (2'S)-1'-(β-D-glucopyranosyloxy)-3',4'-didehydro-1',2'-dihydro-φ,ψ-caroten-2'-ol, is novel and has been given the trivial name 'sioxanthin'. Sioxanthin is a C40 -carotenoid, glycosylated on one end of the molecule and containing an aryl moiety on the opposite end. Glycosylation is unusual among actinomycete carotenoids, and sioxanthin joins a rare group of carotenoids with polar and non-polar head groups. Gene sequence homology predicts that the sioxanthin biosynthetic pathway is present in all of the Salinispora as well as other members of the family Micromonosporaceae. Additionally, this study's investigations of clustering of carotenoid biosynthetic genes in heterotrophic bacteria show that a non-clustered genome arrangement is more common than previously suggested, with nearly half of the investigated genomes showing a non-clustered architecture. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

PubMed

Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

2008-06-01

Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

Single Cell Genome Amplification Accelerates Identification of the Apratoxin Biosynthetic Pathway from a Complex Microbial Assemblage

PubMed Central

Grindberg, Rashel V.; Ishoey, Thomas; Brinza, Dumitru; Esquenazi, Eduardo; Coates, R. Cameron; Liu, Wei-ting; Gerwick, Lena; Dorrestein, Pieter C.; Pevzner, Pavel; Lasken, Roger; Gerwick, William H.

2011-01-01

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites. PMID:21533272

New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallo, A.; Bruno, K. S.; Solfrizzo, M.

2012-09-14

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been obtained in Penicillium species. In Aspergillus species only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase in OTA producing A. carbonarius ITEM 5010 has removed the ability of the fungus to produce OTA. This is themore » first report on the involvement of an nrps gene product in OTA biosynthetic pathway in Aspergillus species. The absence of OTA and ochratoxin α-the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β- the dechloro analog of ochratoxin α- were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius, and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight in the biosynthetic pathway of the toxin.« less

A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

PubMed

Karim, Ashty S; Jewett, Michael C

2016-07-01

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.

PubMed

Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

2006-08-16

Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

Convergence of isoprene and polyketide biosynthetic machinery: isoprenyl-S-carrier proteins in the pksX pathway of Bacillus subtilis.

PubMed

Calderone, Christopher T; Kowtoniuk, Walter E; Kelleher, Neil L; Walsh, Christopher T; Dorrestein, Pieter C

2006-06-13

The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide/polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived beta-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a Delta(2)-isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon beta-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

PubMed Central

Fresquet-Corrales, Sandra; Roque, Edelín; Sarrión-Perdigones, Alejandro; Rochina, Maricruz; López-Gresa, María P.; Díaz-Mula, Huertas M.; Bellés, José M.; Tomás-Barberán, Francisco; Beltrán, José P.

2017-01-01

Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce “bloat-safe” plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed. PMID:28902886

The pyrimidine nucleotide biosynthetic pathway modulates production of biofilm determinants in Escherichia coli.

PubMed

Garavaglia, Marco; Rossi, Elio; Landini, Paolo

2012-01-01

Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions.

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa.

PubMed

Nag, Ambarish; Karpinets, Tatiana V; Chang, Christopher H; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

Enhancing a Pathway-Genome Database (PGDB) to capture subcellular localization of metabolites and enzymes: the nucleotide-sugar biosynthetic pathways of Populus trichocarpa

PubMed Central

Nag, Ambarish; Karpinets, Tatiana V.; Chang, Christopher H.; Bar-Peled, Maor

2012-01-01

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can

Expanding the product profile of a microbial alkane biosynthetic pathway.

PubMed

Harger, Matthew; Zheng, Lei; Moon, Austin; Ager, Casey; An, Ju Hye; Choe, Chris; Lai, Yi-Ling; Mo, Benjamin; Zong, David; Smith, Matthew D; Egbert, Robert G; Mills, Jeremy H; Baker, David; Pultz, Ingrid Swanson; Siegel, Justin B

2013-01-18

Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.

Ultrafast time-resolved carotenoid to-bacteriochlorophyll energy transfer in LH2 complexes from photosynthetic bacteria.

PubMed

Cong, Hong; Niedzwiedzki, Dariusz M; Gibson, George N; LaFountain, Amy M; Kelsh, Rhiannon M; Gardiner, Alastair T; Cogdell, Richard J; Frank, Harry A

2008-08-28

Steady-state and ultrafast time-resolved optical spectroscopic investigations have been carried out at 293 and 10 K on LH2 pigment-protein complexes isolated from three different strains of photosynthetic bacteria: Rhodobacter (Rb.) sphaeroides G1C, Rb. sphaeroides 2.4.1 (anaerobically and aerobically grown), and Rps. acidophila 10050. The LH2 complexes obtained from these strains contain the carotenoids, neurosporene, spheroidene, spheroidenone, and rhodopin glucoside, respectively. These molecules have a systematically increasing number of pi-electron conjugated carbon-carbon double bonds. Steady-state absorption and fluorescence excitation experiments have revealed that the total efficiency of energy transfer from the carotenoids to bacteriochlorophyll is independent of temperature and nearly constant at approximately 90% for the LH2 complexes containing neurosporene, spheroidene, spheroidenone, but drops to approximately 53% for the complex containing rhodopin glucoside. Ultrafast transient absorption spectra in the near-infrared (NIR) region of the purified carotenoids in solution have revealed the energies of the S1 (2(1)Ag-)-->S2 (1(1)Bu+) excited-state transitions which, when subtracted from the energies of the S0 (1(1)Ag-)-->S2 (1(1)Bu+) transitions determined by steady-state absorption measurements, give precise values for the positions of the S1 (2(1)Ag-) states of the carotenoids. Global fitting of the ultrafast spectral and temporal data sets have revealed the dynamics of the pathways of de-excitation of the carotenoid excited states. The pathways include energy transfer to bacteriochlorophyll, population of the so-called S* state of the carotenoids, and formation of carotenoid radical cations (Car*+). The investigation has found that excitation energy transfer to bacteriochlorophyll is partitioned through the S1 (1(1)Ag-), S2 (1(1)Bu+), and S* states of the different carotenoids to varying degrees. This is understood through a consideration of the

Origin of Bacteriochlorophyll a and the Early Diversification of Photosynthesis.

PubMed

Cardona, Tanai

2016-01-01

Photosynthesis originated in the domain Bacteria billions of years ago; however, the identity of the last common ancestor to all phototrophic bacteria remains undetermined and speculative. Here I present the evolution of BchF or 3-vinyl-bacteriochlorophyll hydratase, an enzyme exclusively found in bacteria capable of synthetizing bacteriochlorophyll a. I show that BchF exists in two forms originating from an early divergence, one found in the phylum Chlorobi, including its paralogue BchV, and a second form that was ancestral to the enzyme found in the remaining anoxygenic phototrophic bacteria. The phylogeny of BchF is consistent with bacteriochlorophyll a evolving in an ancestral phototrophic bacterium that lived before the radiation event that gave rise to the phylum Chloroflexi, Chlorobi, Acidobacteria, Proteobacteria, and Gemmatimonadetes, but only after the divergence of Type I and Type II reaction centers. Consequently, it is suggested that the lack of phototrophy in many groups of extant bacteria is a derived trait.

Evidence for anoxygenic photosynthesis from the distribution of bacteriochlorophylls in the Black Sea.

PubMed

Repeta, D J; Simpson, D J; Jorgensen, B B; Jannasch, H W

1989-11-02

The contribution of anoxygenic photosynthesis to carbon cycling in the Black Sea, the world's largest body of anoxic marine water, has been vigorously investigated and debated for over four decades. Penetration of light into the sulphide-containing deep water may result in a zone of anaerobic primary production by photosynthetic bacteria. We report here the results of analyses of photosynthetic pigments in samples of suspended particulate matter collected from two stations in the western basin of the Black Sea. Our data demonstrate high concentrations of a bacterio-chlorophyll at the chemocline, and thus the potential for anoxygenic photosynthesis as a component of primary production in the carbon cycle of the Black Sea. More than 95% of the pigments in the bacteriochlorophyll-maximum are accounted for by a series of aromatic carotenoids and bacteriochlorophylls-e, including a previously unreported geranyl ester of 4-i-butyl bacteriochlorophyll-e. The distribution of pigments is characteristic of the obligate phototrophs Chlorobium phaeobacteroides and C. phaeovibriodes. Total depth-integrated bacteriochlorophyll at one station exceeded total chlorophyll-a in the overlying oxygenated portion of the euphotic zone. We suggest that anoxygenic photosynthesis is a relatively recent phenomenon in the Black Sea initiated by shallowing of the chemocline over the past decade and development of an anoxic layer devoid of O2 and H2S.

Propagation rate constants for the peroxidation of sterols on the biosynthetic pathway to cholesterol.

PubMed

Lamberson, Connor R; Muchalski, Hubert; McDuffee, Kari B; Tallman, Keri A; Xu, Libin; Porter, Ned A

2017-10-01

The free radical chain autoxidation of cholesterol and the oxidation products formed, i.e. oxysterols, have been the focus of intensive study for decades. The peroxidation of sterol precursors to cholesterol such as 7-dehydrocholesterol (7-DHC) and desmosterol as well as their oxysterols has received less attention. The peroxidation of these sterol precursors can become important under circumstances in which genetic conditions or exposures to small molecules leads to an increase of these biosynthetic intermediates in tissues and fluids. 7-DHC, for example, has a propagation rate constant for peroxidation some 200 times that of cholesterol and this sterol is found at elevated levels in a devastating human genetic condition, Smith-Lemli-Opitz syndrome (SLOS). The propagation rate constants for peroxidation of sterol intermediates on the biosynthetic pathway to cholesterol were determined by a competition kinetic method, i.e. a peroxyl radical clock. In this work, propagation rate constants for lathosterol, zymostenol, desmosterol, 7-dehydrodesmosterol and other sterols in the Bloch and Kandutsch-Russell pathways are assigned and these rate constants are related to sterol structural features. Furthermore, potential oxysterols products are proposed for sterols whose oxysterol products have not been determined. Copyright © 2017 Elsevier B.V. All rights reserved.

Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP-l-galactose mutase

PubMed Central

Zhu, Qinghua; Chen, Qi; Song, Yongxiang; Huang, Hongbo; Li, Jun; Ma, Junying; Li, Qinglian; Ju, Jianhua

2017-01-01

Galactose, a monosaccharide capable of assuming two possible configurational isomers (d-/l-), can exist as a six-membered ring, galactopyranose (Galp), or as a five-membered ring, galactofuranose (Galf). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for d-Galf. Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive l-Galf production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-l-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: (i) decipher the unique enzyme, GDP-l-galactose mutase associated with production of two unique d-mannose-derived sugars, and (ii) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of l-Galf-containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies. PMID:28438999

Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP-l-galactose mutase.

PubMed

Zhu, Qinghua; Chen, Qi; Song, Yongxiang; Huang, Hongbo; Li, Jun; Ma, Junying; Li, Qinglian; Ju, Jianhua

2017-05-09

Galactose, a monosaccharide capable of assuming two possible configurational isomers (d-/l-), can exist as a six-membered ring, galactopyranose (Gal p ), or as a five-membered ring, galactofuranose (Gal f ). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for d-Gal f Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive l-Gal f production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-l-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: ( i ) decipher the unique enzyme, GDP-l-galactose mutase associated with production of two unique d-mannose-derived sugars, and ( ii ) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of l-Gal f -containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.

Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus Neurospora crassa Ergothioneine Biosynthetic Pathway

PubMed Central

2015-01-01

Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C–S bond formation and a PLP-mediated C–S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C–S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible. PMID:25275953

Metabolic engineering of biosynthetic pathway for production of renewable biofuels.

PubMed

Singh, Vijai; Mani, Indra; Chaudhary, Dharmendra Kumar; Dhar, Pawan Kumar

2014-02-01

Metabolic engineering is an important area of research that involves editing genetic networks to overproduce a certain substance by the cells. Using a combination of genetic, metabolic, and modeling methods, useful substances have been synthesized in the past at industrial scale and in a cost-effective manner. Currently, metabolic engineering is being used to produce sufficient, economical, and eco-friendly biofuels. In the recent past, a number of efforts have been made towards engineering biosynthetic pathways for large scale and efficient production of biofuels from biomass. Given the adoption of metabolic engineering approaches by the biofuel industry, this paper reviews various approaches towards the production and enhancement of renewable biofuels such as ethanol, butanol, isopropanol, hydrogen, and biodiesel. We have also identified specific areas where more work needs to be done in the future.

Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways.

PubMed

Janevska, Slavica; Tudzynski, Bettina

2018-01-01

The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

Understanding the carotenoid biosynthetic pathway through observation of four color variants of developing watermelon (Citrullus lanatus (Thunb.) Matsum. & Nanai)

USDA-ARS?s Scientific Manuscript database

The carotenoid biosynthetic pathway regulatory mechanisms leading to lycopene accumulation are well defined in the model fruit, tomato (Lycopersicon esculentum L.). The regulatory mechanisms leading to accumulation of other carotenoids and flesh colors, however, are poorly understood. The variety ...

Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga

DTIC Science & Technology

2011-10-17

analysis results. The components of the TAG biosynthetic pathway, including glycerol-3-phosphate acyl- transferase (GPAT), lyso- phosphatidic acid ...acyltransferase (LPAAT), phosphatidic acid phosphatase (PAP), lyso-phosphati- dylcholine acyltransferase (LPAT), and diacylglycerol acyltransfer- ase (DGAT...transfer to position one of G3P results in the formation of lyso- phosphatidic acid (LPA), in a reaction catalyzed by GPAT. Subsequent acyl transfer to

Complete characterization of the seventeen step moenomycin biosynthetic pathway

PubMed Central

Ostash, Bohdan; Doud, Emma; Lin, Cecilie; Ostash, Iryna; Perlstein, Deborah; Fuse, Shinichiro; Wolpert, Manuel; Kahne, Daniel; Walker, Suzanne

2009-01-01

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here we report a comprehensive set of genetic and enzymatic experiments that establish functions for the seventeen moenomycin biosynthetic genes involved in the synthesis moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogs. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogs can be explored. PMID:19640006

Expression of carotenoid biosynthetic pathway genes and changes in carotenoids during ripening in tomato (Lycopersicon esculentum).

PubMed

Namitha, Kanakapura Krishnamurthy; Archana, Surya Narayana; Negi, Pradeep Singh

2011-04-01

To study the expression pattern of carotenoid biosynthetic pathway genes, changes in their expression at different stages of maturity in tomato fruit (cv. Arka Ahuti) were investigated. The genes regulating carotenoid production were quantified by a dot blot method using a DIG (dioxigenin) labelling and detection kit. The results revealed that there was an increase in the levels of upstream genes of the carotenoid biosynthetic pathway such as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase (Lyt B), phytoene synthase (PSY), phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) by 2-4 fold at the breaker stage as compared to leaf. The lycopene and β-carotene content was analyzed by HPLC at different stages of maturity. The lycopene (15.33 ± 0.24 mg per 100 g) and β-carotene (10.37 ± 0.46 mg per 100 g) content were found to be highest at 5 days post-breaker and 10 days post-breaker stage, respectively. The lycopene accumulation pattern also coincided with the color values at different stages of maturity. These studies may provide insight into devising gene-based strategies for enhancing carotenoid accumulation in tomato fruits.

Indole-3-acetic acid in Fusarium graminearum: Identification of biosynthetic pathways and characterization of physiological effects.

PubMed

Luo, Kun; Rocheleau, Hélène; Qi, Peng-Fei; Zheng, You-Liang; Zhao, Hui-Yan; Ouellet, Thérèse

2016-09-01

Fusarium graminearum is a devastating pathogenic fungus causing fusarium head blight (FHB) of wheat. This fungus can produce indole-3-acetic acid (IAA) and a very large amount of IAA accumulates in wheat head tissues during the first few days of infection by F. graminearum. Using liquid culture conditions, we have determined that F. graminearum can use tryptamine (TAM) and indole-3-acetonitrile (IAN) as biosynthetic intermediates to produce IAA. It is the first time that F. graminearum is shown to use the l-tryptophan-dependent TAM and IAN pathways rather than the indole-3-acetamide or indole-3-pyruvic acid pathways to produce IAA. Our experiments also showed that exogenous IAA was metabolized by F. graminearum. Exogenous IAA, TAM, and IAN inhibited mycelial growth; IAA and IAN also affected the hyphae branching pattern and delayed macroconidium germination. IAA and TAM had a small positive effect on the production of the mycotoxin 15-ADON while IAN inhibited its production. Our results showed that IAA and biosynthetic intermediates had a significant effect on F. graminearum physiology and suggested a new area of exploration for fungicidal compounds. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Evolution of a genome-encoded bias in amino acid biosynthetic pathways is a potential indicator of amino acid dynamics in the environment.

PubMed

Fasani, Rick A; Savageau, Michael A

2014-11-01

Overcoming the stress of starvation is one of an organism's most challenging phenotypic responses. Those organisms that frequently survive the challenge, by virtue of their fitness, will have evolved genomes that are shaped by their specific environments. Understanding this genotype-environment-phenotype relationship at a deep level will require quantitative predictive models of the complex molecular systems that link these aspects of an organism's existence. Here, we treat one of the most fundamental molecular systems, protein synthesis, and the amino acid biosynthetic pathways involved in the stringent response to starvation. These systems face an inherent logical dilemma: Building an amino acid biosynthetic pathway to synthesize its product-the cognate amino acid of the pathway-may require that very amino acid when it is no longer available. To study this potential "catch-22," we have created a generic model of amino acid biosynthesis in response to sudden starvation. Our mathematical analysis and computational results indicate that there are two distinctly different outcomes: Partial recovery to a new steady state, or full system failure. Moreover, the cell's fate is dictated by the cognate bias, the number of cognate amino acids in the corresponding biosynthetic pathway relative to the average number of that amino acid in the proteome. We test these implications by analyzing the proteomes of over 1,800 sequenced microbes, which reveals statistically significant evidence of low cognate bias, a genetic trait that would avoid the biosynthetic quandary. Furthermore, these results suggest that the pattern of cognate bias, which is readily derived by genome sequencing, may provide evolutionary clues to an organism's natural environment. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Revisiting sesquiterpene biosynthetic pathways leading to santalene and its analogues: a comprehensive mechanistic study.

PubMed

Jindal, Garima; Sunoj, Raghavan B

2012-10-21

Santalene and bergamotene are the major olefinic sesquiterpenes responsible for the fragrance of sandalwood oil. Herein we report the details of density functional theory investigations on the biosynthetic pathway of this important class of terpenes. The mechanistic study has been found to be effective toward gaining significant new insight into different possibilities for the formation of the key intermediates involved in santalene and bergamotene biosynthesis. The stereoelectronic features of the transition states and intermediates for (i) ring closure of the initial bisabolyl cation, and (ii) skeletal rearrangements in the ensuing bicyclic carbocationic intermediates leading to (-)-epi-β-santalene, (-)-β-santalene, (-)-α-santalene, (+)-epi-β-santalene, exo-β-bergamotene, endo-β-bergamotene, exo-α-bergamotene, and endo-α-bergamotene are presented. Interesting structural features pertaining to certain new carbocationic intermediates (such as b) resulting from the ring closure of bisabolyl cation are discussed. Extensive conformational sampling of all key intermediates along the biosynthetic pathway offered new insight into the role of the isoprenyl side chain conformation in the formation of santalene and its analogues. Although the major bicyclic products in Santalum album appear to arise from the right or left handed helical form of farnesyl pyrophosphate (FPP), different alternatives for their formation are found to be energetically feasible. The interconversion of the exo and endo isomers of bisabolyl cation and a likely epimerization, both with interesting mechanistic implications, are presented. The exo to endo conversion is identified to be energetically more favorable than another pathway emanating from the left handed helical FPP. The role of pyrophosphate (OPP(-)) in the penultimate deprotonation step leading to olefinic sesquiterpenes is also examined.

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin-Fatty Acid Biosynthetic Pathway.

PubMed

Haushalter, Robert W; Phelan, Ryan M; Hoh, Kristina M; Su, Cindy; Wang, George; Baidoo, Edward E K; Keasling, Jay D

2017-04-05

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotin and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.

Evolution and Multifarious Horizontal Transfer of an Alternative Biosynthetic Pathway for the Alternative Polyamine sym-Homospermidine*♦

PubMed Central

Shaw, Frances L.; Elliott, Katherine A.; Kinch, Lisa N.; Fuell, Christine; Phillips, Margaret A.; Michael, Anthony J.

2010-01-01

Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life, and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, sym-homospermidine. We demonstrate that homospermidine synthase (HSS) has evolved vertically, primarily in the α-Proteobacteria, but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes, and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of co-products diaminopropane and N1-aminobutylcadaverine, in addition to sym-homospermidine. We show that sym-homospermidine is required for normal growth of the α-proteobacterium Rhizobium leguminosarum. However, sym-homospermidine can be replaced, for growth restoration, by the structural analogues spermidine and sym-norspermidine, suggesting that the symmetrical or unsymmetrical form and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some α-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine. PMID:20194510

Evolution of a Genome-Encoded Bias in Amino Acid Biosynthetic Pathways Is a Potential Indicator of Amino Acid Dynamics in the Environment

PubMed Central

Fasani, Rick A.; Savageau, Michael A.

2014-01-01

Overcoming the stress of starvation is one of an organism’s most challenging phenotypic responses. Those organisms that frequently survive the challenge, by virtue of their fitness, will have evolved genomes that are shaped by their specific environments. Understanding this genotype–environment–phenotype relationship at a deep level will require quantitative predictive models of the complex molecular systems that link these aspects of an organism’s existence. Here, we treat one of the most fundamental molecular systems, protein synthesis, and the amino acid biosynthetic pathways involved in the stringent response to starvation. These systems face an inherent logical dilemma: Building an amino acid biosynthetic pathway to synthesize its product—the cognate amino acid of the pathway—may require that very amino acid when it is no longer available. To study this potential “catch-22,” we have created a generic model of amino acid biosynthesis in response to sudden starvation. Our mathematical analysis and computational results indicate that there are two distinctly different outcomes: Partial recovery to a new steady state, or full system failure. Moreover, the cell’s fate is dictated by the cognate bias, the number of cognate amino acids in the corresponding biosynthetic pathway relative to the average number of that amino acid in the proteome. We test these implications by analyzing the proteomes of over 1,800 sequenced microbes, which reveals statistically significant evidence of low cognate bias, a genetic trait that would avoid the biosynthetic quandary. Furthermore, these results suggest that the pattern of cognate bias, which is readily derived by genome sequencing, may provide evolutionary clues to an organism’s natural environment. PMID:25118252

The Cremeomycin Biosynthetic Gene Cluster Encodes a Pathway for Diazo Formation.

PubMed

Waldman, Abraham J; Pechersky, Yakov; Wang, Peng; Wang, Jennifer X; Balskus, Emily P

2015-10-12

Diazo groups are found in a range of natural products that possess potent biological activities. Despite longstanding interest in these metabolites, diazo group biosynthesis is not well understood, in part because of difficulties in identifying specific genes linked to diazo formation. Here we describe the discovery of the gene cluster that produces the o-diazoquinone natural product cremeomycin and its heterologous expression in Streptomyces lividans. We used stable isotope feeding experiments and in vitro characterization of biosynthetic enzymes to decipher the order of events in this pathway and establish that diazo construction involves late-stage N-N bond formation. This work represents the first successful production of a diazo-containing metabolite in a heterologous host, experimentally linking a set of genes with diazo formation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin–Fatty Acid Biosynthetic Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haushalter, Robert W.; Phelan, Ryan M.; Hoh, Kristina M.

Dicarboxylic acids are commodity chemicals used in the production of plastics, polyesters, nylons, fragrances, and medications. Bio-based routes to dicarboxylic acids are gaining attention due to environmental concerns about petroleum-based production of these compounds. Some industrial applications require dicarboxylic acids with specific carbon chain lengths, including odd-carbon species. Biosynthetic pathways involving cytochrome P450-catalyzed oxidation of fatty acids in yeast and bacteria have been reported, but these systems produce almost exclusively even-carbon species. Here in this paper we report a novel pathway to odd-carbon dicarboxylic acids directly from glucose in Escherichia coli by employing an engineered pathway combining enzymes from biotinmore » and fatty acid synthesis. Optimization of the pathway will lead to industrial strains for the production of valuable odd-carbon diacids.« less

Perturbations in the Photosynthetic Pigment Status Result in Photooxidation-Induced Crosstalk between Carotenoid and Porphyrin Biosynthetic Pathways

PubMed Central

Park, Joon-Heum; Tran, Lien H.; Jung, Sunyo

2017-01-01

Possible crosstalk between the carotenoid and porphyrin biosynthetic pathways under photooxidative conditions was investigated by using their biosynthetic inhibitors, norflurazon (NF) and oxyfluorfen (OF). High levels of protoporphyrin IX (Proto IX) accumulated in rice plants treated with OF, whereas Proto IX decreased in plants treated with NF. Both NF and OF treatments resulted in greater decreases in MgProto IX, MgProto IX methyl ester, and protochlorophyllide. Activities and transcript levels of most porphyrin biosynthetic enzymes, particularly in the Mg-porphyrin branch, were greatly down-regulated in NF and OF plants. In contrast, the transcript levels of GSA, PPO1, and CHLD as well as FC2 and HO2 were up-regulated in NF-treated plants, while only moderate increases in FC2 and HO2 were observed in the early stage of OF treatment. Phytoene, antheraxanthin, and zeaxanthin showed high accumulation in NF-treated plants, whereas other carotenoid intermediates greatly decreased. Transcript levels of carotenoid biosynthetic genes, PSY1 and PDS, decreased in response to NF and OF, whereas plants in the later stage of NF treatment exhibited up-regulation of BCH and VDE as well as recovery of PDS. However, perturbed porphyrin biosynthesis by OF did not noticeably influence levels of carotenoid metabolites, regardless of the strong down-regulation of carotenoid biosynthetic genes. Both NF and OF plants appeared to provide enhanced protection against photooxidative damage, not only by scavenging of Mg-porphyrins, but also by up-regulating FC2, HO2, and Fe-chelatase, particularly with increased levels of zeaxanthin via up-regulation of BCH and VDE in NF plants. On the other hand, the up-regulation of GSA, PPO1, and CHLD under inhibition of carotenogenic flux may be derived from the necessity to recover impaired chloroplast biogenesis during photooxidative stress. Our study demonstrates that perturbations in carotenoid and porphyrin biosynthesis coordinate the expression

Perturbations in the Photosynthetic Pigment Status Result in Photooxidation-Induced Crosstalk between Carotenoid and Porphyrin Biosynthetic Pathways.

PubMed

Park, Joon-Heum; Tran, Lien H; Jung, Sunyo

2017-01-01

Possible crosstalk between the carotenoid and porphyrin biosynthetic pathways under photooxidative conditions was investigated by using their biosynthetic inhibitors, norflurazon (NF) and oxyfluorfen (OF). High levels of protoporphyrin IX (Proto IX) accumulated in rice plants treated with OF, whereas Proto IX decreased in plants treated with NF. Both NF and OF treatments resulted in greater decreases in MgProto IX, MgProto IX methyl ester, and protochlorophyllide. Activities and transcript levels of most porphyrin biosynthetic enzymes, particularly in the Mg-porphyrin branch, were greatly down-regulated in NF and OF plants. In contrast, the transcript levels of GSA, PPO1 , and CHLD as well as FC2 and HO2 were up-regulated in NF-treated plants, while only moderate increases in FC2 and HO2 were observed in the early stage of OF treatment. Phytoene, antheraxanthin, and zeaxanthin showed high accumulation in NF-treated plants, whereas other carotenoid intermediates greatly decreased. Transcript levels of carotenoid biosynthetic genes, PSY1 and PDS , decreased in response to NF and OF, whereas plants in the later stage of NF treatment exhibited up-regulation of BCH and VDE as well as recovery of PDS . However, perturbed porphyrin biosynthesis by OF did not noticeably influence levels of carotenoid metabolites, regardless of the strong down-regulation of carotenoid biosynthetic genes. Both NF and OF plants appeared to provide enhanced protection against photooxidative damage, not only by scavenging of Mg - porphyrins, but also by up-regulating FC2, HO2 , and Fe-chelatase, particularly with increased levels of zeaxanthin via up-regulation of BCH and VDE in NF plants. On the other hand, the up-regulation of GSA, PPO1 , and CHLD under inhibition of carotenogenic flux may be derived from the necessity to recover impaired chloroplast biogenesis during photooxidative stress. Our study demonstrates that perturbations in carotenoid and porphyrin biosynthesis coordinate the

Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

PubMed Central

2012-01-01

Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH) and fumarase (RoFUM1) were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2) was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1) than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner. PMID:22335940

The Nutrient-Sensing Hexosamine Biosynthetic Pathway as the Hub of Cancer Metabolic Rewiring.

PubMed

Chiaradonna, Ferdinando; Ricciardiello, Francesca; Palorini, Roberta

2018-06-02

Alterations in glucose and glutamine utilizing pathways and in fatty acid metabolism are currently considered the most significant and prevalent metabolic changes observed in almost all types of tumors. Glucose, glutamine and fatty acids are the substrates for the hexosamine biosynthetic pathway (HBP). This metabolic pathway generates the "sensing molecule" UDP- N -Acetylglucosamine (UDP-Glc N Ac). UDP-Glc N Ac is the substrate for the enzymes involved in protein N - and O -glycosylation, two important post-translational modifications (PTMs) identified in several proteins localized in the extracellular space, on the cell membrane and in the cytoplasm, nucleus and mitochondria. Since protein glycosylation controls several key aspects of cell physiology, aberrant protein glycosylation has been associated with different human diseases, including cancer. Here we review recent evidence indicating the tight association between the HBP flux and cell metabolism, with particular emphasis on the post-transcriptional and transcriptional mechanisms regulated by the HBP that may cause the metabolic rewiring observed in cancer. We describe the implications of both protein O - and N -glycosylation in cancer cell metabolism and bioenergetics; focusing our attention on the effect of these PTMs on nutrient transport and on the transcriptional regulation and function of cancer-specific metabolic pathways.

Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors.

PubMed

Suzuki, Hiroyoshi; Yokokura, Junpei; Ito, Tsukasa; Arai, Ryoma; Yokoyama, Chiaki; Toshima, Hiroaki; Nagata, Shinji; Asami, Tadao; Suzuki, Yoshihito

2014-10-01

Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Examination of Triacylglycerol Biosynthetic Pathways via De Novo Transcriptomic and Proteomic Analyses in an Unsequenced Microalga

PubMed Central

Guarnieri, Michael T.; Nag, Ambarish; Smolinski, Sharon L.; Darzins, Al; Seibert, Michael; Pienkos, Philip T.

2011-01-01

Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga. PMID:22043295

Modulation of guanosine nucleotides biosynthetic pathways enhanced GDP-L-fucose production in recombinant Escherichia coli.

PubMed

Lee, Won-Heong; Shin, So-Yeon; Kim, Myoung-Dong; Han, Nam Soo; Seo, Jin-Ho

2012-03-01

Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress.

PubMed

Gupta, Rupali; Singh, Akanksha; Srivastava, Madhumita; Singh, Vivek; Gupta, M M; Pandey, Rakesh

2017-02-03

Plant-associated beneficial microbes have been explored to fulfill the imperative function for plant health. However, their impact on the host secondary metabolite production and nematode disease management remains elusive. Our present work has shown that chitinolytic microbes viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 singly as well as in combination modulated the biosynthetic pathway of bacoside A and systemic defense mechanism against Meloidogyne incognita in Bacopa monnieri. Interestingly, expression of bacoside biosynthetic pathway genes (3-Hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase) were upregulated in plants treated with the microbial combination in the presence as well as in absence of M. incognita stress. These microbes not only augmented bacoside A production (1.5 fold) but also strengthened host resistance via enhancement in chlorophyll a, defense enzymes and phenolic compounds like gallic acid, syringic acid, ferulic acid and cinnamic acid. Furthermore, elevated lignification and callose deposition in the microbial combination treated plants corroborate well with the above findings. Overall, the results provide novel insights into the underlying mechanisms of priming by beneficial microbes and underscore their capacity to trigger bacoside A production in B. monnieri under biotic stress.

Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress

PubMed Central

Gupta, Rupali; Singh, Akanksha; Srivastava, Madhumita; Singh, Vivek; Gupta, M. M.; Pandey, Rakesh

2017-01-01

Plant-associated beneficial microbes have been explored to fulfill the imperative function for plant health. However, their impact on the host secondary metabolite production and nematode disease management remains elusive. Our present work has shown that chitinolytic microbes viz., Chitiniphilus sp. MTN22 and Streptomyces sp. MTN14 singly as well as in combination modulated the biosynthetic pathway of bacoside A and systemic defense mechanism against Meloidogyne incognita in Bacopa monnieri. Interestingly, expression of bacoside biosynthetic pathway genes (3-Hydroxy-3-methylglutaryl coenzyme A reductase, mevalonate diphosphate decarboxylase, and squalene synthase) were upregulated in plants treated with the microbial combination in the presence as well as in absence of M. incognita stress. These microbes not only augmented bacoside A production (1.5 fold) but also strengthened host resistance via enhancement in chlorophyll a, defense enzymes and phenolic compounds like gallic acid, syringic acid, ferulic acid and cinnamic acid. Furthermore, elevated lignification and callose deposition in the microbial combination treated plants corroborate well with the above findings. Overall, the results provide novel insights into the underlying mechanisms of priming by beneficial microbes and underscore their capacity to trigger bacoside A production in B. monnieri under biotic stress. PMID:28157221

Biosynthetic pathways of glycinebetaine in Thalassiosira pseudonana; functional characterization of enzyme catalyzing three-step methylation of glycine.

PubMed

Kageyama, Hakuto; Tanaka, Yoshito; Takabe, Teruhiro

2018-06-01

Betaine (trimethylglycine) is an important compatible solute that accumulates in response to abiotic stresses such as drought and salinity. Biosynthetic pathways of betaine have been extensively studied, but it remains to be clarified on algae. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems. Here we show that the genome sequence of Thalassiosira suggests the presence of two biosynthetic pathways for betaine, via three step methylation of glycine and via two step oxidation of choline. The choline oxidation via choline dehydrogenase was suggested and its sequential characteristics were analyzed. A candidate gene TpORF1 for glycine methylation encodes a protein consisted of 574 amino acids with two putative tandem repeat methyltransferase domains. The TpORF1 was expressed in E. coli, and the purified protein was shown to synthesize betaine via three step methylation of glycine and designated as TpGSDMT. The proteins containing C-terminal half or N-terminal half were expressed in E. coli and exhibited the methyl transferase activities with different substrate specificity for glycine, sarcosine and dimethylglycine. Upregulation of TpGSDMT transcription and betaine levels were observed at high salinity, suggesting the importance of TpGSDMT for salt tolerance in T. pseudonana cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Chloroplast biogenesis 87: Evidence of resonance excitation energy transfer between tetrapyrrole intermediates of the chlorophyll biosynthetic pathway and chlorophyll a.

PubMed

Kolossov, Vladimir L; Kopetz, Karen J; Rebeiz, Constantin A

2003-08-01

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination of chlorophyll (Chl) and thylakoid apoprotein biosynthesis. As a working model for future investigations, we have proposed three Chl-thylakoid apoprotein biosynthesis models, namely, a single-branched Chl biosynthetic pathway (SBP) single-location model, an SBP multilocation model and a multibranched Chl biosynthetic pathway (MBP) sublocation model. Rejection or validation of these models can be probed by determination of resonance excitation energy transfer between various tetrapyrrole intermediates of the Chl biosynthetic pathway and various thylakoid Chl-protein complexes. In this study we describe the detection of resonance energy transfer between protoporphyrin IX (Proto), Mg-Proto and its monomethyl ester (Mp(e)) and divinyl and monovinyl protochlorophyllide a (Pchlide a) and several Chl-protein complexes. Induction of various amounts of tetrapyrrole accumulation in green photoperiodically grown cucumber cotyledons and barley leaves was achieved by dark incubation of excised tissues with delta-aminolevulinic acid (ALA) and various concentrations of 2,2'-dipyridyl for various periods of time. Controls were incubated in distilled water. After plastid isolation, treated and control plastids were diluted in buffered glycerol to the same Chl concentration. Excitation spectra were then recorded at 77 K at emission maxima of about 686, 694 and 738 nm. Resonance excitation energy transfer from Proto, Mp(e) and Pchlide a to Chl-protein complexes emitting at 686, 694 and 738 nm was observed by calculation of treated minus control difference excitation spectra. The occurrence of resonance excitation energy transfer between anabolic tetrapyrroles and Chl-protein complexes appeared as well-defined excitation bands with excitation maxima corresponding to those of Proto, Mp(e) and Pchlide a. Furthermore, it appeared that resonance excitation energy transfer from

[Construction of Corynebacterium crenatum AS 1.542 δ argR and analysis of transcriptional levels of the related genes of arginine biosynthetic pathway].

PubMed

Chen, Xuelan; Tang, Li; Jiao, Haitao; Xu, Feng; Xiong, Yonghua

2013-01-04

ArgR, coded by the argR gene from Corynebacterium crenatum AS 1.542, acts as a negative regulator in arginine biosynthetic pathway. However, the effect of argR on transcriptional levels of the related biosynthetic genes has not been reported. Here, we constructed a deletion mutant of argR gene: C. crenatum AS 1.542 Delta argR using marker-less knockout technology, and compared the changes of transcriptional levels of the arginine biosynthetic genes between the mutant strain and the wild-type strain. We used marker-less knockout technology to construct C. crenatum AS 1.542 Delta argR and analyzed the changes of the relate genes at the transcriptional level using real-time fluorescence quantitative PCR. C. crenatum AS 1.542 Delta argR was successfully obtained and the transcriptional level of arginine biosynthetic genes in this mutant increased significantly with an average of about 162.1 folds. The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR. However, the deletion of this regulator does not result in a clear change in arginine production in the bacteria.

Leveraging microbial biosynthetic pathways for the generation of 'drop-in' biofuels.

PubMed

Zargar, Amin; Bailey, Constance B; Haushalter, Robert W; Eiben, Christopher B; Katz, Leonard; Keasling, Jay D

2017-06-01

Advances in retooling microorganisms have enabled bioproduction of 'drop-in' biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gas-turbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), 'drop-in' biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical properties (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure of ThiM from Vitamin B1 biosynthetic pathway of Staphylococcus aureus - Insights into a novel pro-drug approach addressing MRSA infections

NASA Astrophysics Data System (ADS)

Drebes, Julia; Künz, Madeleine; Windshügel, Björn; Kikhney, Alexey G.; Müller, Ingrid B.; Eberle, Raphael J.; Oberthür, Dominik; Cang, Huaixing; Svergun, Dmitri I.; Perbandt, Markus; Betzel, Christian; Wrenger, Carsten

2016-03-01

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today known to be a substantial threat for global health. Emerging multi-drug resistant bacteria have created a substantial need to identify and discover new drug targets and to develop novel strategies to treat bacterial infections. A promising and so far untapped antibiotic target is the biosynthesis of vitamin B1 (thiamin). Thiamin in its activated form, thiamin pyrophosphate, is an essential co-factor for all organisms. Therefore, thiamin analogous compounds, when introduced into the vitamin B1 biosynthetic pathway and further converted into non-functional co-factors by the bacterium can function as pro-drugs which thus block various co-factor dependent pathways. We characterized one of the key enzymes within the S. aureus vitamin B1 biosynthetic pathway, 5-(hydroxyethyl)-4-methylthiazole kinase (SaThiM; EC 2.7.1.50), a potential target for pro-drug compounds and analyzed the native structure of SaThiM and complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues.

Identification of averantin as an aflatoxin B1 precursor: placement in the biosynthetic pathway.

PubMed Central

Bennett, J W; Lee, L S; Shoss, S M; Boudreaux, G H

1980-01-01

A new blocked mutant of Aspergillus parasiticus produces no detectable aflatoxin B1, but accumulates several polyhydroxyanthraquinones. One of these pigments was identified as averantin. This is the first report of its formation by A. parasiticus. Radiotracer studies with [14C]averantin showed that 15.3% of label from averantin was incorporated into aflatoxin B1. This incorporation was blocked by dichlorvos. With radiotracers and other mutants, averantin was placed after norsolorinic acid and before averufin in the biosynthetic pathway in which the general steps are norsolorinic acid leads to averantin leads to averufin leads to versiconal hemiacetal acetate leads to versicolorin A leads to sterigmatocystin leads to aflatoxin B1. PMID:7377778

Overexpression of the Coq8 Kinase in Saccharomyces cerevisiae coq Null Mutants Allows for Accumulation of Diagnostic Intermediates of the Coenzyme Q6 Biosynthetic Pathway*

PubMed Central

Xie, Letian X.; Ozeir, Mohammad; Tang, Jeniffer Y.; Chen, Jia Y.; Jaquinod, Sylvie-Kieffer; Fontecave, Marc; Clarke, Catherine F.; Pierrel, Fabien

2012-01-01

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q6 biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q6 biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q6. Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q6 biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway. PMID:22593570

Overexpression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway.

PubMed

Xie, Letian X; Ozeir, Mohammad; Tang, Jeniffer Y; Chen, Jia Y; Jaquinod, Sylvie-Kieffer; Fontecave, Marc; Clarke, Catherine F; Pierrel, Fabien

2012-07-06

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q(6) biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q(6) biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q(6). Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q(6) biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.

Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes.

PubMed

Honda, Kohsuke; Maya, Shohei; Omasa, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Ohtake, Hisao

2010-08-02

Six thermophilic enzymes from Thermus thermophilus were used to construct an 'artificial bio-synthetic pathway' for the production of 2-deoxyribose 5-phosphate from fructose. By a simple operation using six recombinant Escherichia coli strains producing the thermophilic enzymes, respectively, fructose was converted to 2-deoxyribose 5-phosphate with a molar yield of 55%. Copyright 2010 Elsevier B.V. All rights reserved.

Leveraging microbial biosynthetic pathways for the generation of ‘drop-in’ biofuels

DOE PAGES

Zargar, Amin; Bailey, Constance B.; Haushalter, Robert W.; ...

2017-04-17

Advances in retooling microorganisms have enabled bioproduction of ‘drop-in’ biofuels, fuels that are compatible with existing spark-ignition, compression-ignition, and gasturbine engines. As the majority of petroleum consumption in the United States consists of gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), ‘drop-in’ biofuels that replace these petrochemical sources are particularly attractive. In this review, we discuss the application of aldehyde decarbonylases to produce gasoline substitutes from fatty acid products, a recently crystallized reductase that could hydrogenate jet fuel precursors from terpene synthases, and the exquisite control of polyketide synthases to produce biofuels with desired physical propertiesmore » (e.g., lower freezing points). With our increased understanding of biosynthetic logic of metabolic pathways, we discuss the unique advantages of fatty acid, terpene, and polyketide synthases for the production of bio-based gasoline, diesel and jet fuel.« less

In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana.

PubMed

Saifi, Monica; Nasrullah, Nazima; Ahmad, Malik Mobeen; Ali, Athar; Khan, Jawaid A; Abdin, M Z

2015-09-01

miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Towards a palaeosalinity proxy: hydrogen isotopic fractionation between source water and lipids produced via different biosynthetic pathways in haptophyte algae

NASA Astrophysics Data System (ADS)

Chivall, David; M'Boule, Daniela; Heinzelmann, Sandra M.; Kasper, Sebastian; Sinke-Schoen, Daniëlle; Sininnghe-Damsté, Jaap S.; Schouten, Stefan; van der Meer, Marcel T. J.

2014-05-01

Palaeosalinity is one of the most important oceanographic parameters that cannot currently be quantified with reasonable accuracy from sedimentary records. Hydrogen isotopic fractionation between water and alkenones is dependent, amongst other factors, upon the salinity in which alkenone-producing haptophyte algae grow and is represented by the fractionation factor, α, increasing with salinity.1 As such, the hydrogen isotopic composition of alkenones is emerging as a palaeosalinity proxy. Understanding the mechanism behind the sensitivity of fractionation to salinity is important for the correct application of the proxy, however this mechanism is currently unknown. Here we present hydrogen isotopic compositions of lipids produced via different biosynthetic pathways from batch cultures of Emiliania huxleyi CCMP 1516 and Isochrysis galbana CCMP 1323 grown over a range of salinities and discuss the possible sources of the sensitivity of hydrogen isotope fractionation to salinity. α for C37 alkenones (produced via an unknown biosynthetic pathway but assumed to be acetogenic; e.g.2) and that for C14:0, C16:0, and C18:1 fatty acids (acetogenic) from exponential growth phase I. galbana show a similar sensitivity to salinity, increasing at 0.0013-0.0019 per salinity unit (S-1). Meanwhile, in exponential growth phase E. huxleyi, α for C37 alkenones and α for brassicasterol (mevalonate pathway) increase at 0.0015-0.0022 S-1, but α for phytol (methylerythritol pathway) shows no significant relationship with salinity. These results suggest that fractionation is sensitive to salinity for lipids formed both in the chloroplast and cytosol. They also suggest that the sensitivity may either originate in glyceralde-3-phosphate or pyruvate but is then lost through hydrogen exchange with cell water during sugar rearrangements in the methylerythritol pathway or sensitivity originates with the production and consumption of acetate. References Schouten, S., Ossebaar, J., Schreiber

Specific inhibition of antenna bacteriochlorophyll synthesis in Chlorobium vibrioforme by anesthetic gases.

PubMed Central

Ormerod, J G; Nesbakken, T; Beale, S I

1990-01-01

The green sulfur bacterium Chlorobium vibrioforme contains two types of bacteriochlorophyll (Bchl). The minor pigment, Bchl a, is associated primarily with the cell membrane and its reaction centers; and the major light-harvesting antenna pigment, Bchl d, is found primarily in the chlorosomes, which are attached to the inner surface of the cell membrane. Anesthetic gases, such as N2O, ethylene, and acetylene, were found to inhibit the synthesis of Bchl d, but not of Bchl a, thus allowing the cells to grow at high light intensities with a greatly diminished content of antenna pigment. Chlorosomes were absent or sparse in inhibited cells. Porphyrins accumulated in the inhibited cells. The major one was identified as the Bchl precursor magnesium-protoporphyrin IX monomethyl ester (Mg-PPME) by comparative absorption and fluorescence spectroscopy and thin-layer chromatography of the porphyrin and its derivatives with those of authentic protoporphyrin IX. Small amounts of Mg-PPME were present in control cells, but the addition of inhibitor caused a rapid increase in the Mg-PPME concentration, accompanying the inhibition of Bchl d synthesis. Cells grown in the presence of ethephon (as a source of ethylene) and allowed to stand in dim light for long periods accumulated large amounts of PPME and other porphyrins and excreted or released porphyrins, which accumulated as a brown precipitate in the culture. Inhibition of Bchl d synthesis was relieved upon removal of the inhibitor. These results suggest that the gases act at a step in pigment biosynthesis that affects the utilization of Mg-PPME for isocyclic ring formation. Synthesis of Bchl d and Bchl a may be differentially affected by the gases because of compartmentation of their biosynthetic apparatus or because competition for precursors favors Bchl a synthesis. An ethephon-resistant mutant strain was isolated by selection for growth in dim, long-wavelength light. The mutant cells were also resistant to acetylene, but not

Protein-induced geometric constraints and charge transfer in bacteriochlorophyll-histidine complexes in LH2.

PubMed

Wawrzyniak, Piotr K; Alia, A; Schaap, Roland G; Heemskerk, Mattijs M; de Groot, Huub J M; Buda, Francesco

2008-12-14

Bacteriochlorophyll-histidine complexes are ubiquitous in nature and are essential structural motifs supporting the conversion of solar energy into chemically useful compounds in a wide range of photosynthesis processes. A systematic density functional theory study of the NMR chemical shifts for histidine and for bacteriochlorophyll-a-histidine complexes in the light-harvesting complex II (LH2) is performed using the BLYP functional in combination with the 6-311++G(d,p) basis set. The computed chemical shift patterns are consistent with available experimental data for positive and neutral(tau) (N(tau) protonated) crystalline histidines. The results for the bacteriochlorophyll-a-histidine complexes in LH2 provide evidence that the protein environment is stabilizing the histidine close to the Mg ion, thereby inducing a large charge transfer of approximately 0.5 electronic equivalent. Due to this protein-induced geometric constraint, the Mg-coordinated histidine in LH2 appears to be in a frustrated state very different from the formal neutral(pi) (N(pi) protonated) form. This finding could be important for the understanding of basic functional mechanisms involved in tuning the electronic properties and exciton coupling in LH2.

Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms.

PubMed

Coesel, Sacha; Oborník, Miroslav; Varela, Joao; Falciatore, Angela; Bowler, Chris

2008-08-06

Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP) enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several components of the

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

PubMed

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

Expression of resolvin D1 biosynthetic pathways in salivary epithelium.

PubMed

Leigh, N J; Nelson, J W; Mellas, R E; Aguirre, A; Baker, O J

2014-03-01

Resolvins are potent anti-inflammatory mediators derived from ω-3 fatty acids. Results from our previous studies indicated that resolvin D1 (RvD1) blocks pro-inflammatory responses in salivary glands. Furthermore, RvD1 enhances salivary epithelial integrity, demonstrating its potential use for the restoration of salivary gland function in Sjögren's syndrome (SS). We investigated whether the RvD1 biosynthetic machinery (e.g., cytosolic phospholipase A2, calcium-independent phospholipase A2, 12/15 and 5-lipoxygenase) is expressed in mouse submandibular glands (mSMG), using qPCR and Western blot analyses. Additionally, we determined the localization of RvD1 biosynthetic machinery in mSMG and human minor salivary glands (hMSG), with and without SS, using confocal microscopy. Finally, we measured RvD1 levels in cell supernatants from mSMG cell cultures and freshly isolated mSMG cells, with and without SS, using ELISA. Our results indicate that: (1) RvD1 machinery is expressed in mouse and human salivary glands; (2) polar distribution of RvD1 biosynthetic machinery is lost in hMSG with SS; (3) RvD1 levels in mSMG cell culture supernatants increased with time; and (4) RvD1 levels in mSMG cell supernatants, with and without SS, were similar. These studies demonstrate that the RvD1 biosynthesis machinery is expressed and functional in salivary glands with and without SS.

Functional Conservation of Coenzyme Q Biosynthetic Genes among Yeasts, Plants, and Humans

PubMed Central

Hayashi, Kazuhiro; Ogiyama, Yuki; Yokomi, Kazumasa; Nakagawa, Tsuyoshi; Kaino, Tomohiro; Kawamukai, Makoto

2014-01-01

Coenzyme Q (CoQ) is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3–9) that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana) to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants. PMID:24911838

Biosynthetic engineering of nonribosomal peptide synthetases.

PubMed

Kries, Hajo

2016-09-01

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Fluorescent probes for tracking the transfer of iron–sulfur cluster and other metal cofactors in biosynthetic reaction pathways

DOE PAGES

Vranish, James N.; Russell, William K.; Yu, Lusa E.; ...

2014-12-05

Iron–sulfur (Fe–S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe–S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe–S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe–2S] and [4Fe–4S] clusters), ligand environments ([2Fe–2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe–S clustermore » transfer reactions are monitored between two Fdx molecules that have identical Fe–S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe–2S]–DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe–S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe–S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. Lastly, we anticipate that this cluster detection methodology will transform the study of Fe–S cluster pathways and potentially other metal cofactor biosynthetic pathways.« less

The study of flavonolignan association patterns in fruits of diverging Silybum marianum (L.) Gaertn. chemotypes provides new insights into the silymarin biosynthetic pathway.

PubMed

Martinelli, Tommaso; Whittaker, Anne; Benedettelli, Stefano; Carboni, Andrea; Andrzejewska, Jadwiga

2017-12-01

Silymarin is the phytochemical with medicinal properties extracted from Silybum marianum (L.) Gaertn. fruits. Yet, little information is available about silymarin biosynthesis. Moreover, the generally accepted pathway, formulated thus far, is not in agreement with actual experimental measurements on flavonolignan contents. The present work analyses flavonolignan and taxifolin content in 201 S. marianum samples taking into consideration a wide phenotypic variability. Two stable chemotypes were identified: one characterized by both high silychristin and silybin content (chemotype A) and another by a high silydianin content (chemotype B). Through the correlation analysis of samples divided according to chemotype, it was possible to construct a simplified silymarin biosynthetic pathway that is sufficiently versatile in explaining experimental results responding to the actually unresolved questions about this process. The proposed pathway highlights that three separate and equally sized metabolite pools exist, namely: diastereoisomers A (silybin A plus isosilybin A), diastereoisomers B (silybin B plus isosilybin B) and silychristin. In both A and B diastereoisomers pools, isosilybin A and isosilybin B always represent a given amount of the metabolite flux through the specific metabolite pool suggesting the possible involvement of dirigent protein-like enzymes. We suggest that chemotype B possesses a complete silymarin biosynthetic pathway in which silydianin biosynthesis is enzymatically controlled. On the contrary, chemotype A is probably a natural mutant unable to biosynthesize silydianin. The present simplified pathway for silymarin biosynthesis will constitute an important tool for the further understanding of the reactions that drive flavonolignan biosynthesis in S. marianum. Copyright © 2017 Elsevier Ltd. All rights reserved.

Producing the Ethylene Signal: Regulation and Diversification of Ethylene Biosynthetic Enzymes1

PubMed Central

Booker, Matthew A.; DeLong, Alison

2015-01-01

Strictly controlled production of ethylene gas lies upstream of the signaling activities of this crucial regulator throughout the plant life cycle. Although the biosynthetic pathway is enzymatically simple, the regulatory circuits that modulate signal production are fine tuned to allow integration of responses to environmental and intrinsic cues. Recently identified posttranslational mechanisms that control ethylene production converge on one family of biosynthetic enzymes and overlay several independent reversible phosphorylation events and distinct mediators of ubiquitin-dependent protein degradation. Although the core pathway is conserved throughout seed plants, these posttranslational regulatory mechanisms may represent evolutionarily recent innovations. The evolutionary origins of the pathway and its regulators are not yet clear; outside the seed plants, numerous biochemical and phylogenetic questions remain to be addressed. PMID:26134162

Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Joon-Heum; Jung, Sunyo

In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F{sub v}/F{sub m}. NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced bymore » photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. - Highlights: • Two modes of photooxidation by carotenoid and tetrapyrrole biosynthetic inhibitors. • We examine differential alterations in chloroplast function and plastid signaling. • NF and OF cause differential alterations in chloroplast ultrastructure and function. • Photooxidation coordinates photosynthetic gene expression from nucleus and plastid.« less

Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling

PubMed Central

Zhan, Chuansong; Li, Xiaohua; Zhao, Zeying; Yang, Tewu; Wang, Xuekui; Luo, Biaobiao; Zhang, Qiyun; Hu, Yanru; Hu, Xuebo

2016-01-01

Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. Conclusion: A

Sex pheromone biosynthetic pathways are conserved between moths and the butterfly Bicyclus anynana

PubMed Central

Liénard, Marjorie A; Wang, Hong-Lei; Lassance, Jean-Marc; Löfstedt, Christer

2014-01-01

Although phylogenetically nested within the moths, butterflies have diverged extensively in a number of life history traits. Whereas moths rely greatly on chemical signals, visual advertisement is the hallmark of mate finding in butterflies. In the context of courtship, however, male chemical signals are widespread in both groups although they likely have multiple evolutionary origins. Here, we report that in males of the butterfly Bicyclus anynana, courtship scents are produced de novo via biosynthetic pathways shared with females of many moth species. We show that two of the pheromone components that play a major role in mate choice, namely the (Z)-9-tetradecenol and hexadecanal, are produced through the activity of a fatty acyl Δ11-desaturase and two specialized alcohol-forming fatty acyl reductases. Our study provides the first evidence of conservation and sharing of ancestral genetic modules for the production of FA-derived pheromones over a long evolutionary timeframe thereby reconciling mate communication in moths and butterflies. PMID:24862548

Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate Biosynthetic Pathway in the Brain.

PubMed

Zhu, Hongying; Wang, Ning; Yao, Lei; Chen, Qi; Zhang, Ran; Qian, Junchao; Hou, Yiwen; Guo, Weiwei; Fan, Sijia; Liu, Siling; Zhao, Qiaoyun; Du, Feng; Zuo, Xin; Guo, Yujun; Xu, Yan; Li, Jiali; Xue, Tian; Zhong, Kai; Song, Xiaoyuan; Huang, Guangming; Xiong, Wei

2018-06-14

Sunlight exposure is known to affect mood, learning, and cognition. However, the molecular and cellular mechanisms remain elusive. Here, we show that moderate UV exposure elevated blood urocanic acid (UCA), which then crossed the blood-brain barrier. Single-cell mass spectrometry and isotopic labeling revealed a novel intra-neuronal metabolic pathway converting UCA to glutamate (GLU) after UV exposure. This UV-triggered GLU synthesis promoted its packaging into synaptic vesicles and its release at glutamatergic terminals in the motor cortex and hippocampus. Related behaviors, like rotarod learning and object recognition memory, were enhanced after UV exposure. All UV-induced metabolic, electrophysiological, and behavioral effects could be reproduced by the intravenous injection of UCA and diminished by the application of inhibitor or short hairpin RNA (shRNA) against urocanase, an enzyme critical for the conversion of UCA to GLU. These findings reveal a new GLU biosynthetic pathway, which could contribute to some of the sunlight-induced neurobehavioral changes. Copyright © 2018 Elsevier Inc. All rights reserved.

Excitation energy transfer from the bacteriochlorophyll Soret band to carotenoids in the LH2 light-harvesting complex from Ectothiorhodospira haloalkaliphila is negligible.

PubMed

Razjivin, A P; Lukashev, E P; Kompanets, V O; Kozlovsky, V S; Ashikhmin, A A; Chekalin, S V; Moskalenko, A A; Paschenko, V Z

2017-09-01

Pathways of intramolecular conversion and intermolecular electronic excitation energy transfer (EET) in the photosynthetic apparatus of purple bacteria remain subject to debate. Here we experimentally tested the possibility of EET from the bacteriochlorophyll (BChl) Soret band to the singlet S 2 level of carotenoids using femtosecond pump-probe measurements and steady-state fluorescence excitation and absorption measurements in the near-ultraviolet and visible spectral ranges. The efficiency of EET from the Soret band of BChl to S 2 of the carotenoids in light-harvesting complex LH2 from the purple bacterium Ectothiorhodospira haloalkaliphila appeared not to exceed a few percent.

Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core.

PubMed

Hoshino, Tsutomu

2011-09-01

Violacein is a natural violet pigment produced by several gram-negative bacteria, including Chromobacterium violaceum, Janthinobacterium lividum, and Pseudoalteromonas tunicata D2, among others. This pigment has potential medical applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs. The structure of violacein consists of three units: a 5-hydroxyindole, an oxindole, and a 2-pyrrolidone. The biosynthetic origins of hydrogen, nitrogen, and carbon in the pyrrolidone nucleus were established by feeding experiments using various stable isotopically labeled tryptophans (Trps). Pro-S hydrogen of CH(2) at the 3-position of Trp is retained during biosynthesis. The nitrogen atom is exclusively from the α-amino group, and the skeletal carbon atoms originate from the side chains of the two Trp molecules. All three oxygen atoms in the violacein core are derived from molecular oxygen. The most interesting biosynthetic mechanism is the 1,2-shift of the indole nucleus on the left side of the violacein scaffold. The alternative Trp molecule is directly incorporated into the right side of the violacein core. This indole shift has been observed only in violacein biosynthesis, despite the large number of natural products having been isolated. There were remarkable advances in biosynthetic studies in 2006-2008. During the 3 years, most of the intermediates and the complete pathway were established. Two independent processes are involved: the enzymatic process catalyzed by the five proteins VioABCDE or the alternative nonenzymatic oxidative decarboxylation reactions. The X-ray crystallographic structure of VioE that mediates the indole rearrangement reaction was recently identified, and the mechanism of the indole shift is discussed here.

Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

PubMed

Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

2015-09-30

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling.

PubMed

Park, Joon-Heum; Jung, Sunyo

2017-01-22

In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F v /F m . NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Pheromone biosynthetic pathways in the moths Heliothis subflexa and Heliothis virescens.

PubMed

Choi, Man-Yeon; Groot, Astrid; Jurenka, Russell A

2005-06-01

Sex pheromones of many moth species have relatively simple structures consisting of a hydrocarbon chain with a functional group and one to several double bonds. These sex pheromones are derived from fatty acids through specific biosynthetic pathways. We investigated the incorporation of deuterium-labeled tetradecanoic, hexadecanoic, and octadecanoic acid precursors into pheromone components of Heliothis subflexa and Heliothis virescens. The two species utilize (Z)11-hexadecenal as the major pheromone component, which is produced by Delta11 desaturation of hexadecanoic acid. H. subflexa also produced (Z)11-hexadecanol and (Z)-11-hexadecenyl acetate via Delta11 desaturation. In H. subflexa, octadecanoic acid was used to biosynthesize the minor pheromone components (Z)9-hexadecenal, (Z)9-hexadecenol, and (Z)9-hexadecenyl acetate. These minor components are produced by Delta11 desaturation of octadecanoic acid followed by one round of chain-shortening. In contrast, H. virescens used hexadecanoic acid as a substrate to form (Z)11-hexadecenal and (Z)11-hexadecenol and hexadecenal. H. virescens also produced (Z)9-tetradecenal by Delta11 desaturation of the hexadecanoic acid followed by one round of chain-shortening and reduction. Tetradecanoic acid was not utilized as a precursor to form Z9-14:Ald in H. virescens. This labeling pattern indicates that the Delta11 desaturase is the only active desaturase present in the pheromone gland cells of both species.

"Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis.

PubMed

Sato, Naoki; Awai, Koichiro

2017-11-01

Lipid biosynthesis within the chloroplast, or more generally plastids, was conventionally called "prokaryotic pathway," which produces glycerolipids bearing C18 acids at the sn-1 position and C16 acids at the sn-2 position, as in cyanobacteria such as Anabaena and Synechocystis. This positional specificity is determined during the synthesis of phosphatidate, which is a precursor to diacylglycerol, the acceptor of galactose for the synthesis of galactolipids. The first acylation at sn-1 is catalyzed by glycerol-3-phosphate acyltransferase (GPAT or GPT), whereas the second acylation at sn-2 is performed by lysophosphatidate acyltransferase (LPAAT, AGPAT, or PlsC). Here we present comprehensive phylogenomic analysis of the origins of various acyltransferases involved in the synthesis of phosphatidate, as well as phosphatidate phosphatases in the chloroplasts. The results showed that the enzymes involved in the two steps of acylation in cyanobacteria and chloroplasts are entirely phylogenetically unrelated despite a previous report stating that the chloroplast LPAAT (ATS2) and cyanobacterial PlsC were sister groups. Phosphatidate phosphatases were separated into eukaryotic and prokaryotic clades, and the chloroplast enzymes were not of cyanobacterial origin, in contrast with another previous report. These results indicate that the lipid biosynthetic pathway in the chloroplasts or plastids did not originate from the cyanobacterial endosymbiont and is not "prokaryotic" in the context of endosymbiotic theory of plastid origin. This is another line of evidence for the discontinuity of plastids and cyanobacteria, which has been suggested in the glycolipid biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Triterpenoid Saponin Biosynthetic Pathway Profiling and Candidate Gene Mining of the Ilex asprella Root Using RNA-Seq

PubMed Central

Zheng, Xiasheng; Xu, Hui; Ma, Xinye; Zhan, Ruoting; Chen, Weiwen

2014-01-01

Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins. PMID:24722569

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

DOE PAGES

Javidpour, Pouya; Deutsch, Samuel; Mutalik, Vivek K.; ...

2016-03-14

Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present studymore » was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Javidpour, Pouya; Deutsch, Samuel; Mutalik, Vivek K.

Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present studymore » was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

PubMed Central

Javidpour, Pouya; Deutsch, Samuel; Mutalik, Vivek K.; Hillson, Nathan J.; Petzold, Christopher J.; Keasling, Jay D.; Beller, Harry R.

2016-01-01

Ladderanes are hydrocarbon chains with three or five linearly concatenated cyclobutane rings that are uniquely produced as membrane lipid components by anammox (anaerobic ammonia-oxidizing) bacteria. By virtue of their angle and torsional strain, ladderanes are unusually energetic compounds, and if produced biochemically by engineered microbes, could serve as renewable, high-energy-density jet fuel components. The biochemistry and genetics underlying the ladderane biosynthetic pathway are unknown, however, previous studies have identified a pool of 34 candidate genes from the anammox bacterium, Kuenenia stuttgartiensis, some or all of which may be involved with ladderane fatty acid biosynthesis. The goal of the present study was to establish a systematic means of testing the candidate genes from K. stuttgartiensis for involvement in ladderane biosynthesis through heterologous expression in E. coli under anaerobic conditions. This study describes an efficient means of assembly of synthesized, codon-optimized candidate ladderane biosynthesis genes in synthetic operons that allows for changes to regulatory element sequences, as well as modular assembly of multiple operons for simultaneous heterologous expression in E. coli (or potentially other microbial hosts). We also describe in vivo functional tests of putative anammox homologs of the phytoene desaturase CrtI, which plays an important role in the hypothesized ladderane pathway, and a method for soluble purification of one of these enzymes. This study is, to our knowledge, the first experimental effort focusing on the role of specific anammox genes in the production of ladderanes, and lays the foundation for future efforts toward determination of the ladderane biosynthetic pathway. Our substantial, but far from comprehensive, efforts at elucidating the ladderane biosynthetic pathway were not successful. We invite the scientific community to take advantage of the considerable synthetic biology resources and

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants.

PubMed

Ruiz-López, Noemi; Sayanova, Olga; Napier, Johnathan A; Haslam, Richard P

2012-04-01

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens.

PubMed

Gulick, Andrew M

2017-08-02

Covering: up to 2017.Natural products are important secondary metabolites produced by bacterial and fungal species that play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion. Because of the pharmaceutical activity of many NRPS products, much effort has gone into the exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS pathways have been identified and characterized from both terrestrial and marine bacterial sources. Recently, several NRPS pathways in human commensal bacterial species have been identified that produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these organisms has prompted calls from multiple international agencies to identify novel antibacterial targets and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains several NRPS biosynthetic gene clusters. While some have been well characterized and produce known natural products with important biological roles in microbial physiology, others have yet to be investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel NRPS products may lead to a better understanding of the chemical communication used by human pathogens and potentially to the discovery of novel therapeutic approaches.

Alteration of the coenzyme A biosynthetic pathway in neurodegeneration with brain iron accumulation syndromes.

PubMed

Venco, Paola; Dusi, Sabrina; Valletta, Lorella; Tiranti, Valeria

2014-08-01

NBIA (neurodegeneration with brain iron accumulation) comprises a heterogeneous group of neurodegenerative diseases having as a common denominator, iron overload in specific brain areas, mainly basal ganglia and globus pallidus. In the past decade a bunch of disease genes have been identified, but NBIA pathomechanisms are still not completely clear. PKAN (pantothenate kinase-associated neurodegeneration), an autosomal recessive disorder with progressive impairment of movement, vision and cognition, is the most common form of NBIA. It is caused by mutations in the PANK2 (pantothenate kinase 2) gene, coding for a mitochondrial enzyme that phosphorylates vitamin B5 in the first reaction of the CoA (coenzyme A) biosynthetic pathway. A distinct form of NBIA, denominated CoPAN (CoA synthase protein-associated neurodegeneration), is caused by mutations in the CoASY (CoA synthase) gene coding for a bifunctional mitochondrial enzyme, which catalyses the final steps of CoA biosynthesis. These two inborn errors of CoA metabolism further support the concept that dysfunctions in CoA synthesis may play a crucial role in the pathogenesis of NBIA.

Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

PubMed

Netzer, Roman; Stafsnes, Marit H; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

2010-11-01

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides.

PubMed

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

NASA Astrophysics Data System (ADS)

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N.; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

Characterization of gossypol biosynthetic pathway

PubMed Central

Tian, Xiu; Ruan, Ju-Xin; Huang, Jin-Quan; Fang, Xin; Chen, Zhi-Wen; Hong, Hui; Wang, Ling-Jian; Mao, Ying-Bo; Lu, Shan; Zhang, Tian-Zhen; Chen, Xiao-Ya

2018-01-01

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion. PMID:29784821

Comparative transcriptomic analysis of key genes involved in flavonoid biosynthetic pathway and identification of a flavonol synthase from Artemisia annua L.

PubMed

Liu, S; Liu, L; Tang, Y; Xiong, S; Long, J; Liu, Z; Tian, N

2017-07-01

The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

Identification of early fumonisin biosynthetic intermediates by inactivation of the FUM6 gene in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

Fumonisins are polyketide mycotoxins produced by the maize pathogen Fusarium verticillioides and are associated with multiple human and animal diseases. A fumonisin biosynthetic pathway has been proposed, but structures of early pathway intermediates have not been demonstrated. The F. verticillioide...

Biosynthetic Genes for the Tetrodecamycin Antibiotics.

PubMed

Gverzdys, Tomas; Nodwell, Justin R

2016-07-15

We recently described 13-deoxytetrodecamycin, a new member of the tetrodecamycin family of antibiotics. A defining feature of these molecules is the presence of a five-membered lactone called a tetronate ring. By sequencing the genome of a producer strain, Streptomyces sp. strain WAC04657, and searching for a gene previously implicated in tetronate ring formation, we identified the biosynthetic genes responsible for producing 13-deoxytetrodecamycin (the ted genes). Using the ted cluster in WAC04657 as a reference, we found related clusters in three other organisms: Streptomyces atroolivaceus ATCC 19725, Streptomyces globisporus NRRL B-2293, and Streptomyces sp. strain LaPpAH-202. Comparing the four clusters allowed us to identify the cluster boundaries. Genetic manipulation of the cluster confirmed the involvement of the ted genes in 13-deoxytetrodecamycin biosynthesis and revealed several additional molecules produced through the ted biosynthetic pathway, including tetrodecamycin, dihydrotetrodecamycin, and another, W5.9, a novel molecule. Comparison of the bioactivities of these four molecules suggests that they may act through the covalent modification of their target(s). The tetrodecamycins are a distinct subgroup of the tetronate family of secondary metabolites. Little is known about their biosynthesis or mechanisms of action, making them an attractive subject for investigation. In this paper we present the biosynthetic gene cluster for 13-deoxytetrodecamycin in Streptomyces sp. strain WAC04657. We identify related clusters in several other organisms and show that they produce related molecules. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Biosynthetic Genes for the Tetrodecamycin Antibiotics

PubMed Central

Gverzdys, Tomas

2016-01-01

ABSTRACT We recently described 13-deoxytetrodecamycin, a new member of the tetrodecamycin family of antibiotics. A defining feature of these molecules is the presence of a five-membered lactone called a tetronate ring. By sequencing the genome of a producer strain, Streptomyces sp. strain WAC04657, and searching for a gene previously implicated in tetronate ring formation, we identified the biosynthetic genes responsible for producing 13-deoxytetrodecamycin (the ted genes). Using the ted cluster in WAC04657 as a reference, we found related clusters in three other organisms: Streptomyces atroolivaceus ATCC 19725, Streptomyces globisporus NRRL B-2293, and Streptomyces sp. strain LaPpAH-202. Comparing the four clusters allowed us to identify the cluster boundaries. Genetic manipulation of the cluster confirmed the involvement of the ted genes in 13-deoxytetrodecamycin biosynthesis and revealed several additional molecules produced through the ted biosynthetic pathway, including tetrodecamycin, dihydrotetrodecamycin, and another, W5.9, a novel molecule. Comparison of the bioactivities of these four molecules suggests that they may act through the covalent modification of their target(s). IMPORTANCE The tetrodecamycins are a distinct subgroup of the tetronate family of secondary metabolites. Little is known about their biosynthesis or mechanisms of action, making them an attractive subject for investigation. In this paper we present the biosynthetic gene cluster for 13-deoxytetrodecamycin in Streptomyces sp. strain WAC04657. We identify related clusters in several other organisms and show that they produce related molecules. PMID:27137499

Tryptophan biosynthetic enzymes of Staphylococcus aureus.

PubMed

Proctor, A R; Kloos, W E

1973-04-01

Tryptophan biosynthetic enzymes were assayed in various tryptophan mutants of Staphylococcus aureus strain 655 and the wild-type parent. All mutants, except trpB mutants, lacked only the activity corresponding to the particular biosynthetic block, as suggested previously by analysis of accumulated intermediates and auxonography. Tryptophan synthetase A was not detected in extracts of either trpA or trpB mutants but appeared normal in other mutants. Mutants in certain other classes exhibited partial loss of another particular tryptophan enzyme activity. Tryptophan synthetase B activity was not detected in cell extract preparations but was detected in whole cells. The original map order proposed for the S. aureus tryptophan gene cluster was clarified by the definition of trpD (phosphoribosyl transferase(-)) and trpF (phosphoribosyl anthranilate isomerase(-)) mutants. These mutants were previously unresolved and designated as trp(DF) mutants (anthranilate accumulators). Phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase enzymes were separable by molecular sieve chromatography, suggesting that these functions are coded by separate loci. Molecular sieve chromatography failed to reveal aggregates involving anthranilate synthetase, phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole-3-glycerol phosphate synthetase, and this procedure provided an estimate of the molecular weights of these enzymes. Tryptophan was shown to repress synthesis of all six tryptophan biosynthetic enzymes, and derepression of all six activities was incident upon tryptophan starvation. Tryptophan inhibited the activity of anthranilate synthetase, the first enzyme of the pathway.

Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production

PubMed Central

Remali, Juwairiah; Sarmin, Nurul ‘Izzah Mohd; Ng, Chyan Leong; Tiong, John J.L.; Aizat, Wan M.; Keong, Loke Kok

2017-01-01

Background Streptomyces are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy) carbonyl) phenazine-1-carboxylic acid (HCPCA) extracted from Streptomyces kebangsaanensis, an endophyte isolated from the ethnomedicinal Portulaca oleracea. Methods The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s) believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of S. kebangsaanensis in producing other useful secondary metabolites. Results The S. kebangsaanensis genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35%) consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis. Discussion The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of S. kebangsaanensis for producing various antibiotics and secondary metabolites. PMID:29201559

Investigation of early molybdopterin biosynthetic intermediates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wuebbens, M.M.; Rajagopalan, K.V.

1991-03-11

Little information is available regarding the early steps in the biosynthetic pathway of molybdopterin (MPT). In order to explore these early reactions, and in particular to investigate the origin of the ring and side chain carbons of MPT, a metabolic approach employing the incorporation of {sup 14}C label was chosen. This method was facilitated by the recent purification and characterization of desulfomolybdopterin 2{prime},4{prime}-cyclic phosphate, the precursor which is converted directly to active molybdopterin in Escherichia coli by the addition of vicinal sulfurs to the side chain. This labile precursor readily oxidizes to Compound Z, a stable 6-alkyl pterin which retainsmore » all of the carbon atoms present in molybdopterin. Compound Z, rather than molybdopterin itself was chosen as the end product for labeling due to its overproduction in some MPT-deficient strains, as well as its stability and ease of purification. The authors report here the isolation of {sup 14}C-labelled Compound Z from E.coli chlN cells cultured in minimal media supplemented with U-{sup 14}C guanosine. Successive cleavage of the side chain carbons by permanganate treatment and UV light produced a decrease in the specific radioactivity of the resulting pterins. These data indicate that the early portion of the molybdopterin biosynthetic pathway may be similar to that of the bioactive pterins folate and biopterin, both of which are derived from guanosine triphosphate.« less

A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

PubMed

Pi, Borui; Yu, Dongliang; Dai, Fangwei; Song, Xiaoming; Zhu, Congyi; Li, Hongye; Yu, Yunsong

2015-01-01

Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

A Genomics Based Discovery of Secondary Metabolite Biosynthetic Gene Clusters in Aspergillus ustus

PubMed Central

Pi, Borui; Yu, Dongliang; Dai, Fangwei; Song, Xiaoming; Zhu, Congyi; Li, Hongye; Yu, Yunsong

2015-01-01

Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic. PMID:25706180

Carotenoid Biosynthetic Pathways Are Regulated by a Network of Multiple Cascades of Alternative Sigma Factors in Azospirillum brasilense Sp7.

PubMed

Rai, Ashutosh Kumar; Dubey, Ashutosh Prakash; Kumar, Santosh; Dutta, Debashis; Mishra, Mukti Nath; Singh, Bhupendra Narain; Tripathi, Anil Kumar

2016-11-01

Carotenoids constitute an important component of the defense system against photooxidative stress in bacteria. In Azospirillum brasilense Sp7, a nonphotosynthetic rhizobacterium, carotenoid synthesis is controlled by a pair of extracytoplasmic function sigma factors (RpoEs) and their cognate zinc-binding anti-sigma factors (ChrRs). Its genome harbors two copies of the gene encoding geranylgeranyl pyrophosphate synthase (CrtE), the first critical step in the carotenoid biosynthetic pathway in bacteria. Inactivation of each of two crtE paralogs found in A. brasilense caused reduction in carotenoid content, suggesting their involvement in carotenoid synthesis. However, the effect of crtE1 deletion was more pronounced than that of crtE2 deletion. Out of the five paralogs of rpoH in A. brasilense, overexpression of rpoH1 and rpoH2 enhanced carotenoid synthesis. Promoters of crtE2 and rpoH2 were found to be dependent on RpoH2 and RpoE1, respectively. Using a two-plasmid system in Escherichia coli, we have shown that the crtE2 gene of A. brasilense Sp7 is regulated by two cascades of sigma factors: one consisting of RpoE1and RpoH2 and the other consisting of RpoE2 and RpoH1. In addition, expression of crtE1 was upregulated indirectly by RpoE1 and RpoE2. This study shows, for the first time in any carotenoid-producing bacterium, that the regulation of carotenoid biosynthetic pathway involves a network of multiple cascades of alternative sigma factors. Carotenoids play a very important role in coping with photooxidative stress in prokaryotes and eukaryotes. Although extracytoplasmic function (ECF) sigma factors are known to directly regulate the expression of carotenoid biosynthetic genes in bacteria, regulation of carotenoid biosynthesis by one or multiple cascades of sigma factors had not been reported. This study provides the first evidence of the involvement of multiple cascades of sigma factors in the regulation of carotenoid synthesis in any bacterium by showing the

Carotenoid Biosynthetic Pathways Are Regulated by a Network of Multiple Cascades of Alternative Sigma Factors in Azospirillum brasilense Sp7

PubMed Central

Rai, Ashutosh Kumar; Dubey, Ashutosh Prakash; Kumar, Santosh; Dutta, Debashis; Mishra, Mukti Nath; Singh, Bhupendra Narain

2016-01-01

ABSTRACT Carotenoids constitute an important component of the defense system against photooxidative stress in bacteria. In Azospirillum brasilense Sp7, a nonphotosynthetic rhizobacterium, carotenoid synthesis is controlled by a pair of extracytoplasmic function sigma factors (RpoEs) and their cognate zinc-binding anti-sigma factors (ChrRs). Its genome harbors two copies of the gene encoding geranylgeranyl pyrophosphate synthase (CrtE), the first critical step in the carotenoid biosynthetic pathway in bacteria. Inactivation of each of two crtE paralogs found in A. brasilense caused reduction in carotenoid content, suggesting their involvement in carotenoid synthesis. However, the effect of crtE1 deletion was more pronounced than that of crtE2 deletion. Out of the five paralogs of rpoH in A. brasilense, overexpression of rpoH1 and rpoH2 enhanced carotenoid synthesis. Promoters of crtE2 and rpoH2 were found to be dependent on RpoH2 and RpoE1, respectively. Using a two-plasmid system in Escherichia coli, we have shown that the crtE2 gene of A. brasilense Sp7 is regulated by two cascades of sigma factors: one consisting of RpoE1and RpoH2 and the other consisting of RpoE2 and RpoH1. In addition, expression of crtE1 was upregulated indirectly by RpoE1 and RpoE2. This study shows, for the first time in any carotenoid-producing bacterium, that the regulation of carotenoid biosynthetic pathway involves a network of multiple cascades of alternative sigma factors. IMPORTANCE Carotenoids play a very important role in coping with photooxidative stress in prokaryotes and eukaryotes. Although extracytoplasmic function (ECF) sigma factors are known to directly regulate the expression of carotenoid biosynthetic genes in bacteria, regulation of carotenoid biosynthesis by one or multiple cascades of sigma factors had not been reported. This study provides the first evidence of the involvement of multiple cascades of sigma factors in the regulation of carotenoid synthesis in any

Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

PubMed

Ipekoğlu, Emre M; Göçmen, Koray; Öz, Mehmet T; Gürgan, Muazzez; Yücel, Meral

2017-03-01

Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A natural plasmid uniquely encodes two biosynthetic pathways creating a potent anti-MRSA antibiotic.

PubMed

Fukuda, Daisuke; Haines, Anthony S; Song, Zhongshu; Murphy, Annabel C; Hothersall, Joanne; Stephens, Elton R; Gurney, Rachel; Cox, Russell J; Crosby, John; Willis, Christine L; Simpson, Thomas J; Thomas, Christopher M

2011-03-31

Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin. High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via "mutasynthesis" that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates. Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.

Spliced X-box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway

PubMed Central

Wang, Zhao V.; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L.; Morales, Cyndi R.; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A.; Rothermel, Beverly A.; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P.A.; Ferdous, Anwarul; Gillette, Thomas G.; Scherer, Philipp E.; Hill, Joseph A.

2014-01-01

SUMMARY The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis, by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. PMID:24630721

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

PubMed

Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

2014-03-13

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Ying; Bai, Silei; Liu, Jingjing

Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-framemore » gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.« less

The evolutionary life cycle of the polysaccharide biosynthetic gene cluster based on the Sphingomonadaceae.

PubMed

Wu, Mengmeng; Huang, Haidong; Li, Guoqiang; Ren, Yi; Shi, Zhong; Li, Xiaoyan; Dai, Xiaohui; Gao, Ge; Ren, Mengnan; Ma, Ting

2017-04-21

Although clustering of genes from the same metabolic pathway is a widespread phenomenon, the evolution of the polysaccharide biosynthetic gene cluster remains poorly understood. To determine the evolution of this pathway, we identified a scattered production pathway of the polysaccharide sanxan by Sphingomonas sanxanigenens NX02, and compared the distribution of genes between sphingan-producing and other Sphingomonadaceae strains. This allowed us to determine how the scattered sanxan pathway developed, and how the polysaccharide gene cluster evolved. Our findings suggested that the evolution of microbial polysaccharide biosynthesis gene clusters is a lengthy cyclic process comprising cluster 1 → scatter → cluster 2. The sanxan biosynthetic pathway proved the existence of a dispersive process. We also report the complete genome sequence of NX02, in which we identified many unstable genetic elements and powerful secretion systems. Furthermore, nine enzymes for the formation of activated precursors, four glycosyltransferases, four acyltransferases, and four polymerization and export proteins were identified. These genes were scattered in the NX02 genome, and the positive regulator SpnA of sphingans synthesis could not regulate sanxan production. Finally, we concluded that the evolution of the sanxan pathway was independent. NX02 evolved naturally as a polysaccharide producing strain over a long-time evolution involving gene acquisitions and adaptive mutations.

Regulatory role of hexosamine biosynthetic pathway on hepatic cancer stem cell marker CD133 under low glucose conditions

NASA Astrophysics Data System (ADS)

Lin, Shu-Hai; Liu, Tengfei; Ming, Xiaoyan; Tang, Zhi; Fu, Li; Schmitt-Kopplin, Philippe; Kanawati, Basem; Guan, Xin-Yuan; Cai, Zongwei

2016-02-01

Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.

Terminal steps of bacteriochlorophyll a phytol formation in purple photosynthetic bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shioi, Y.; Sasa, T.

1984-04-01

Four chemically different bacteriochlorophylls (Bchls) a esterified with geranylgeraniol, dihydrogeranyl-geraniol, tetrahydrogeraniol, and phytol have been detected by high-pressure liquid chromatography in cell extracts from Rhodopseudomonas sphaeroides and Chromatium vinosum. Bchl a containing phytol is the principal component, and the other three Bchls a comprise about 4% of the total Bchls a in stationary-phase cells of R. sphaeroides and C. vinosum. The high levels of the minor pigments occur in the beginning of Bchl a phytol formation, indicating that they are not degradation products, but intermediates of Bchl a phytol formation.

An Integrated Metabolomic and Genomic Mining Workflow To Uncover the Biosynthetic Potential of Bacteria

PubMed Central

Maansson, Maria; Vynne, Nikolaj G.; Klitgaard, Andreas; Nybo, Jane L.; Melchiorsen, Jette; Nguyen, Don D.; Sanchez, Laura M.; Ziemert, Nadine; Dorrestein, Pieter C.

2016-01-01

ABSTRACT Microorganisms are a rich source of bioactives; however, chemical identification is a major bottleneck. Strategies that can prioritize the most prolific microbial strains and novel compounds are of great interest. Here, we present an integrated approach to evaluate the biosynthetic richness in bacteria and mine the associated chemical diversity. Thirteen strains closely related to Pseudoalteromonas luteoviolacea isolated from all over the Earth were analyzed using an untargeted metabolomics strategy, and metabolomic profiles were correlated with whole-genome sequences of the strains. We found considerable diversity: only 2% of the chemical features and 7% of the biosynthetic genes were common to all strains, while 30% of all features and 24% of the genes were unique to single strains. The list of chemical features was reduced to 50 discriminating features using a genetic algorithm and support vector machines. Features were dereplicated by tandem mass spectrometry (MS/MS) networking to identify molecular families of the same biosynthetic origin, and the associated pathways were probed using comparative genomics. Most of the discriminating features were related to antibacterial compounds, including the thiomarinols that were reported from P. luteoviolacea here for the first time. By comparative genomics, we identified the biosynthetic cluster responsible for the production of the antibiotic indolmycin, which could not be predicted with standard methods. In conclusion, we present an efficient, integrative strategy for elucidating the chemical richness of a given set of bacteria and link the chemistry to biosynthetic genes. IMPORTANCE We here combine chemical analysis and genomics to probe for new bioactive secondary metabolites based on their pattern of distribution within bacterial species. We demonstrate the usefulness of this combined approach in a group of marine Gram-negative bacteria closely related to Pseudoalteromonas luteoviolacea, which is a

Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

PubMed

Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N; Barny, Marie-Anne

2014-06-27

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.

PubMed

Currin, Andrew; Dunstan, Mark S; Johannissen, Linus O; Hollywood, Katherine A; Vinaixa, Maria; Jervis, Adrian J; Swainston, Neil; Rattray, Nicholas J W; Gardiner, John M; Kell, Douglas B; Takano, Eriko; Toogood, Helen S; Scrutton, Nigel S

2018-03-02

The realization of a synthetic biology approach to microbial (1 R ,2 S ,5 R )-( - )-menthol ( 1 ) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing ( R )-(+)-pulegone (( R )- 2 ) from (+)- cis -isopulegone ( 3 ). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate 1 from 3 . This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.

Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts.

PubMed

Hugueney, P; Badillo, A; Chen, H C; Klein, A; Hirschberg, J; Camara, B; Kuntz, M

1995-09-01

The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non-green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to beta-carotene when expressed in Escherichia coli. The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species-specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene beta-cyclase activity when expressed in E. coli. A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re-channeling of linear carotenoids into beta-cyclic carotenoids in C. annuum ripening fruits.

Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster

PubMed Central

2012-01-01

Background The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(M)BOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8) form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5) belonging to the same CYP71C subfamily. The origin of this cluster is unknown. Results We show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase) and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region. Conclusions These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2) at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster. PMID:22577841

A R2R3-MYB Transcription Factor from Epimedium sagittatum Regulates the Flavonoid Biosynthetic Pathway

PubMed Central

Lv, Haiyan; Luo, Ming; Zeng, Shaohua; Pattanaik, Sitakanta; Yuan, Ling; Wang, Ying

2013-01-01

Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants. PMID:23936468

Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster.

PubMed

Dutartre, Leslie; Hilliou, Frédérique; Feyereisen, René

2012-05-11

The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(M)BOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8) form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5) belonging to the same CYP71C subfamily. The origin of this cluster is unknown. We show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase) and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region. These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2) at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.

Genome mining-directed activation of a silent angucycline biosynthetic gene cluster in Streptomyces chattanoogensis.

PubMed

Zhou, Zhenxing; Xu, Qingqing; Bu, Qingting; Guo, Yuanyang; Liu, Shuiping; Liu, Yu; Du, Yiling; Li, Yongquan

2015-02-09

Genomic sequencing of actinomycetes has revealed the presence of numerous gene clusters seemingly capable of natural product biosynthesis, yet most clusters are cryptic under laboratory conditions. Bioinformatics analysis of the completely sequenced genome of Streptomyces chattanoogensis L10 (CGMCC 2644) revealed a silent angucycline biosynthetic gene cluster. The overexpression of a pathway-specific activator gene under the constitutive ermE* promoter successfully triggered the expression of the angucycline biosynthetic genes. Two novel members of the angucycline antibiotic family, chattamycins A and B, were further isolated and elucidated. Biological activity assays demonstrated that chattamycin B possesses good antitumor activities against human cancer cell lines and moderate antibacterial activities. The results presented here provide a feasible method to activate silent angucycline biosynthetic gene clusters to discover potential new drug leads. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Complete Proteomic-Based Enzyme Reaction and Inhibition Kinetics Reveal How Monolignol Biosynthetic Enzyme Families Affect Metabolic Flux and Lignin in Populus trichocarpa[W

PubMed Central

Wang, Jack P.; Naik, Punith P.; Chen, Hsi-Chuan; Shi, Rui; Lin, Chien-Yuan; Liu, Jie; Shuford, Christopher M.; Li, Quanzi; Sun, Ying-Hsuan; Tunlaya-Anukit, Sermsawat; Williams, Cranos M.; Muddiman, David C.; Ducoste, Joel J.; Sederoff, Ronald R.; Chiang, Vincent L.

2014-01-01

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants. PMID:24619611

Vibronic coupling explains the ultrafast carotenoid-to-bacteriochlorophyll energy transfer in natural and artificial light harvesters

NASA Astrophysics Data System (ADS)

Perlík, Václav; Seibt, Joachim; Cranston, Laura J.; Cogdell, Richard J.; Lincoln, Craig N.; Savolainen, Janne; Šanda, František; Mančal, Tomáš; Hauer, Jürgen

2015-06-01

The initial energy transfer steps in photosynthesis occur on ultrafast timescales. We analyze the carotenoid to bacteriochlorophyll energy transfer in LH2 Marichromatium purpuratum as well as in an artificial light-harvesting dyad system by using transient grating and two-dimensional electronic spectroscopy with 10 fs time resolution. We find that Förster-type models reproduce the experimentally observed 60 fs transfer times, but overestimate coupling constants, which lead to a disagreement with both linear absorption and electronic 2D-spectra. We show that a vibronic model, which treats carotenoid vibrations on both electronic ground and excited states as part of the system's Hamiltonian, reproduces all measured quantities. Importantly, the vibronic model presented here can explain the fast energy transfer rates with only moderate coupling constants, which are in agreement with structure based calculations. Counterintuitively, the vibrational levels on the carotenoid electronic ground state play the central role in the excited state population transfer to bacteriochlorophyll; resonance between the donor-acceptor energy gap and the vibrational ground state energies is the physical basis of the ultrafast energy transfer rates in these systems.

Physical Mapping of bchG, orf427, and orf177 in the Photosynthesis Gene Cluster of Rhodobacter sphaeroides: Functional Assignment of the Bacteriochlorophyll Synthetase Gene

PubMed Central

Addlesee, Hugh A.; Fiedor, Leszek; Hunter, C. Neil

2000-01-01

The purple photosynthetic bacterium Rhodobacter sphaeroides has within its genome a cluster of photosynthesis-related genes approximately 41 kb in length. In an attempt to identify genes involved in the terminal esterification stage of bacteriochlorophyll biosynthesis, a previously uncharacterized 5-kb region of this cluster was sequenced. Four open reading frames (ORFs) were identified, and each was analyzed by transposon mutagenesis. The product of one of these ORFs, bchG, shows close homologies with (bacterio)chlorophyll synthetases, and mutants in this gene were found to accumulate bacteriopheophorbide, the metal-free derivative of the bacteriochlorophyll precursor bacteriochlorophyllide, suggesting that bchG is responsible for the esterification of bacteriochlorophyllide with an alcohol moiety. This assignment of function to bchG was verified by the performance of assays demonstrating the ability of BchG protein, heterologously synthesized in Escherichia coli, to esterify bacteriochlorophyllide with geranylgeranyl pyrophosphate in vitro, thereby generating bacteriochlorophyll. This step is pivotal to the assembly of a functional photosystem in R. sphaeroides, a model organism for the study of structure-function relationships in photosynthesis. A second gene, orf177, is a member of a large family of isopentenyl diphosphate isomerases, while sequence homologies suggest that a third gene, orf427, may encode an assembly factor for photosynthetic complexes. The function of the remaining ORF, bchP, is the subject of a separate paper (H. Addlesee and C. N. Hunter, J. Bacteriol. 181:7248–7255, 1999). An operonal arrangement of the genes is proposed. PMID:10809697

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli

PubMed Central

Ma, Tian; Zhou, Yuanjie; Li, Xiaowei; Zhu, Fayin; Cheng, Yongbo; Liu, Yi; Deng, Zixin

2015-01-01

Abstract As a highly valued keto‐carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α‐Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole‐genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio‐Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high‐efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4‐fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future. PMID:26580858

Carotenoid-bacteriochlorophyll energy transfer in LH2 complexes studied with 10-fs time resolution.

PubMed

Polli, Dario; Cerullo, Giulio; Lanzani, Guglielmo; De Silvestri, Sandro; Hashimoto, Hideki; Cogdell, Richard J

2006-04-01

In this report, we present a study of carotenoid-bacteriochlorophyll energy transfer processes in two peripheral light-harvesting complexes (known as LH2) from purple bacteria. We use transient absorption spectroscopy with approximately 10 fs temporal resolution, which is necessary to observe the very fast energy relaxation processes. By comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it has been possible to directly evaluate their energy transfer efficiency to the bacteriochlorophylls. In the case of okenone in the LH2 complex from Chromatium purpuratum, we obtained an energy transfer efficiency of etaET2=63+/-2.5% from the optically active excited state (S2) and etaET1=61+/-2% from the optically dark state (S1); for rhodopin glucoside contained in the LH2 complex from Rhodopseudomonas acidophila these values become etaET2=49.5+/-3.5% and etaET1=5.1+/-1%. The measurements also enabled us to observe vibrational energy relaxation in the carotenoids' S1 state and real-time collective vibrational coherence initiated by the ultrashort pump pulses. Our results are important for understanding the dynamics of early events of photosynthesis and relating it to the structural arrangement of the chromophores.

Unexpectedly high bacteriochlorophyll a concentrations in neotropical tank bromeliads.

PubMed

Lehours, Anne-Catherine; Jeune, Anne-Hélène Le; Aguer, Jean-Pierre; Céréghino, Régis; Corbara, Bruno; Kéraval, Benoit; Leroy, Céline; Perrière, Fanny; Jeanthon, Christian; Carrias, Jean-François

2016-06-06

The contribution of bacteriochlorophyll a (BChl a) to photosynthetically driven electron transport is generally low in aquatic and terrestrial systems. Here, we provide evidence that anoxygenic bacterial phototrophy is widespread and substantial in water retained by tank bromeliads of a primary rainforest in French Guiana. An analysis of the water extracted from 104 randomly selected tank bromeliads using infrared fluorimetry suggested the overall presence of abundant anoxygenic phototrophic bacterial populations. We found that purple bacteria dominated these populations responsible for unusually high BChl a/chlorophyll a ratios (>50%). Our data suggest that BChl a-based phototrophy in tank bromeliads can have significant effects on the ecology of tank-bromeliad ecosystems and on the carbon and energy fluxes in Neotropical forests. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Cloning and Characterization of a Putative R2R3 MYB Transcriptional Repressor of the Rosmarinic Acid Biosynthetic Pathway from Salvia miltiorrhiza

PubMed Central

Zhang, Shuncang; Ma, Pengda; Yang, Dongfeng; Li, Wenjing; Liang, Zongsuo; Liu, Yan; Liu, Fenghua

2013-01-01

Salvia miltiorrhiza Bunge is one of the most renowned traditional medicinal plants in China. Phenolic acids that are derived from the rosmarinic acid pathway, such as rosmarinic acid and salvianolic acid B, are important bioactive components in S. miltiorrhiza. Accumulations of these compounds have been reported to be induced by various elicitors, while little is known about transcription factors that function in their biosynthetic pathways. We cloned a subgroup 4 R2R3 MYB transcription factor gene (SmMYB39) from S. miltiorrhiza and characterized its roles through overexpression and RNAi-mediated silencing. As the results showed, the content of 4-coumaric acid, rosmarinic acid, salvianolic acid B, salvianolic acid A and total phenolics was dramatically decreased in SmMYB39-overexpressing S. miltiorrhiza lines while being enhanced by folds in SmMYB39-RNAi lines. Quantitative real-time PCR and enzyme activities analyses showed that SmMYB39 negatively regulated transcripts and enzyme activities of 4-hydroxylase (C4H) and tyrosine aminotransferase (TAT). These data suggest that SmMYB39 is involved in regulation of rosmarinic acid pathway and acts as a repressor through suppressing transcripts of key enzyme genes. PMID:24039895

Biochemical analysis of the biosynthetic pathway of an anticancer tetracycline SF2575.

PubMed

Pickens, Lauren B; Kim, Woncheol; Wang, Peng; Zhou, Hui; Watanabe, Kenji; Gomi, Shuichi; Tang, Yi

2009-12-09

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4'-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogues. In this study, we identified, sequenced, and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features, are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant-group addition is C-9 glycosylation, C-4 salicylation, and O-4' angelylcylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity toward substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group toward structure-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto

Construction and engineering of large biochemical pathways via DNA assembler

PubMed Central

Shao, Zengyi; Zhao, Huimin

2015-01-01

Summary DNA assembler enables rapid construction and engineering of biochemical pathways in a one-step fashion by exploitation of the in vivo homologous recombination mechanism in Saccharomyces cerevisiae. It has many applications in pathway engineering, metabolic engineering, combinatorial biology, and synthetic biology. Here we use two examples including the zeaxanthin biosynthetic pathway and the aureothin biosynthetic gene cluster to describe the key steps in the construction of pathways containing multiple genes using the DNA assembler approach. Methods for construct design, pathway assembly, pathway confirmation, and functional analysis are shown. The protocol for fine genetic modifications such as site-directed mutagenesis for engineering the aureothin gene cluster is also illustrated. PMID:23996442

Role and regulation of coordinately expressed de novo purine biosynthetic enzymes PPAT and PAICS in lung cancer.

PubMed

Goswami, Moloy T; Chen, Guoan; Chakravarthi, Balabhadrapatruni V S K; Pathi, Satya S; Anand, Sharath K; Carskadon, Shannon L; Giordano, Thomas J; Chinnaiyan, Arul M; Thomas, Dafydd G; Palanisamy, Nallasivam; Beer, David G; Varambally, Sooryanarayana

2015-09-15

Cancer cells exhibit altered metabolism including aerobic glycolysis that channels several glycolytic intermediates into de novo purine biosynthetic pathway. We discovered increased expression of phosphoribosyl amidotransferase (PPAT) and phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) enzymes of de novo purine biosynthetic pathway in lung adenocarcinomas. Transcript analyses from next-generation RNA sequencing and gene expression profiling studies suggested that PPAT and PAICS can serve as prognostic biomarkers for aggressive lung adenocarcinoma. Immunohistochemical analysis of PAICS performed on tissue microarrays showed increased expression with disease progression and was significantly associated with poor prognosis. Through gene knockdown and over-expression studies we demonstrate that altering PPAT and PAICS expression modulates pyruvate kinase activity, cell proliferation and invasion. Furthermore we identified genomic amplification and aneuploidy of the divergently transcribed PPAT-PAICS genomic region in a subset of lung cancers. We also present evidence for regulation of both PPAT and PAICS and pyruvate kinase activity by L-glutamine, a co-substrate for PPAT. A glutamine antagonist, 6-Diazo-5-oxo-L-norleucine (DON) blocked glutamine mediated induction of PPAT and PAICS as well as reduced pyruvate kinase activity. In summary, this study reveals the regulatory mechanisms by which purine biosynthetic pathway enzymes PPAT and PAICS, and pyruvate kinase activity is increased and exposes an existing metabolic vulnerability in lung cancer cells that can be explored for pharmacological intervention.

Characterization of novel bacteriochlorophyll-a-containing red filaments from alkaline hot springs in Yellowstone National Park.

PubMed

Boomer, S M; Pierson, B K; Austinhirst, R; Castenholz, R W

2000-09-01

Novel red, filamentous, gliding bacteria formed deep red layers in several alkaline hot springs in Yellowstone National Park. Filaments contained densely layered intracellular membranes and bacteriochlorophyll a. The in vivo absorption spectrum of the red layer filaments was distinct from other phototrophs, with unusual bacteriochlorophyll a signature peaks in the near-infrared (IR) region (807 nm and 911 nm). These absorption peaks were similar to the wavelengths penetrating to the red layer of the mats as measured with in situ spectroradiometry. The filaments also demonstrated maximal photosynthetic uptake of radiolabeled carbon sources at these wavelengths. The red layer filaments displayed anoxygenic photoheterotrophy, as evidenced by the specific incorporation of acetate, not bicarbonate, and by the absence of oxygen production. Photoheterotrophy was unaffected by sulfide and oxygen, but was diminished by high-intensity visible light. Near-IR radiation supported photoheterotrophy. Morphologically and spectrally similar filaments were observed in several springs in Yellowstone National Park, including Octopus Spring. Taken together, these data suggest that the red layer filaments are most similar to the photoheterotroph, Heliothrix oregonensis. Notable differences include mat position and coloration, absorption spectra, and prominent intracellular membranes.

Vibronic coupling explains the ultrafast carotenoid-to-bacteriochlorophyll energy transfer in natural and artificial light harvesters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perlík, Václav; Seibt, Joachim; Šanda, František

The initial energy transfer steps in photosynthesis occur on ultrafast timescales. We analyze the carotenoid to bacteriochlorophyll energy transfer in LH2 Marichromatium purpuratum as well as in an artificial light-harvesting dyad system by using transient grating and two-dimensional electronic spectroscopy with 10 fs time resolution. We find that Förster-type models reproduce the experimentally observed 60 fs transfer times, but overestimate coupling constants, which lead to a disagreement with both linear absorption and electronic 2D-spectra. We show that a vibronic model, which treats carotenoid vibrations on both electronic ground and excited states as part of the system’s Hamiltonian, reproduces all measuredmore » quantities. Importantly, the vibronic model presented here can explain the fast energy transfer rates with only moderate coupling constants, which are in agreement with structure based calculations. Counterintuitively, the vibrational levels on the carotenoid electronic ground state play the central role in the excited state population transfer to bacteriochlorophyll; resonance between the donor-acceptor energy gap and the vibrational ground state energies is the physical basis of the ultrafast energy transfer rates in these systems.« less

Biosynthetic Origin of Hygromycin A

PubMed Central

Habib, El-Sayed E.; Scarsdale, J. Neel; Reynolds, Kevin A.

2003-01-01

Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus, is an inhibitor of bacterial ribosomal peptidyl transferase. The antibiotic binds to the ribosome in a distinct but overlapping manner with other antibiotics and offers a different template for generation of new agents effective against multidrug-resistant pathogens. Reported herein are the results from a series of stable-isotope-incorporation studies demonstrating the biosynthetic origins of the three distinct structural moieties which comprise hygromycin A. Incorporation of [1-13C]mannose and intact incorporation of d-[1,2-13C2]glucose into the 6-deoxy-5-keto-d-arabino-hexofuranose moiety are consistent with a pathway in which mannose is converted to an activated l-fucose, via a 4-keto-6-deoxy-d-mannose intermediate, with a subsequent unusual mutation of the pyranose to the corresponding furanose. The aminocyclitol moiety was labeled by d-[1,2-13C2]glucose in a manner consistent with formation of myo-inositol and a subsequent unprecedented oxidation and transamination of the C-2 hydroxyl group to generate neo-inosamine-2. Incorporation of [carboxy-13C]-4-hydroxybenzoic acid and intact incorporation of [2,3-13C2]propionate are consistent with a polyketide synthase-type decarboxylation condensation to generate the 3,4-dihydroxy-α-methylcinnamic acid moiety of hygromycin A. No labeling of hygromycin A was observed when [3-13C]tyrosine, [3-13C]phenylalanine, or [carboxy-13C]benzoic acid was used, suggesting that the 4-hydroxybenzoic acid is derived directly from chorismic acid. Consistent with this hypothesis was the observation that hygromycin A titers could be reduced by addition of N-(phosphonomethyl)-glycine (an inhibitor of chorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoic acid. The convergent biosynthetic pathway established for hygromycin A offers significant versatility for applying the techniques of combinatorial and directed biosynthesis to production of new

Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

PubMed

Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

2016-07-01

Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

Molecular variation of the nonribosomal peptide-polyketide siderophore yersiniabactin through biosynthetic and metabolic engineering.

PubMed

Ahmadi, Mahmoud Kamal; Fawaz, Samar; Fang, Lei; Yu, Zhipeng; Pfeifer, Blaine A

2016-05-01

The production of the mixed nonribosomal peptide-polyketide natural product yersiniabactin (Ybt) has been established using E. coli as a heterologous host. In this study, precursor-directed biosynthesis was used to generate five new analogs of Ybt, demonstrating the flexibility of the heterologous system and the biosynthetic process in allowing compound diversity. A combination of biosynthetic and cellular engineering was then used to influence the production metrics of the resulting analogs. First, the cellular levels and activity of FadL, a hydrocarbon transport protein, were tested for subsequent influence upon exogenous precursor uptake and Ybt analog production with a positive correlation observed between FadL over-production and analog formation. Next, a Ybt biosynthetic editing enzyme was removed from the heterologous system which decreased native compound production but increased analog formation. A final series of experiments enhanced endogenous anthranilate towards complete pathway formation of the associated analog which showed a selective ability to bind gold. © 2015 Wiley Periodicals, Inc.

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

PubMed

Ma, Tian; Zhou, Yuanjie; Li, Xiaowei; Zhu, Fayin; Cheng, Yongbo; Liu, Yi; Deng, Zixin; Liu, Tiangang

2016-02-01

As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the α-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future. © 2015 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Applied evolutionary theories for engineering of secondary metabolic pathways.

PubMed

Bachmann, Brian O

2016-12-01

An expanded definition of 'secondary metabolism' is emerging. Once the exclusive provenance of naturally occurring organisms, evolved over geological time scales, secondary metabolism increasingly encompasses molecules generated via human engineered biocatalysts and biosynthetic pathways. Many of the tools and strategies for enzyme and pathway engineering can find origins in evolutionary theories. This perspective presents an overview of selected proposed evolutionary strategies in the context of engineering secondary metabolism. In addition to the wealth of biocatalysts provided via secondary metabolic pathways, improving the understanding of biosynthetic pathway evolution will provide rich resources for methods to adapt to applied laboratory evolution. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Draft Genome Sequence of Actinokineospora bangkokensis 44EHWT Reveals the Biosynthetic Pathway of the Antifungal Thailandin Compounds with Unusual Butylmalonyl-CoA Extender Units.

PubMed

Greule, Anja; Intra, Bungonsiri; Flemming, Stephan; Rommel, Marcel G E; Panbangred, Watanalai; Bechthold, Andreas

2016-11-23

We report the draft genome sequence of Actinokineospora bangkokensis 44EHW T , the producer of the antifungal polyene compounds, thailandins A and B. The sequence contains 7.45 Mb, 74.1% GC content and 35 putative gene clusters for the biosynthesis of secondary metabolites. There are three gene clusters encoding large polyketide synthases of type I. Annotation of the ORF functions and targeted gene disruption enabled us to identify the cluster for thailandin biosynthesis. We propose a plausible biosynthetic pathway for thailandin, where the unusual butylmalonyl-CoA extender unit is incorporated and results in an untypical side chain.

Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway.

PubMed

Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan; Han, Li; Huang, Xueshi; He, Jing

2016-04-22

Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Rational engineering of p-hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway.

PubMed

Chen, Zhenya; Shen, Xiaolin; Wang, Jian; Wang, Jia; Yuan, Qipeng; Yan, Yajun

2017-11-01

Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant

PubMed Central

Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong

2016-01-01

Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that’s highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in Flavonoid Biosynthetic Pathway

PubMed Central

Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

2015-01-01

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants. PMID:25742495

Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

PubMed

Sun, Wei; Meng, Xiangyu; Liang, Lingjie; Jiang, Wangshu; Huang, Yafei; He, Jing; Hu, Haiyan; Almqvist, Jonas; Gao, Xiang; Wang, Li

2015-01-01

Chalcone synthase (CHS) catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1) encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

PubMed

Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

2017-03-01

The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1- 14 C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1- 14 C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

PubMed Central

Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

2007-01-01

Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

Raman spectrum of bacteriochlorophyll a in the S1 state

NASA Astrophysics Data System (ADS)

Nishizawa, Ei-ichi; Hashimoto, Hideki; Koyama, Yasushi

1991-07-01

The S 1 Raman spectrum of bacteriochlorophyll a is reported for the first time. A one-color experiment using the 351 nm picosecond pulses (duration 50 ps and repetition 1 kHz) for tetrahydrofuran solution detected a transient species, which showed distinct Raman lines at 1567, 1409 and 1320 cm -1 and weak profiles around 1169, 1092, 1051 and 794 cm -1. The other one-color experiment using the 355 nm nanosecond pulses (duration 12 ns and repetition 10 Hz) detected the T 1 species reported previously showing Raman lines at 1578 and 1330 cm -1. Thus, the newly identified transient species, which was pumped and probed within 50 ps, is assigned to S 1.

Mapping and comparative proteomic analysis of the starch biosynthetic pathway in rice by 2D PAGE/MS.

PubMed

Chang, Tao-Shan; Liu, Chih-Wei; Lin, Yu-Ling; Li, Chao-Yi; Wang, Arthur Z; Chien, Min-Wei; Wang, Chang-Sheng; Lai, Chien-Chen

2017-11-01

Our results not only provide a comprehensive overview of the starch biosynthetic pathway in the developing endosperm but also reveal some important protein markers that regulate the synthesis of starch. In human diets, rice (Oryza sativa L.) is an important source of starch, a substantial amount of which is accumulated in developing endosperm. A better understanding of the complicated pathways involved in starch biosynthesis is needed to improve the yield and quality of rice and other cereal crops through breeding. One pure line rice mutant, SA0419, was induced from a wild-type rice, TNG67, by sodium azide mutagenesis; therefore, TNG67 and SA0419 share the same genetic background. SA0419 is, however, a unique glutinous rice with a lower amylose content (8%) than that of TNG67 (20%), and the grains of SA0419 develop earlier and faster than those of TNG67. In this study, we used a comparative proteomic analysis to identify the differentially expressed proteins that may explain the differences in starch biosynthesis and the characteristics of TNG67 and SA0419. A gel-based proteomic approach was applied to profile the expressed proteome in the developing endosperm of these two rice varieties by nano-LC/MS/MS. Several over-expressed proteins were found in SA0419, such as plastidial ADP-glucose pyrophosphorylase (AGPase), phosphoglucomutase (PGM), pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP), 6-phosphofructokinase (PFK), pyruvate phosphate dikinase (PPDK), starch branching enzymes (SBE) and starch debranching enzyme (SDBE), with those proteins mainly being involved in the pathways of starch metabolism and PPDK-mediated gluconeogenesis. Those over-expressed enzymes may contribute to the relatively early development, similar starch accumulation and rapid grain filling of SA0419 as compared with TNG67. This study provides a detailed biochemical description of starch biosynthesis and related information regarding a unique starch mutant that may assist future

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

PubMed

Nozaki, T; Asai, T; Sanchez, L B; Kobayashi, S; Nakazawa, M; Takeuchi, T

1999-11-05

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

Chlorophyll Biosynthesis Gene Evolution Indicates Photosystem Gene Duplication, Not Photosystem Merger, at the Origin of Oxygenic Photosynthesis

PubMed Central

Sousa, Filipa L.; Shavit-Grievink, Liat; Allen, John F.; Martin, William F.

2013-01-01

An open question regarding the evolution of photosynthesis is how cyanobacteria came to possess the two reaction center (RC) types, Type I reaction center (RCI) and Type II reaction center (RCII). The two main competing theories in the foreground of current thinking on this issue are that either 1) RCI and RCII are related via lineage divergence among anoxygenic photosynthetic bacteria and became merged in cyanobacteria via an event of large-scale lateral gene transfer (also called "fusion" theories) or 2) the two RC types are related via gene duplication in an ancestral, anoxygenic but protocyanobacterial phototroph that possessed both RC types before making the transition to using water as an electron donor. To distinguish between these possibilities, we studied the evolution of the core (bacterio)chlorophyll biosynthetic pathway from protoporphyrin IX (Proto IX) up to (bacterio)chlorophyllide a. The results show no dichotomy of chlorophyll biosynthesis genes into RCI- and RCII-specific chlorophyll biosynthetic clades, thereby excluding models of fusion at the origin of cyanobacteria and supporting the selective-loss hypothesis. By considering the cofactor demands of the pathway and the source genes from which several steps in chlorophyll biosynthesis are derived, we infer that the cell that first synthesized chlorophyll was a cobalamin-dependent, heme-synthesizing, diazotrophic anaerobe. PMID:23258841

Chlorophyll biosynthesis gene evolution indicates photosystem gene duplication, not photosystem merger, at the origin of oxygenic photosynthesis.

PubMed

Sousa, Filipa L; Shavit-Grievink, Liat; Allen, John F; Martin, William F

2013-01-01

An open question regarding the evolution of photosynthesis is how cyanobacteria came to possess the two reaction center (RC) types, Type I reaction center (RCI) and Type II reaction center (RCII). The two main competing theories in the foreground of current thinking on this issue are that either 1) RCI and RCII are related via lineage divergence among anoxygenic photosynthetic bacteria and became merged in cyanobacteria via an event of large-scale lateral gene transfer (also called "fusion" theories) or 2) the two RC types are related via gene duplication in an ancestral, anoxygenic but protocyanobacterial phototroph that possessed both RC types before making the transition to using water as an electron donor. To distinguish between these possibilities, we studied the evolution of the core (bacterio)chlorophyll biosynthetic pathway from protoporphyrin IX (Proto IX) up to (bacterio)chlorophyllide a. The results show no dichotomy of chlorophyll biosynthesis genes into RCI- and RCII-specific chlorophyll biosynthetic clades, thereby excluding models of fusion at the origin of cyanobacteria and supporting the selective-loss hypothesis. By considering the cofactor demands of the pathway and the source genes from which several steps in chlorophyll biosynthesis are derived, we infer that the cell that first synthesized chlorophyll was a cobalamin-dependent, heme-synthesizing, diazotrophic anaerobe.

OsWOX3A is involved in negative feedback regulation of the gibberellic acid biosynthetic pathway in rice (Oryza sativa).

PubMed

Cho, Sung-Hwan; Kang, Kiyoon; Lee, Sang-Hwa; Lee, In-Jung; Paek, Nam-Chon

2016-03-01

The plant-specific WUSCHEL-related homeobox (WOX) nuclear proteins have important roles in the transcriptional regulation of many developmental processes. Among the rice (Oryza sativa) WOX proteins, a loss of OsWOX3A function in narrow leaf2 (nal2) nal3 double mutants (termed nal2/3) causes pleiotropic effects, such as narrow and curly leaves, opened spikelets, narrow grains, more tillers, and fewer lateral roots, but almost normal plant height. To examine OsWOX3A function in more detail, transgenic rice overexpressing OsWOX3A (OsWOX3A-OX) were generated; unexpectedly, all of them consistently exhibited severe dwarfism with very short and wide leaves, a phenotype that resembles that of gibberellic acid (GA)-deficient or GA-insensitive mutants. Exogenous GA3 treatment fully rescued the developmental defects of OsWOX3A-OX plants, suggesting that constitutive overexpression of OsWOX3A downregulates GA biosynthesis. Quantitative analysis of GA intermediates revealed significantly reduced levels of GA20 and bioactive GA1 in OsWOX3A-OX, possibly due to downregulation of the expression of KAO, which encodes ent-kaurenoic acid oxidase, a GA biosynthetic enzyme. Yeast one-hybrid and electrophoretic mobility shift assays revealed that OsWOX3A directly interacts with the KAO promoter. OsWOX3A expression is drastically and temporarily upregulated by GA3 and downregulated by paclobutrazol, a blocker of GA biosynthesis. These data indicate that OsWOX3A is a GA-responsive gene and functions in the negative feedback regulation of the GA biosynthetic pathway for GA homeostasis to maintain the threshold levels of endogenous GA intermediates throughout development. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Effect of tamoxifen on the sphingolipid biosynthetic pathway in the different intraerythrocytic stages of the apicomplexa Plasmodium falciparum.

PubMed

Piñero, Tamara A; Landoni, Malena; Duschak, Vilma G; Katzin, Alejandro M; Couto, Alicia S

2018-03-18

Parasites of the genus Plasmodium responsible for Malaria are obligate intracellular pathogens residing in mammalian red blood cells, hepatocytes, or mosquito midgut epithelial cells. Regarding that detailed knowledge on the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites is scarce, different stages of Plasmodium falciparum were treated with tamoxifen in order to evaluate the effects of this drug on the glycosphingolipid biosynthesis. Thin layer chromatography, High performance reverse phase chromatography and UV-MALDI-TOF mass spectrometry were the tools used for the analysis. In the ring forms, the increase of NBD-phosphatidyl inositol biosynthesis was notorious but differences at NBD-GlcCer levels were undetectable. In trophozoite forms, an abrupt decrease of NBD-acylated GlcDHCer and NBD-GlcDHCer in addition to an increase of NBD-PC biosynthesis was observed. On the contrary, in schizonts, tamoxifen seems not to be producing substantial changes in lipid biosynthesis. Our findings indicate that in this parasite, tamoxifen is exerting an inhibitory action on Glucosylceramidesynthase and sphingomyelin synthase levels. Moreover, regarding that Plasmodium does not biosynthesize inositolphosphoceramides, the accumulation of phosphatidylinositol should indicate an inhibitory action on glycosylinositol phospholipid synthesis. Copyright © 2018 Elsevier Inc. All rights reserved.

[Influence of LDAO on the conformation and release of bacteriochlorophyll of peripheral light-harvesting complex (LH2) from Rhodobacter azotoformans].

PubMed

Zhao, Gen-gui; Dong, Yan-min; Yang, Su-ping; Jiao, Nian-zhi; Qu, Yin-bo

2010-10-01

The aim of this study is to reveal the interaction relationships between lauryl dimethylamine N-oxide (LDAO) and peripheral light-harvesting complex (LH2) as well as the influence of LDAO on structure and function of LH2. In the present work, the effects of LDAO on the conformation and release processes of bacteriochlorophyll (BChl) of LH2 when incubated under different temperature and pH in the presence and absence of LDAO were investigated by spectroscopy. The results indicated that (1) the presence of LDAO resulted in alterations in the conformation, alpha-helix content, and spectra of Tyr and B850 band of LH2 at room temperature and pH 8.0. Moreover, energy transfer efficiency of LH2 was enhanced markedly in the presence of LDAO. (2) At 60 degrees C, both the B800 and B850 band of LH2 were released and transited into free BChl at pH 8.0. However, the release rates of bacteriochlorophylls of B800 and B850 band from LH2 were slowed down and the release processes were changed when incubated in the presence of LDAO. Hence, the stability of LH2 was improved in the presence of LDAO. (3) The accelerated release processes of bacteriochlorophylls of B800 and B850 band of LH2 were induced to transform into bacteriopheophytin (BPhe) and free BChl by LDAO in strong acid and strong alkalic solution at room temperature. However, the kinetic patterns of the B800 and B850 band were remarkably different. The release and self-assemble processes of B850 in LH2 were observed in strong acid solution without LDAO. Therefore, the different release behaviors of B800 and B850 of LH2 are induced by LDAO under different extreme environmental conditions.

Characterization of Cyanobacterial Hydrocarbon Composition and Distribution of Biosynthetic Pathways

PubMed Central

Coates, R. Cameron; Podell, Sheila; Korobeynikov, Anton; Lapidus, Alla; Pevzner, Pavel; Sherman, David H.; Allen, Eric E.; Gerwick, Lena; Gerwick, William H.

2014-01-01

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both. PMID:24475038

R2R3 MYB transcription factors: key regulators of the flavonoid biosynthetic pathway in grapevine.

PubMed

Czemmel, Stefan; Heppel, Simon C; Bogs, Jochen

2012-06-01

Flavonoids compose one of the most abundant and important subgroups of secondary metabolites with more than 6,000 compounds detected so far in higher plants. They are found in various compositions and concentrations in nearly all plant tissues. Besides the attraction of pollinators and dispersers to fruits and flowers, flavonoids also protect against a plethora of stresses including pathogen attack, wounding and UV irradiation. Flavonoid content and composition of fruits such as grapes, bilberries, strawberries and apples as well as food extracts such as green tea, wine and chocolate have been associated with fruit quality including taste, colour and health-promoting effects. To unravel the beneficial potentials of flavonoids on fruit quality, research has been focused recently on the molecular basis of flavonoid biosynthesis and regulation in economically important fruit-producing plants such as grapevine (Vitis vinifera L.). Transcription factors and genes encoding biosynthetic enzymes have been characterized, studies that set a benchmark for future research on the regulatory networks controlling flavonoid biosynthesis and diversity. This review summarizes recent advances in the knowledge of regulatory cascades involved in flavonoid biosynthesis in grapevine. Transcriptional regulation of flavonoid biosynthesis during berry development is highlighted, with a particular focus on MYB transcription factors as molecular clocks, key regulators and powerful biotechnological tools to identify novel pathway enzymes to optimize flavonoid content and composition in grapes.

Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains

USDA-ARS?s Scientific Manuscript database

In order to determine the genetic basis for loss of fumonisin B¬2 (FB2) biosynthesis in FB2 non-producing A. niger strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified in FB2-produci...

Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1.

PubMed

Fujii, Ritsuko; Shimonaka, Shozo; Uchida, Naoko; Gardiner, Alastair T; Cogdell, Richard J; Sugisaki, Mitsuru; Hashimoto, Hideki

2008-01-01

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109-127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.

Integration of Fermentation and Organic Synthesis: Studies of Roquefortine C and Biosynthetic Derivatives

NASA Astrophysics Data System (ADS)

Gober, Claire Marie

Roquefortine C is one of the most ubiquitous indoline alkaloids of fungal origin. It has been isolated from over 30 different species of Penicillium fungi and has garnered attention in recent years for its role as a biosynthetic precursor to the triazaspirocyclic natural products glandicoline B, meleagrin, and oxaline. The triazaspirocyclic motif, which encompasses three nitrogen atoms attached to one quaternary carbon forming a spirocyclic scaffold, is a unique chemical moiety that has been shown to impart a wide array of biological activity, from anti-bacterial activity and antiproliferative activity against cancer cell lines to anti-biofouling against marine organisms. Despite the promise of these compounds in the pharmaceutical and materials industries, few syntheses of triazaspirocycles exist in the literature. The biosynthesis of roquefortine C-derived triazaspirocycles, however, provides inspiration for the synthesis of these compounds, namely through a nitrone-promoted transannular rearrangement. This type of internal rearrangement has never been carried out synthetically and would provide an efficient stereoselective synthesis of triazaspirocycles. This work encompasses efforts towards elucidating the biosynthetic pathway of roquefortine C-derived triazaspirocycles as well as synthetic efforts towards the construction of triazaspirocycles. Chapter 1 will discuss a large-scale fermentation procedure for the production of roquefortine C from Penicillium crustosum. Chapters 2 and 3 explore (through enzymatic and synthetic means, respectively) the formation of the key indoline nitrone moiety required for the proposed transannular rearrangement. Finally, chapter 4 will discuss synthetic efforts towards the synthesis of triazaspirocycles. This work has considerably enhanced our understanding of the roquefortine C biosynthetic pathway and the unique chemistry of this natural product, and our efforts towards the synthesis of triazaspirocycles will facilitate the

Chloroplast biogenesis 89: development of analytical tools for probing the biosynthetic topography of photosynthetic membranes by determination of resonance excitation energy transfer distances separating metabolic tetrapyrrole donors from chlorophyll a acceptors.

PubMed

Kopetz, Karen J; Kolossov, Vladimir L; Rebeiz, Constantin A

2004-06-15

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination and regulation of the chlorophyll (Chl) and thylakoid apoprotein biosynthetic pathways. As a working hypothesis we have recently proposed three different Chl-thylakoid apoprotein biosynthesis models: a single-branched Chl biosynthetic pathway (SBP)-single location model, a SBP-multilocation model, and a multibranched Chl biosynthetic pathway (MBP)-sublocation model. The detection of resonance excitation energy transfer between tetrapyrrole precursors of Chl, and several Chl-protein complexes, has made it possible to test the validity of the proposed Chl-thylakoid apoprotein biosynthesis models by resonance excitation energy transfer determinations. In this work, resonance excitation energy transfer techniques that allow the determination of distances separating tetrapyrrole donors from Chl-protein acceptors in green plants by using readily available electronic spectroscopic instrumentation are developed. It is concluded that the calculated distances are compatible with the MBP-sublocation model and incompatible with the operation of the SBP-single location Chl-protein biosynthesis model.

Spectral heterogeneity and carotenoid-to-bacteriochlorophyll energy transfer in LH2 light-harvesting complexes from Allochromatium vinosum.

PubMed

Magdaong, Nikki M; LaFountain, Amy M; Hacking, Kirsty; Niedzwiedzki, Dariusz M; Gibson, George N; Cogdell, Richard J; Frank, Harry A

2016-02-01

Photosynthetic organisms produce a vast array of spectral forms of antenna pigment-protein complexes to harvest solar energy and also to adapt to growth under the variable environmental conditions of light intensity, temperature, and nutrient availability. This behavior is exemplified by Allochromatium (Alc.) vinosum, a photosynthetic purple sulfur bacterium that produces different types of LH2 light-harvesting complexes in response to variations in growth conditions. In the present work, three different spectral forms of LH2 from Alc. vinosum, B800-820, B800-840, and B800-850, were isolated, purified, and examined using steady-state absorption and fluorescence spectroscopy, and ultrafast time-resolved absorption spectroscopy. The pigment composition of the LH2 complexes was analyzed by high-performance liquid chromatography, and all were found to contain five carotenoids: lycopene, anhydrorhodovibrin, spirilloxanthin, rhodopin, and rhodovibrin. Spectral reconstructions of the absorption and fluorescence excitation spectra based on the pigment composition revealed significantly more spectral heterogeneity in these systems compared to LH2 complexes isolated from other species of purple bacteria. The data also revealed the individual carotenoid-to-bacteriochlorophyll energy transfer efficiencies which were correlated with the kinetic data from the ultrafast transient absorption spectroscopic experiments. This series of LH2 complexes allows a systematic exploration of the factors that determine the spectral properties of the bound pigments and control the rate and efficiency of carotenoid-to-bacteriochlorophyll energy transfer.

“Prokaryotic Pathway” Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis

PubMed Central

Awai, Koichiro

2017-01-01

Abstract Lipid biosynthesis within the chloroplast, or more generally plastids, was conventionally called “prokaryotic pathway,” which produces glycerolipids bearing C18 acids at the sn-1 position and C16 acids at the sn-2 position, as in cyanobacteria such as Anabaena and Synechocystis. This positional specificity is determined during the synthesis of phosphatidate, which is a precursor to diacylglycerol, the acceptor of galactose for the synthesis of galactolipids. The first acylation at sn-1 is catalyzed by glycerol-3-phosphate acyltransferase (GPAT or GPT), whereas the second acylation at sn-2 is performed by lysophosphatidate acyltransferase (LPAAT, AGPAT, or PlsC). Here we present comprehensive phylogenomic analysis of the origins of various acyltransferases involved in the synthesis of phosphatidate, as well as phosphatidate phosphatases in the chloroplasts. The results showed that the enzymes involved in the two steps of acylation in cyanobacteria and chloroplasts are entirely phylogenetically unrelated despite a previous report stating that the chloroplast LPAAT (ATS2) and cyanobacterial PlsC were sister groups. Phosphatidate phosphatases were separated into eukaryotic and prokaryotic clades, and the chloroplast enzymes were not of cyanobacterial origin, in contrast with another previous report. These results indicate that the lipid biosynthetic pathway in the chloroplasts or plastids did not originate from the cyanobacterial endosymbiont and is not “prokaryotic” in the context of endosymbiotic theory of plastid origin. This is another line of evidence for the discontinuity of plastids and cyanobacteria, which has been suggested in the glycolipid biosynthesis. PMID:29145606

Assembly of lipase and P450 fatty acid decarboxylase to constitute a novel biosynthetic pathway for production of 1-alkenes from renewable triacylglycerols and oils.

PubMed

Yan, Jinyong; Liu, Yi; Wang, Cong; Han, Bingnan; Li, Shengying

2015-01-01

Biogenic hydrocarbons (biohydrocarbons) are broadly accepted to be the ideal 'drop-in' biofuel alternative to petroleum-based fuels due to their highly similar chemical composition and physical characteristics. The biological production of aliphatic hydrocarbons is largely dependent on engineering of the complicated enzymatic network surrounding fatty acid biosynthesis. In this work, we developed a novel system for bioproduction of terminal fatty alkenes (1-alkenes) from renewable and low-cost triacylglycerols (TAGs) based on the lipase hydrolysis coupled to the P450 catalyzed decarboxylation. This artificial biosynthetic pathway was constituted using both cell-free systems including purified enzymes or cell-free extracts, and cell-based systems including mixed resting cells or growing cells. The issues of high cost of fatty acid feedstock and complicated biosynthesis network were addressed by replacement of the de novo biosynthesized fatty acids with the fed cheap TAGs. This recombinant tandem enzymatic pathway consisting of the Thermomyces lanuginosus lipase (Tll) and the P450 fatty acid decarboxylase OleTJE resulted in the production of 1-alkenes from purified TAGs or natural oils with 6.7 to 46.0% yields. Since this novel hydrocarbon-producing pathway only requires two catalytically efficient enzymatic steps, it may hold great potential for industrial application by fulfilling the large-scale and cost-effective conversion of renewable TAGs into biohydrocarbons. This work highlights the power of designing and implementing an artificial pathway for production of advanced biofuels.

Transformation with Oncogenic Ras and the Simian Virus 40 T Antigens Induces Caspase-Dependent Sensitivity to Fatty Acid Biosynthetic Inhibition

PubMed Central

Xu, Shihao; Spencer, Cody M.

2015-01-01

ABSTRACT Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here, we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens and oncogenic Ras12V, affect fatty acid metabolism. Our results indicate that SV40/Ras12V-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (mTOR) dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras12V-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed breast epithelial cells through transduction with oncogenic Ras12V. Similar to what was observed in the transformed fibroblasts, the Ras12V-induced sensitivity to fatty acid biosynthetic inhibition was independent of the proliferative status and could be attenuated by supplementing the medium with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis. IMPORTANCE Viral oncoproteins and cellular mutations

Biosynthetic inorganic chemistry.

PubMed

Lu, Yi

2006-08-25

Inorganic chemistry and biology can benefit greatly from each other. Although synthetic and physical inorganic chemistry have been greatly successful in clarifying the role of metal ions in biological systems, the time may now be right to utilize biological systems to advance coordination chemistry. One such example is the use of small, stable, easy-to-make, and well-characterized proteins as ligands to synthesize novel inorganic compounds. This biosynthetic inorganic chemistry is possible thanks to a number of developments in biology. This review summarizes the progress in the synthesis of close models of complex metalloproteins, followed by a description of recent advances in using the approach for making novel compounds that are unprecedented in either inorganic chemistry or biology. The focus is mainly on synthetic "tricks" learned from biology, as well as novel structures and insights obtained. The advantages and disadvantages of this biosynthetic approach are discussed.

The Structure of L-Tyrosine 2,3-Aminomutase frmo the C-1027 Enediyne Antitumor Antibiotic Biosynthetic Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christianson,C.; Montavon, T.; Van Lanen, S.

2007-01-01

The SgcC4 L-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-{beta}-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate L-tyrosine to generate (S)-{beta}-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Heremore » we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the L-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.« less

Elucidation of Enzymatic Mechanism of Phenazine Biosynthetic Protein PhzF Using QM/MM and MD Simulations

PubMed Central

Liu, Fei; Zhao, Yi-Lei; Wang, Xiaolei; Hu, Hongbo; Peng, Huasong; Wang, Wei; Wang, Jing-Fang; Zhang, Xuehong

2015-01-01

The phenazine biosynthetic pathway is of considerable importance for the pharmaceutical industry. The pathway produces two products: phenazine-1,6-dicarboxylic acid and phenazine-1-carboxylic acid. PhzF is an isomerase that catalyzes trans-2,3-dihydro-3-hydroxyanthranilic acid isomerization and plays an essential role in the phenazine biosynthetic pathway. Although the PhzF crystal structure has been determined recently, an understanding of the detailed catalytic mechanism and the roles of key catalytic residues are still lacking. In this study, a computational strategy using a combination of molecular modeling, molecular dynamics simulations, and quantum mechanics/molecular mechanics simulations was used to elucidate these important issues. The Apo enzyme, enzyme–substrate complexes with negatively charged Glu45, enzyme–transition state analog inhibitor complexes with neutral Glu45, and enzyme–product complexes with negatively charged Glu45 structures were optimized and modeled using a 200 ns molecular dynamics simulation. Residues such as Gly73, His74, Asp208, Gly212, Ser213, and water, which play important roles in ligand binding and the isomerization reaction, were comprehensively investigated. Our results suggest that the Glu45 residue at the active site of PhzF acts as a general base/acid catalyst during proton transfer. This study provides new insights into the detailed catalytic mechanism of PhzF and the results have important implications for PhzF modification. PMID:26414009

Localisation and origin of the bacteriochlorophyll-derived photosensitizer in the retina of the deep-sea dragon fish Malacosteus niger

PubMed Central

Douglas, Ronald H.; Genner, Martin J.; Hudson, Alan G.; Partridge, Julian C.; Wagner, Hans-Joachim

2016-01-01

Most deep-sea fish have a single visual pigment maximally sensitive at short wavelengths, approximately matching the spectrum of both downwelling sunlight and bioluminescence. However, Malcosteus niger produces far-red bioluminescence and its longwave retinal sensitivity is enhanced by red-shifted visual pigments, a longwave reflecting tapetum and, uniquely, a bacteriochlorophyll-derived photosensitizer. The origin of the photosensitizer, however, remains unclear. We investigated whether the bacteriochlorophyll was produced by endosymbiotic bacteria within unusual structures adjacent to the photoreceptors that had previously been described in this species. However, microscopy, elemental analysis and SYTOX green staining provided no evidence for such localised retinal bacteria, instead the photosensitizer was shown to be distributed throughout the retina. Furthermore, comparison of mRNA from the retina of Malacosteus to that of the closely related Pachystomias microdon (which does not contain a bacterichlorophyll-derived photosensitzer) revealed no genes of bacterial origin that were specifically up-regulated in Malacosteus. Instead up-regulated Malacosteus genes were associated with photosensitivity and may relate to its unique visual ecology and the chlorophyll-based visual system. We also suggest that the unusual longwave-reflecting, astaxanthin-based, tapetum of Malacosteus may protect the retina from the potential cytotoxicity of such a system. PMID:27996027

Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives

PubMed Central

Moses, Tessa; Papadopoulou, Kalliope K.

2014-01-01

Saponins are widely distributed plant natural products with vast structural and functional diversity. They are typically composed of a hydrophobic aglycone, which is extensively decorated with functional groups prior to the addition of hydrophilic sugar moieties, to result in surface-active amphipathic compounds. The saponins are broadly classified as triterpenoids, steroids or steroidal glycoalkaloids, based on the aglycone structure from which they are derived. The saponins and their biosynthetic intermediates display a variety of biological activities of interest to the pharmaceutical, cosmetic and food sectors. Although their relevance in industrial applications has long been recognized, their role in plants is underexplored. Recent research on modulating native pathway flux in saponin biosynthesis has demonstrated the roles of saponins and their biosynthetic intermediates in plant growth and development. Here, we review the literature on the effects of these molecules on plant physiology, which collectively implicate them in plant primary processes. The industrial uses and potential of saponins are discussed with respect to structure and activity, highlighting the undoubted value of these molecules as therapeutics. PMID:25286183

Metabolic Flux Between Unsaturated and Saturated Fatty Acids is Controlled by the FabA:FabB Ratio in the Fully Reconstituted Fatty Acid Biosynthetic Pathway of E. coli#

PubMed Central

Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

2013-01-01

The entire fatty acid biosynthetic pathway from Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from fourteen purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H into the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multi-enzyme system. At steady state, a maximum turnover rate of 0.5 s−1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. By altering these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximum turnover rate of the pathway. Our reconstituted system provides a powerful tool to understand and engineer rate-limiting and regulatory steps in this complex and practically significant metabolic pathway. PMID:24147979

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) and serine biosynthetic pathway genes are co-ordinately increased during anabolic agent-induced skeletal muscle growth.

PubMed

Brown, D M; Williams, H; Ryan, K J P; Wilson, T L; Daniel, Z C T R; Mareko, M H D; Emes, R D; Harris, D W; Jones, S; Wattis, J A D; Dryden, I L; Hodgman, T C; Brameld, J M; Parr, T

2016-06-28

We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20 ppm in feed) or Reporcin (recombinant growth hormone; GH; 10 mg/48 hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p = 0.002) and muscle weights (Vastus Lateralis: p < 0.001; Semitendinosus: p = 0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p < 0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p < 0.05) and 7 (p < 0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p < 0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth.

Structural Insights Into the Evolutionary Paths of Oxylipin Biosynthetic Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, D.-S.; Nioche, P.; Hamberg, M.

2009-05-20

The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactionsmore » with an aromatic {pi}-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata.« less

Biosynthetic Potential-Based Strain Prioritization for Natural Product Discovery: A Showcase for Diterpenoid-Producing Actinomycetes

PubMed Central

2015-01-01

Natural products remain the best sources of drugs and drug leads and serve as outstanding small-molecule probes to dissect fundamental biological processes. A great challenge for the natural product community is to discover novel natural products efficiently and cost effectively. Here we report the development of a practical method to survey biosynthetic potential in microorganisms, thereby identifying the most promising strains and prioritizing them for natural product discovery. Central to our approach is the innovative preparation, by a two-tiered PCR method, of a pool of pathway-specific probes, thereby allowing the survey of all variants of the biosynthetic machineries for the targeted class of natural products. The utility of the method was demonstrated by surveying 100 strains, randomly selected from our actinomycete collection, for their biosynthetic potential of four classes of natural products, aromatic polyketides, reduced polyketides, nonribosomal peptides, and diterpenoids, identifying 16 talented strains. One of the talented strains, Streptomyces griseus CB00830, was finally chosen to showcase the discovery of the targeted classes of natural products, resulting in the isolation of three diterpenoids, six nonribosomal peptides and related metabolites, and three polyketides. Variations of this method should be applicable to the discovery of other classes of natural products. PMID:24484381

Flg22-Triggered Immunity Negatively Regulates Key BR Biosynthetic Genes.

PubMed

Jiménez-Góngora, Tamara; Kim, Seong-Ki; Lozano-Durán, Rosa; Zipfel, Cyril

2015-01-01

In plants, activation of growth and activation of immunity are opposing processes that define a trade-off. In the past few years, the growth-promoting hormones brassinosteroids (BR) have emerged as negative regulators of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), promoting growth at the expense of defense. The crosstalk between BR and PTI signaling was described as negative and unidirectional, since activation of PTI does not affect several analyzed steps in the BR signaling pathway. In this work, we describe that activation of PTI by the bacterial PAMP flg22 results in the reduced expression of BR biosynthetic genes. This effect does not require BR perception or signaling, and occurs within 15 min of flg22 treatment. Since the described PTI-induced repression of gene expression may result in a reduction in BR biosynthesis, the crosstalk between PTI and BR could actually be negative and bidirectional, a possibility that should be taken into account when considering the interaction between these two pathways.

Allocate carbon for a reason: priorities are reflected in the ¹³C/¹²C ratios of plant lipids synthesized via three independent biosynthetic pathways.

PubMed

Zhou, Youping; Stuart-Williams, Hilary; Grice, Kliti; Kayler, Zachary E; Zavadlav, Saša; Vogts, Angela; Rommerskirchen, Florian; Farquhar, Graham D; Gessler, Arthur

2015-03-01

It has long been theorized that carbon allocation, in addition to the carbon source and to kinetic isotopic effects associated with a particular lipid biosynthetic pathway, plays an important role in shaping the carbon isotopic composition ((13)C/(12)C) of lipids (Park and Epstein, 1961). If the latter two factors are properly constrained, valuable information about carbon allocation during lipid biosynthesis can be obtained from carbon isotope measurements. Published work of Chikaraishi et al. (2004) showed that leaf lipids isotopic shifts from bulk leaf tissue Δδ(13)C(bk-lp) (defined as δ(13)C(bulkleaftissue)-δ(13)C(lipid)) are pathway dependent: the acetogenic (ACT) pathway synthesizing fatty lipids has the largest isotopic shift, the mevalonic acid (MVA) pathway synthesizing sterols the lowest and the phytol synthesizing 1-deoxy-D-xylulose 5-phosphate (DXP) pathway gives intermediate values. The differences in Δδ(13)C(bk-lp) between C3 and C4 plants Δδ(13)C(bk-lp,C4-C3) are also pathway-dependent: Δδ(13)C(ACT)(bk-lp,C4-C3) > Δδ(13)C(DXP(bk-lp,C4-C3) > Δδ(13)C(MVA)(bk-lp,C4-C3). These pathway-dependent differences have been interpreted as resulting from kinetic isotopic effect differences of key but unspecified biochemical reactions involved in lipids biosynthesis between C3 and C4 plants. After quantitatively considering isotopic shifts caused by (dark) respiration, export-of-carbon (to sink tissues) and photorespiration, we propose that the pathway-specific differences Δδ(13)C(bk-lp,C4-C3) can be successfully explained by C4-C3 carbon allocation (flux) differences with greatest flux into the ACT pathway and lowest into the MVA pathways (when flux is higher, isotopic shift relative to source is smaller). Highest carbon allocation to the ACT pathway appears to be tied to the most stringent role of water-loss-minimization by leaf waxes (composed mainly of fatty lipids) while the lowest carbon allocation to the MVA pathway can be largely explained

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

PubMed Central

Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

2014-01-01

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae*

PubMed Central

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; Shirasaki, Dyna I.; Wang, Charles; Blaby-Haas, Crysten E.; Merchant, Sabeeha S.; Loo, Joseph A.; Clarke, Catherine F.

2015-01-01

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11. PMID:25631044

Metabolic pathway reconstruction of eugenol to vanillin bioconversion in Aspergillus niger

PubMed Central

Srivastava, Suchita; Luqman, Suaib; Khan, Feroz; Chanotiya, Chandan S; Darokar, Mahendra P

2010-01-01

Identification of missing genes or proteins participating in the metabolic pathways as enzymes are of great interest. One such class of pathway is involved in the eugenol to vanillin bioconversion. Our goal is to develop an integral approach for identifying the topology of a reference or known pathway in other organism. We successfully identify the missing enzymes and then reconstruct the vanillin biosynthetic pathway in Aspergillus niger. The procedure combines enzyme sequence similarity searched through BLAST homology search and orthologs detection through COG & KEGG databases. Conservation of protein domains and motifs was searched through CDD, PFAM & PROSITE databases. Predictions regarding how proteins act in pathway were validated experimentally and also compared with reported data. The bioconversion of vanillin was screened on UV-TLC plates and later confirmed through GC and GC-MS techniques. We applied a procedure for identifying missing enzymes on the basis of conserved functional motifs and later reconstruct the metabolic pathway in target organism. Using the vanillin biosynthetic pathway of Pseudomonas fluorescens as a case study, we indicate how this approach can be used to reconstruct the reference pathway in A. niger and later results were experimentally validated through chromatography and spectroscopy techniques. PMID:20978605

The oxylipin pathway in Arabidopsis.

PubMed

Creelman, Robert A; Mulpuri, Rao

2002-01-01

Oxylipins are acyclic or cyclic oxidation products derived from the catabolism of fatty acids which regulate many defense and developmental pathways in plants. The dramatic increase in the volume of publications and reviews on these compounds since 1997 documents the increasing interest in this compound and its role in plants. Research on this topic has solidified our understanding of the chemistry and biosynthetic pathways for oxylipin production. However, more information is still needed on how free fatty acids are produced and the role of beta-oxidation in the biosynthetic pathway for oxylipins. It is also becoming apparent that oxylipin content and composition changes during growth and development and during pathogen or insect attack. Oxylipins such as jasmonic acid (JA) or 12-oxo-phytodienoic acid modulate the expression of numerous genes and influence specific aspects of plant growth, development and responses to abiotic and biotic stresses. Although oxylipins are believed to act alone, several examples were presented to illustrate that JA-induced responses are modulated by the type and the nature of crosstalk with other signaling molecules such as ethylene and salicylic acid. How oxylipins cause changes in gene expression and instigate a physiological response is becoming understood with the isolation of mutations in both positive and negative regulators in the jasmonate signaling pathway and the use of cDNA microarrays.

The Oxylipin Pathway in Arabidopsis

PubMed Central

Creelman, Robert A.; Mulpuri, Rao

2002-01-01

Oxylipins are acyclic or cyclic oxidation products derived from the catabolism of fatty acids which regulate many defense and developmental pathways in plants. The dramatic increase in the volume of publications and reviews on these compounds since 1997 documents the increasing interest in this compound and its role in plants. Research on this topic has solidified our understanding of the chemistry and biosynthetic pathways for oxylipin production. However, more information is still needed on how free fatty acids are produced and the role of beta-oxidation in the biosynthetic pathway for oxylipins. It is also becoming apparent that oxylipin content and composition changes during growth and development and during pathogen or insect attack. Oxylipins such as jasmonic acid (JA) or 12-oxo-phytodienoic acid modulate the expression of numerous genes and influence specific aspects of plant growth, development and responses to abiotic and biotic stresses. Although oxylipins are believed to act alone, several examples were presented to illustrate that JA-induced responses are modulated by the type and the nature of crosstalk with other signaling molecules such as ethylene and salicylic acid. How oxylipins cause changes in gene expression and instigate a physiological response is becoming understood with the isolation of mutations in both positive and negative regulators in the jasmonate signaling pathway and the use of cDNA microarrays. PMID:22303193

antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters

PubMed Central

Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A; Müller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko

2015-01-01

Abstract Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software. PMID:25948579

Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network

PubMed Central

Widhalm, Joshua R.; Gutensohn, Michael; Yoo, Heejin; Adebesin, Funmilayo; Qian, Yichun; Guo, Longyun; Jaini, Rohit; Lynch, Joseph H.; McCoy, Rachel M.; Shreve, Jacob T.; Thimmapuram, Jyothi; Rhodes, David; Morgan, John A.; Dudareva, Natalia

2015-01-01

In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network. PMID:26356302

Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network

DOE Office of Scientific and Technical Information (OSTI.GOV)

Widhalm, Joshua R.; Gutensohn, Michael; Yoo, Heejin

In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles,more » as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network.« less

Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network

DOE PAGES

Widhalm, Joshua R.; Gutensohn, Michael; Yoo, Heejin; ...

2015-09-10

In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles,more » as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network.« less

Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway

PubMed Central

2011-01-01

Background Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. Results In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in

Expression profile of small RNAs in Acacia mangium secondary xylem tissue with contrasting lignin content - potential regulatory sequences in monolignol biosynthetic pathway.

PubMed

Ong, Seong Siang; Wickneswari, Ratnam

2011-11-30

Lignin, after cellulose, is the second most abundant biopolymer accounting for approximately 15-35% of the dry weight of wood. As an important component during wood formation, lignin is indispensable for plant structure and defense. However, it is an undesirable component in the pulp and paper industry. Removal of lignin from cellulose is costly and environmentally hazardous process. Tremendous efforts have been devoted to understand the role of enzymes and genes in controlling the amount and composition of lignin to be deposited in the cell wall. However, studies on the impact of downregulation and overexpression of monolignol biosynthesis genes in model species on lignin content, plant fitness and viability have been inconsistent. Recently, non-coding RNAs have been discovered to play an important role in regulating the entire monolignol biosynthesis pathway. As small RNAs have critical functions in various biological process during wood formation, small RNA profiling is an important tool for the identification of complete set of differentially expressed small RNAs between low lignin and high lignin secondary xylem. In line with this, we have generated two small RNAs libraries from samples with contrasting lignin content using Illumina GAII sequencer. About 10 million sequence reads were obtained in secondary xylem of Am48 with high lignin content (41%) and a corresponding 14 million sequence reads were obtained in secondary xylem of Am54 with low lignin content (21%). Our results suggested that A. mangium small RNAs are composed of a set of 12 highly conserved miRNAs families found in plant miRNAs database, 82 novel miRNAs and a large proportion of non-conserved small RNAs with low expression levels. The predicted target genes of those differentially expressed conserved and non-conserved miRNAs include transcription factors associated with regulation of the lignin biosynthetic pathway genes. Some of these small RNAs play an important role in epigenetic silencing

CYP76M7 Is an ent-Cassadiene C11α-Hydroxylase Defining a Second Multifunctional Diterpenoid Biosynthetic Gene Cluster in Rice[W][OA

PubMed Central

Swaminathan, Sivakumar; Morrone, Dana; Wang, Qiang; Fulton, D. Bruce; Peters, Reuben J.

2009-01-01

Biosynthetic gene clusters are common in microbial organisms, but rare in plants, raising questions regarding the evolutionary forces that drive their assembly in multicellular eukaryotes. Here, we characterize the biochemical function of a rice (Oryza sativa) cytochrome P450 monooxygenase, CYP76M7, which seems to act in the production of antifungal phytocassanes and defines a second diterpenoid biosynthetic gene cluster in rice. This cluster is uniquely multifunctional, containing enzymatic genes involved in the production of two distinct sets of phytoalexins, the antifungal phytocassanes and antibacterial oryzalides/oryzadiones, with the corresponding genes being subject to distinct transcriptional regulation. The lack of uniform coregulation of the genes within this multifunctional cluster suggests that this was not a primary driving force in its assembly. However, the cluster is dedicated to specialized metabolism, as all genes in the cluster are involved in phytoalexin metabolism. We hypothesize that this dedication to specialized metabolism led to the assembly of the corresponding biosynthetic gene cluster. Consistent with this hypothesis, molecular phylogenetic comparison demonstrates that the two rice diterpenoid biosynthetic gene clusters have undergone independent elaboration to their present-day forms, indicating continued evolutionary pressure for coclustering of enzymatic genes encoding components of related biosynthetic pathways. PMID:19825834

antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters.

PubMed

Weber, Tilmann; Blin, Kai; Duddela, Srikanth; Krug, Daniel; Kim, Hyun Uk; Bruccoleri, Robert; Lee, Sang Yup; Fischbach, Michael A; Müller, Rolf; Wohlleben, Wolfgang; Breitling, Rainer; Takano, Eriko; Medema, Marnix H

2015-07-01

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schenck, Craig A.; Holland, Cynthia K.; Schneider, Matthew R.

L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that definesmore » TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.« less

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae

DOE PAGES

Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; ...

2015-01-28

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less

Ultra-broadband 2D electronic spectroscopy of carotenoid-bacteriochlorophyll interactions in the LH1 complex of a purple bacterium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maiuri, Margherita; Department of Chemistry, Princeton University, Washington Road, Princeton, New Jersey 08544; Réhault, Julien

We investigate the excitation energy transfer (EET) pathways in the photosynthetic light harvesting 1 (LH1) complex of purple bacterium Rhodospirillum rubrum with ultra-broadband two-dimensional electronic spectroscopy (2DES). We employ a 2DES apparatus in the partially collinear geometry, using a passive birefringent interferometer to generate the phase-locked pump pulse pair. This scheme easily lends itself to two-color operation, by coupling a sub-10 fs visible pulse with a sub-15-fs near-infrared pulse. This unique pulse combination allows us to simultaneously track with extremely high temporal resolution both the dynamics of the photoexcited carotenoid spirilloxanthin (Spx) in the visible range and the EET betweenmore » the Spx and the B890 bacterio-chlorophyll (BChl), whose Q{sub x} and Q{sub y} transitions peak at 585 and 881 nm, respectively, in the near-infrared. Global analysis of the one-color and two-color 2DES maps unravels different relaxation mechanisms in the LH1 complex: (i) the initial events of the internal conversion process within the Spx, (ii) the parallel EET from the first bright state S{sub 2} of the Spx towards the Q{sub x} state of the B890, and (iii) the internal conversion from Q{sub x} to Q{sub y} within the B890.« less

Stability and properties of quasi-stable conformational states in the LH2 light-harvesting complex of Rbl. acidophilus bacteria formed by hexacoordination of bacteriochlorophyll a magnesium atom

NASA Astrophysics Data System (ADS)

Belov, Aleksandr S.; Khokhlov, Daniil V.; Glebov, Ilya O.; Poddubnyy, Vladimir V.; Eremin, Vadim V.

2017-06-01

Single-molecule spectroscopic experiments on several light-harvesting complexes revealed the existence of a set of metastable conformational states with different spectroscopic properties and lifetimes spanning from milliseconds to tens of seconds. In the absence of explicit structural data, a number of probable structural changes underlying the observed spectroscopic shifts were proposed. We examine the donor-acceptor interaction between the magnesium atom and the acetyl group of the adjacent bacteriochlorophylls a as a possible origin of metastable conformational states in the LH2 light-harvesting complex of Rbl. acidophilus bacteria. The results of QM/MM and molecular dynamics simulations show that such ligation can occur at room temperature and leads to one metastable coordination bond per pair of bacteriochlorophylls in the B850 ring. According to the results of Poisson-TrESP modeling, such coordination lowers the energies of the excited states of the complex by up to 163 cm-1 which causes red spectral shift of the B850 band.

Antibiotics from Gram-negative bacteria: a comprehensive overview and selected biosynthetic highlights.

PubMed

Masschelein, J; Jenner, M; Challis, G L

2017-07-01

Covering: up to 2017The overwhelming majority of antibiotics in clinical use originate from Gram-positive Actinobacteria. In recent years, however, Gram-negative bacteria have become increasingly recognised as a rich yet underexplored source of novel antimicrobials, with the potential to combat the looming health threat posed by antibiotic resistance. In this article, we have compiled a comprehensive list of natural products with antimicrobial activity from Gram-negative bacteria, including information on their biosynthetic origin(s) and molecular target(s), where known. We also provide a detailed discussion of several unusual pathways for antibiotic biosynthesis in Gram-negative bacteria, serving to highlight the exceptional biocatalytic repertoire of this group of microorganisms.

Microbial expression of alkaloid biosynthetic enzymes for characterization of their properties.

PubMed

Minami, Hiromichi; Ikezawa, Nobuhiro; Sato, Fumihiko

2010-01-01

A wide variety of secondary metabolites are produced in higher plants. These metabolites are synthesized in specific organs/cells at certain developmental stages and/or under specific environmental conditions. Since these biosynthetic activities are rather restricted and difficult to detect, the biochemical characterization of biosynthetic enzymes involved in secondary metabolism has been limited compared to those involved in primary metabolism. Recently, however, progress in tissue culture and molecular biology has made it easier to study biosynthetic enzymes. Here we describe protocols for expressing some biosynthetic enzymes in Escherichia coli expression systems, since this system is both efficient and cost-effective. First, we describe a standard system for expressing biosynthetic enzymes as a soluble protein under the T7 promoter of the pET expression system in E. coli. In addition, the successful expression of cytochrome P450 in E. coli in an active soluble form with N-terminal modification is discussed, since P450 is the critical enzyme in secondary metabolite biosynthesis.

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.

PubMed

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-05-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.

Biosynthetic and functional color-scent associations in flowers of Papaver nudicaule and its impact on pollinators.

PubMed

Martinez-Harms, Jaime; Warskulat, Anne-Christin; Dudek, Bettina; Kunert, Grit; Lorenz, Sybille; Hansson, Bill S; Schneider, Bernd

2018-04-26

Despite the increasing evidence for biosynthetic connections between flower pigments and volatiles, examples of such relationships in polymorphic plant species remains limited. Here, we investigated color-scent associations in flowers from Papaver nudicaule (Papaveraceae). We determined the spectral reflectance and the scent composition of flowers of four color cultivars. We found that pigments and volatiles occur in specific combinations in flowers of P. nudicaule. The presence of indole in the bouquets is strongly associated with the occurrence of yellow pigments called nudicaulins, for which indole is one of the final biosynthetic precursors. While yellow flowers emit an excess of indole, orange flowers consume it during nudicaulin production and lack the substance in their bouquet. Using the honeybee, Apis mellifera, we evaluated how color and scent affect the discrimination of these flowers by pollinators. Honeybees were able to discriminate artificial odor mixtures resembling the natural flower odors. Bees trained with stimuli combining colors and odors showed an improved discrimination performance. Our results indicate that the indole moiety of nudicaulins and emitted indole might be products of the same biochemical pathway. We propose that conserved pathways account for the evolution of color-scent associations in P. nudicaule and that these associations positively affect flower constancy of pollinators. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

PubMed

Menendez-Bravo, Simón; Comba, Santiago; Sabatini, Martín; Arabolaza, Ana; Gramajo, Hugo

2014-07-01

Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Averufanin is an aflatoxin B1 precursor between averantin and averufin in the biosynthetic pathway.

PubMed Central

McCormick, S P; Bhatnagar, D; Lee, L S

1987-01-01

Wild-type Aspergillus parasiticus produces, in addition to the colorless aflatoxins, a number of pigmented secondary metabolites. Examination of these pigments demonstrated that a major component was an anthraquinone, averufanin. Radiolabeling studies with [14C]averufanin showed that 23% of the label was incorporated into aflatoxin B1 by the wild type and that 31% of the label was incorporated into O-methylsterigmatocystin by a non-aflatoxin-producing isolate. In similar studies with blocked mutants of A. parasiticus the 14C label from averufanin was accumulated in averufin (72%) and versicolorin A (54%) but not averantin. The results demonstrate that averufanin is a biosynthetic precursor of aflatoxin B1 between averantin and averufin. PMID:3103529

Modification of Monolignol Biosynthetic Pathway in Jute: Different Gene, Different Consequence

PubMed Central

Shafrin, Farhana; Ferdous, Ahlan Sabah; Sarkar, Suprovath Kumar; Ahmed, Rajib; Amin, Al-; Hossain, Kawsar; Sarker, Mrinmoy; Rencoret, Jorge; Gutiérrez, Ana; del Rio, Jose C.; Sanan-Mishra, Neeti; Khan, Haseena

2017-01-01

Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16–25% reduction in acid insoluble lignin for the whole stem and ~13–14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition. PMID:28051165

Novel tryptophan metabolic pathways in auxin biosynthesis in silkworm.

PubMed

Yokoyama, Chiaki; Takei, Mami; Kouzuma, Yoshiaki; Nagata, Shinji; Suzuki, Yoshihito

2017-08-01

In the course of our study of the biosynthetic pathway of auxin, a class of phytohormones, in insects, we proposed the biosynthetic pathway tryptophan (Trp)→indole-3-acetaldoxime (IAOx)→indole-3-acetadehyde (IAAld)→indole-3-acetic acid (IAA). In this study, we identified two branches in the metabolic pathways in the silkworm, possibly affecting the efficiency of IAA production: Trp→indole-3-pyruvic acid→indole-3-lactic acid and IAAld→indole-3-ethanol. We also determined the apparent conversion activities (2.05×10 -7 UmL -1 for Trp→IAA, 1.30×10 -5 UmL -1 for IAOx→IAA, and 3.91×10 -1 UmL -1 for IAAld→IAA), which explain why IAOx and IAAld are barely detectable as either endogenous compounds or metabolites of their precursors. The failure to detect IAAld, even in the presence of an inhibitor of the conversion IAAld→IAA, is explained by a switch in the conversion from IAAld→IAA to IAAld→IEtOH. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nonpigmented and Bacteriochlorophyll-Containing Bradyrhizobia Isolated from Aeschynomene indica

PubMed Central

van Berkum, P.; Tully, R. E.; Keister, D. L.

1995-01-01

The legume genus Aeschynomene is unusual, since many species develop stem nodules and the bradyrhizobia isolated from these nodules produce bacteriochlorophyll (Bchl). Evidence is presented that the bradyrhizobia of Aeschynomene indica have wide distribution throughout the world, since A. indica was nodulated when grown in 58 soils collected in 14 different countries. Only 38 of 79 isolates tested synthesized Bchl and carotenoids during heterotrophic growth. Nine isolates produced Bchl constitutively, and cultures were pigmented after growth in the dark. The other isolates required light for Bchl production. The DNA from seven pigmented and three nonpigmented bradyrhizobia hybridized with a DNA probe containing the genes for the photosynthetic apparatus of Rhodobacter capsulatus, but DNA from two other nonpigmented isolates did not hybridize with this probe. A relationship between pigmentation in culture and symbiotic phenotype was not evident, since bradyrhizobia of both Bchl phenotypes nodulated stems of A. indica and formed nitrogen-fixing symbioses. Several isolates, which were ineffective on A. indica, probably do belong to the proposed cross-inoculation group 3 (D. Alazard, Appl. Environ. Microbiol. 50:732-734, 1985), since they did not nodulate Aeschynomene americana or Macroptilium atropurpureum. Since it has been suggested that extant rhizobia arose from photosynthetic ancestors (J. I. Sprent, p. 45-54, in P. M. Gresshoff, L. E. Roth, G. Stacey, and W. E. Newton, ed., Nitrogen Fixation: Achievements and Objectives, 1990), we propose that the nonpigmented isolates may represent an extant lineage of an intermediate evolutionary stage. PMID:16534933

Mutual control of axial and equatorial ligands: model studies with [Ni]-bacteriochlorophyll-a.

PubMed

Yerushalmi, Roie; Noy, Dror; Baldridge, Kim K; Scherz, Avigdor

2002-07-17

Modification of the metal's electronic environment by ligand association and dissociation in metalloenzymes is considered cardinal to their catalytic activity. We have recently presented a novel system that utilizes the bacteriochlorophyll (BChl) macrocycle as a ligand and reporter. This system allows for charge mobilization in the equatorial plane and experimental estimate of changes in the electronic charge density around the metal with no modification of the metal's chemical environment. The unique spectroscopy, electrochemistry and coordination chemistry of [Ni]-bacteriochlorophyll ([Ni]-BChl) enable us to follow directly fine details and steps involved in the function of the metal redox center. This approach is utilized here whereby electro-chemical reduction of [Ni]-BChl to the monoanion [Ni]-BChl(-) results in reversible dissociation of biologically relevant axial ligands. Similar ligand dissociation was previously detected upon photoexcitation of [Ni]-BChl (Musewald, C.; Hartwich, G.; Lossau, H.; Gilch, P.; Pollinger-Dammer, F.; Scheer, H.; Michel-Beyerle, M. E. J. Phys. Chem. B 1999, 103, 7055-7060 and Noy, D.; Yerushalmi, R.; Brumfeld, V.; Ashur, I.; Baldridge, K. K.; Scheer, H.; Scherz, A. J. Am. Chem. Soc. 2000, 122, 3937-3944). The electrochemical measurements and quantum mechanical (QM) calculations performed here for the neutral, singly reduced, monoligated, and singly reduced, monoligated [Ni]-BChl suggest the following: (a) Electroreduction, although resulting in a pi anion [Ni]-BChl(-) radical, causes electron density migration to the [Ni]-BChl core. (b) Reduction of nonligated [Ni]-BChl does not change the macrocycle conformation, whereas axial ligation results in a dramatic expansion of the metal core and a flattening of the highly ruffled macrocycle conformation. (c) In both the monoanion and singly excited [Ni]-BChl ([Ni]-BChl*), the frontier singly occupied molecular orbital (SOMO) has a small but nonnegligible metal character. Finally, (d

Presence of exclusively bacteriochlorophyll-c containing substrain in the culture of green sulfur photosynthetic bacterium Chlorobium vibrioforme strain NCIB 8327 producing bacteriochlorophyll-d.

PubMed

Saga, Yoshitaka; Oh-oka, Hirozo; Hayashi, Takashi; Tamiaki, Hitoshi

2003-12-01

The light-dependent composition change of light harvesting bacteriochlorophyll(BChl)s in the present culture of a green sulfur photosynthetic bacterium Chlorobium (Chl.) vibrioforme f. sp. thiosulfatophilum strain NCIB 8327 was investigated by visible absorption spectroscopy and HPLC analyses. When the culture was repeatedly grown in liquid media under a low light condition, both the Soret and Qy absorption bands of the in vivo spectrum were shifted to longer wavelengths. Analysis of the extracted pigments by HPLC revealed that the ratio of the amount of BChl-c to that of BChl-d molecules gradually increased during repeated cultivation. In contrast, when the culture grown under a low light intensity was transferred to a high light condition and continued to be grown, the absorption bands were shifted to shorter wavelengths and the ratio of BChls-c/d decreased finally to the almost original value. Colonies were prepared on solid agar media from the liquid culture containing both BChls-c and d, which was grown under a low light intensity. Each colony obtained was found to contain either BChl-c or d, but not both of them. Two types of cells isolated in this study were derived from the same clone, judged from their genetic analyses. The variation of pigment composition in our liquid culture observed here could be ascribed to the difference of growth rates between two substrains containing BChl-c and BChl-d, respectively, depending on light conditions.

Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla x E. grandis leads to improved sugar release

DOE PAGES

Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; ...

2015-08-27

In this study, lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.

Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD

PubMed Central

Vit, Allegra; Misson, Laëtitia; Blankenfeldt, Wulf; Seebeck, Florian Peter

2014-01-01

Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosyn­thesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-l-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit. PMID:24817736

Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nara, Takeshi, E-mail: tnara@juntendo.ac.jp; Hashimoto, Muneaki; Hirawake, Hiroko

2012-02-03

Highlights: Black-Right-Pointing-Pointer An Escherichia coli strain co-expressing CPSII, ATC, and DHO of Trypanosoma cruzi was constructed. Black-Right-Pointing-Pointer Molecular interactions between CPSII, ATC, and DHO of T. cruzi were demonstrated. Black-Right-Pointing-Pointer CPSII bound with both ATC and DHO. Black-Right-Pointing-Pointer ATC bound with both CPSII and DHO. Black-Right-Pointing-Pointer A functional tri-enzyme complex might precede the establishment of the fused enzyme. -- Abstract: The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusionmore » enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.« less

Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

PubMed

Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

2017-11-30

The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications.

PubMed

Chua, Jie Shi; Kuberan, Balagurunathan

2017-11-21

. However, the structure-activity relationship has long been cryptic. Nonetheless, xylosides have been designed to increase HS priming, modified to inhibit endogenous GAG production without priming, and engineered to be more biologically relevant. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. They are small, orally bioavailable, easily excreted, and utilize the host cell biosynthetic machinery to assemble GAGs that are likely nonimmunogenic. Various xylosides have been shown, in different biological systems, to have anticoagulant effects, selectively kill tumor cells, abrogate angiogenic and metastatic pathways, promote angiogenesis and neuronal growth, and affect embryonic development. However, most of these studies utilized the commercially available one or two β-D-xylosides and focused on the impact of endogenous proteoglycan-bound GAG inhibition on biological activity. Nevertheless, the manipulation of cell behavior as a result of stabilizing growth factor signaling with xyloside-primed GAGs is also reckonable but underexplored. Recent advances in the use of molecular modeling and docking simulations to understand the structure-activity relationships of xylosides have opened up the possibility of a more rational aglycone design to achieve a desirable biological outcome through selective priming and inhibitory activities. We envision these advances will encourage more researchers to explore these fascinating xylosides, harness the GAG biosynthetic machinery for a wider range of biomedical applications, and accelerate the successful transition of xyloside-based therapeutics from bench to bedside.

Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry.

PubMed

Liu, Fenghong; Wang, Lei; Gu, Liang; Zhao, Wei; Su, Hongyan; Cheng, Xianhao

2015-12-01

In our preliminary study, the ripe fruits of two highbush blueberry (Vaccinium corymbosum L.) cultivars, cv 'Berkeley' and cv 'Bluecrop', were found to contain different levels of ascorbic acid. However, factors responsible for these differences are still unknown. In the present study, ascorbic acid content in fruits was compared with expression profiles of ascorbic acid biosynthetic and recycling genes between 'Bluecrop' and 'Berkeley' cultivars. The results indicated that the l-galactose pathway was the predominant route of ascorbic acid biosynthesis in blueberry fruits. Moreover, higher expression levels of the ascorbic acid biosynthetic genes GME, GGP, and GLDH, as well as the recycling genes MDHAR and DHAR, were associated with higher ascorbic acid content in 'Bluecrop' compared with 'Berkeley', which indicated that a higher efficiency ascorbic acid biosynthesis and regeneration was likely to be responsible for the higher ascorbic acid accumulation in 'Bluecrop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proposal for probing energy transfer pathway by single-molecule pump-dump experiment.

PubMed

Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

2016-06-09

The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices.

Proposal for probing energy transfer pathway by single-molecule pump-dump experiment

NASA Astrophysics Data System (ADS)

Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

2016-06-01

The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices.

Proposal for probing energy transfer pathway by single-molecule pump-dump experiment

PubMed Central

Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

2016-01-01

The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices. PMID:27277702

Ammosamides Unveil Novel Biosynthetic Machinery.

PubMed

Colosimo, Dominic A; MacMillan, John B

2016-12-22

In this issue of Cell Chemical Biology, Jordan and Moore (2016) present a thorough biosynthetic analysis of ammosamides, a bacterial natural product. The work highlights the previously unknown overlap between two natural products families: pyrroloquinoline alkaloids and ribosomally synthesized posttranslationally modified peptides (RiPPs). Copyright © 2016. Published by Elsevier Ltd.

Real-Time Kinetic Probes Support Monothiol Glutaredoxins As Intermediate Carriers in Fe-S Cluster Biosynthetic Pathways.

PubMed

Vranish, James N; Das, Deepika; Barondeau, David P

2016-11-18

Iron-sulfur (Fe-S) clusters are protein cofactors that are required for many essential cellular functions. Fe-S clusters are synthesized and inserted into target proteins by an elaborate biosynthetic process. The insensitivity of most Fe-S assembly and transfer assays requires high concentrations for components and places major limits on reaction complexity. Recently, fluorophore labels were shown to be effective at reporting cluster content for Fe-S proteins. Here, the incorporation of this labeling approach allowed the design and interrogation of complex Fe-S cluster biosynthetic reactions that mimic in vivo conditions. A bacterial Fe-S assembly complex, composed of the cysteine desulfurase IscS and scaffold protein IscU, was used to generate [2Fe-2S] clusters for transfer to mixtures of putative intermediate carrier and acceptor proteins. The focus of this study was to test whether the monothiol glutaredoxin, Grx4, functions as an obligate [2Fe-2S] carrier protein in the Fe-S cluster distribution network. Interestingly, [2Fe-2S] clusters generated by the IscS-IscU complex transferred to Grx4 at rates comparable to previous assays using uncomplexed IscU as a cluster source in chaperone-assisted transfer reactions. Further, we provide evidence that [2Fe-2S]-Grx4 delivers clusters to multiple classes of Fe-S targets via direct ligand exchange in a process that is both dynamic and reversible. Global fits of cluster transfer kinetics support a model in which Grx4 outcompetes terminal target proteins for IscU-bound [2Fe-2S] clusters and functions as an intermediate cluster carrier. Overall, these studies demonstrate the power of chemically conjugated fluorophore reporters for unraveling mechanistic details of biological metal cofactor assembly and distribution networks.

Novel pathway of 3-hydroxyanthranilic acid formation in limazepine biosynthesis reveals evolutionary relation between phenazines and pyrrolobenzodiazepines.

PubMed

Pavlikova, Magdalena; Kamenik, Zdenek; Janata, Jiri; Kadlcik, Stanislav; Kuzma, Marek; Najmanova, Lucie

2018-05-17

Natural pyrrolobenzodiazepines (PBDs) form a large and structurally diverse group of antitumour microbial metabolites produced through complex pathways, which are encoded within biosynthetic gene clusters. We sequenced the gene cluster of limazepines and proposed their biosynthetic pathway based on comparison with five available gene clusters for the biosynthesis of other PBDs. Furthermore, we tested two recombinant proteins from limazepine biosynthesis, Lim5 and Lim6, with the expected substrates in vitro. The reactions monitored by LC-MS revealed that limazepine biosynthesis involves a new way of 3-hydroxyanthranilic acid formation, which we refer to as the chorismate/DHHA pathway and which represents an alternative to the kynurenine pathway employed for the formation of the same precursor in the biosynthesis of other PBDs. The chorismate/DHHA pathway is presumably also involved in the biosynthesis of PBD tilivalline, several natural products unrelated to PBDs, and its part is shared also with phenazine biosynthesis. The similarities between limazepine and phenazine biosynthesis indicate tight evolutionary links between these groups of compounds.

Hyaluronan Production Regulates Metabolic and Cancer Stem-like Properties of Breast Cancer Cells via Hexosamine Biosynthetic Pathway-coupled HIF-1 Signaling*

PubMed Central

Chanmee, Theerawut; Ontong, Pawared; Izumikawa, Tomomi; Higashide, Miho; Mochizuki, Nobutoshi; Chokchaitaweesuk, Chatchadawalai; Khansai, Manatsanan; Nakajima, Kazuki; Kakizaki, Ikuko; Kongtawelert, Prachya; Taniguchi, Naoyuki; Itano, Naoki

2016-01-01

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling. PMID:27758869

Solvation effect of bacteriochlorophyll excitons in light-harvesting complex LH2.

PubMed

Urboniene, V; Vrublevskaja, O; Trinkunas, G; Gall, A; Robert, B; Valkunas, L

2007-09-15

We have characterized the influence of the protein environment on the spectral properties of the bacteriochlorophyll (Bchl) molecules of the peripheral light-harvesting (or LH2) complex from Rhodobacter sphaeroides. The spectral density functions of the pigments responsible for the 800 and 850 nm electronic transitions were determined from the temperature dependence of the Bchl absorption spectra in different environments (detergent micelles and native membranes). The spectral density function is virtually independent of the hydrophobic support that the protein experiences. The reorganization energy for the B850 Bchls is 220 cm(-1), which is almost twice that of the B800 Bchls, and its Huang-Rhys factor reaches 8.4. Around the transition point temperature, and at higher temperatures, both the static spectral inhomogeneity and the resonance interactions become temperature-dependent. The inhomogeneous distribution function of the transitions exhibits less temperature dependence when LH2 is embedded in membranes, suggesting that the lipid phase protects the protein. However, the temperature dependence of the fluorescence spectra of LH2 cannot be fitted using the same parameters determined from the analysis of the absorption spectra. Correct fitting requires the lowest exciton states to be additionally shifted to the red, suggesting the reorganization of the exciton spectrum.

Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum.

PubMed Central

Kahn, R A; Bak, S; Svendsen, I; Halkier, B A; Møller, B L

1997-01-01

A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-alpha-dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled. PMID:9414567

Molecular Cloning and Functional Characterization of Three Distinct N-Methyltransferases Involved in the Caffeine Biosynthetic Pathway in Coffee Plants1

PubMed Central

Uefuji, Hirotaka; Ogita, Shinjiro; Yamaguchi, Yube; Koizumi, Nozomu; Sano, Hiroshi

2003-01-01

Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps. In the present study, three types of cDNAs encoding N-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated as CaXMT1, CaMXMT2, and CaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with a Km value of 78 μm, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a Km of 251 μm, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a Km of 1,222 μm. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1 and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified three N-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine. PMID:12746542

Molecular Genetic Characterization of Terreic Acid Pathway in Aspergillus terreus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Chun-Jun; Sun, Wei-wen; Bruno, Kenneth S.

Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, in combined with bioinformatic analyses, allowed us to propose a biosynthetic pathway for terreic acid. Lastly, defining the pathway and the genes involved will facilitate the engineering of this molecule with interesting antimicrobial and antitumor bioactivities.

Molecular Genetic Characterization of Terreic Acid Pathway in Aspergillus terreus

DOE PAGES

Guo, Chun-Jun; Sun, Wei-wen; Bruno, Kenneth S.; ...

2014-09-29

Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, in combined with bioinformatic analyses, allowed us to propose a biosynthetic pathway for terreic acid. Lastly, defining the pathway and the genes involved will facilitate the engineering of this molecule with interesting antimicrobial and antitumor bioactivities.

Comparison between chlorosomes containing bacteriochlorophyll-c and chlorosomes containing bacteriochlorophyll-d isolated from two substrains of green sulfur photosynthetic bacterium Chlorobium vibrioforme NCIB 8327.

PubMed

Saga, Yoshitaka; Tamiaki, Hitoshi

2004-07-19

Chlorosomes containing bacteriochlorophyll(BChl)-c and those containing BChl-d were isolated from two substrains of Chlorobium vibrioforme f. sp. thiosulfatophilum NCIB 8327, respectively. The two types of chlorosomes were investigated from the following aspect, what kinds of effects the molecular structure of chlorosomal BChls had on structural and spectroscopic properties of in vivo self-aggregates in chlorosomes without alteration of the other components such as chlorosomal proteins and lipids; both chlorosomes were expected to have the same components except for light-harvesting BChls. In their visible absorption spectra, the differences of Soret and Q(y) peak positions between BChl-c containing and BChl-d containing chlorosomes were similar to the differences between monomeric BChl-c and d. An inverse S-shaped CD signal in the Q(y) region of BChl-d containing chlorosomes was 1.4 times larger than that of BChl-c containing chlorosomes, when the Q(y) absorbance of the two chlorosomes was almost the same. This implies that the excitonic interaction of BChl-d is larger than that of BChl-c in natural chlorosomes. Resonance Raman spectroscopy showed that BChl self-assemblies in both chlorosomes were essentially formed by the same local structural interaction among 3(1)-hydroxy group, 13-keto group, and central magnesium. BChl-d self-aggregates in chlorosomes were more tolerant of 1-hexanol than in vivo BChl-c aggregates, suggesting that the molecular structure of BChl-d provided more stable self-assemblies than BChl-c in natural chlorosomes.

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

PubMed

Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, María Angeles; Sabater-Jara, Ana Belén

2018-07-01

Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight -1 ) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

PubMed

Lucena, Miguel C; Carvalho-Cruz, Patricia; Donadio, Joana L; Oliveira, Isadora A; de Queiroz, Rafaela M; Marinho-Carvalho, Monica M; Sola-Penna, Mauro; de Paula, Iron F; Gondim, Katia C; McComb, Mark E; Costello, Catherine E; Whelan, Stephen A; Todeschini, Adriane R; Dias, Wagner B

2016-06-17

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Modulation of the Isoprenoid/Cholesterol Biosynthetic Pathway During Neuronal Differentiation In Vitro.

PubMed

Cartocci, Veronica; Segatto, Marco; Di Tunno, Ilenia; Leone, Stefano; Pfrieger, Frank W; Pallottini, Valentina

2016-09-01

During differentiation, neurons acquire their typical shape and functional properties. At present, it is unclear, whether this important developmental step involves metabolic changes. Here, we studied the contribution of the mevalonate (MVA) pathway to neuronal differentiation using the mouse neuroblastoma cell line N1E-115 as experimental model. Our results show that during differentiation, the activity of 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR), a key enzyme of MVA pathway, and the level of Low Density Lipoprotein receptor (LDLr) decrease, whereas the level of LDLr-related protein-1 (LRP1) and the dimerization of Scavanger Receptor B1 (SRB-1) rise. Pharmacologic inhibition of HMGR by simvastatin accelerated neuronal differentiation by modulating geranylated proteins. Collectively, our data suggest that during neuronal differentiation, the activity of the MVA pathway decreases and we postulate that any interference with this process impacts neuronal morphology and function. Therefore, the MVA pathway appears as an attractive pharmacological target to modulate neurological and metabolic symptoms of developmental neuropathologies. J. Cell. Biochem. 117: 2036-2044, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Early Wound Morbidity after Open Ventral Hernia Repair with Biosynthetic or Polypropylene Mesh.

PubMed

Sahoo, Sambit; Haskins, Ivy N; Huang, Li-Ching; Krpata, David M; Derwin, Kathleen A; Poulose, Benjamin K; Rosen, Michael J

2017-10-01

Recently introduced slow-resorbing biosynthetic and non-resorbing macroporous polypropylene meshes are being used in hernias with clean-contaminated and contaminated wounds. However, information about the use of biosynthetic meshes and their outcomes compared with polypropylene meshes in clean-contaminated and contaminated cases is lacking. Here we evaluate the use of biosynthetic mesh and polypropylene mesh in elective open ventral hernia repair (OVHR) and investigate differences in early wound morbidity after OVHR within clean-contaminated and contaminated cases. All elective, OVHR with biosynthetic mesh or uncoated polypropylene mesh from January 2013 through October 2016 were identified within the Americas Hernia Society Quality Collaborative. Association of mesh type with 30-day wound events in clean-contaminated or contaminated wounds was investigated using a 1:3 propensity-matched analysis. Biosynthetic meshes were used in 8.5% (175 of 2,051) of elective OVHR, with the majority (57.1%) used in low-risk or comorbid clean cases. Propensity-matched analysis in clean-contaminated and contaminated cases showed no significant difference between biosynthetic mesh and polypropylene mesh groups for 30-day surgical site occurrences (20.7% vs 16.7%; p = 0.49) or unplanned readmission (13.8% vs 9.8%; p = 0.4). However, surgical site infections (22.4% vs 10.9%; p = 0.03), surgical site occurrences requiring procedural intervention (24.1% vs 13.2%; p = 0.049), and reoperation rates (13.8% vs 4.0%; p = 0.009) were significantly higher in the biosynthetic group. Biosynthetic mesh appears to have higher rates of 30-day wound morbidity compared with polypropylene mesh in elective OVHR with clean-contaminated or contaminated wounds. Additional post-market analysis is needed to provide evidence defining best mesh choices, location, and surgical technique for repairing contaminated ventral hernias. Copyright © 2017 American College of Surgeons. Published by Elsevier Inc

Ligand-receptor co-evolution shaped the jasmonate pathway in land plants.

PubMed

Monte, Isabel; Ishida, Sakiko; Zamarreño, Angel M; Hamberg, Mats; Franco-Zorrilla, José M; García-Casado, Gloria; Gouhier-Darimont, Caroline; Reymond, Philippe; Takahashi, Kosaku; García-Mina, José M; Nishihama, Ryuichi; Kohchi, Takayuki; Solano, Roberto

2018-05-01

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

Integrating computational methods to retrofit enzymes to synthetic pathways.

PubMed

Brunk, Elizabeth; Neri, Marilisa; Tavernelli, Ivano; Hatzimanikatis, Vassily; Rothlisberger, Ursula

2012-02-01

Microbial production of desired compounds provides an efficient framework for the development of renewable energy resources. To be competitive to traditional chemistry, one requirement is to utilize the full capacity of the microorganism to produce target compounds with high yields and turnover rates. We use integrated computational methods to generate and quantify the performance of novel biosynthetic routes that contain highly optimized catalysts. Engineering a novel reaction pathway entails addressing feasibility on multiple levels, which involves handling the complexity of large-scale biochemical networks while respecting the critical chemical phenomena at the atomistic scale. To pursue this multi-layer challenge, our strategy merges knowledge-based metabolic engineering methods with computational chemistry methods. By bridging multiple disciplines, we provide an integral computational framework that could accelerate the discovery and implementation of novel biosynthetic production routes. Using this approach, we have identified and optimized a novel biosynthetic route for the production of 3HP from pyruvate. Copyright © 2011 Wiley Periodicals, Inc.

Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis of Bacteriochlorophyll a in Rhodobacter sphaeroides and Its Zinc and Copper Analogue Pigments

NASA Astrophysics Data System (ADS)

Calvano, Cosima Damiana; Ventura, Giovanni; Trotta, Massimo; Bianco, Giuliana; Cataldi, Tommaso R. I.; Palmisano, Francesco

2017-01-01

Bacteriochlorophyll a ( BChl a), a photosynthetic pigment performing the same functions of chlorophylls in plants, features a bacteriochlorin macrocycle ring (18 π electrons) with two reduced pyrrole rings along with a hydrophobic terpenoid side chain (i.e., the phytol residue). Chlorophylls analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is not so straightforward since pheophytinization (i.e., release of the central metal ion) and cleavage of the phytol-ester linkage are invariably observed by employing protonating matrices such as 2,5-dihydroxybenzoic acid, sinapinic acid, and α-cyano-4-hydroxycinnamic acid. Using BChl a from Rhodobacter sphaeroides R26 strain as a model system, different electron-transfer (ET) secondary reaction matrices, leading to the formation of almost stable radical ions in both positive ([M]+•) and negative ([M]-•) ionization modes at m/z 910.55, were evaluated. Compared with ET matrices such as trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 2,2':5',2''-terthiophene (TER), anthracene (ANT), and 9,10-diphenylanthracene (DP-ANT), 1,5-diaminonaphthalene (DAN) was found to provide the highest ionization yield with a negligible fragmentation. DAN also displayed excellent ionization properties for two metal ion-substituted bacteriochlorophylls, (i.e., Zn- and Cu-BChl a at m/z 950.49 and 949.49), respectively. MALDI MS/MS of both radical charged molecular species provide complementary information, thus making analyte identification more straightforward.

Evolution of the Structure and Chromosomal Distribution of Histidine Biosynthetic Genes

NASA Astrophysics Data System (ADS)

Fani, Renato; Mori, Elena; Tamburini, Elena; Lazcano, Antonio

1998-10-01

A database of more than 100 histidine biosynthetic genes from different organisms belonging to the three primary domains has been analyzed, including those found in the now completely sequenced genomes of Haemophilus influenzae, Mycoplasma genitalium, Synechocystis sp., Methanococcus jannaschii, and Saccharomyces cerevisiae. The ubiquity of his genes suggests that it is a highly conserved pathway that was probably already present in the last common ancestor of all extant life. The chromosomal distribution of the his genes shows that the enterobacterial histidine operon structure is not the only possible organization, and that there is a diversity of gene arrays for the his pathway. Analysis of the available sequences shows that gene fusions (like those involved in the origin of the Escherichia coli and Salmonella typhimurium hisIE and hisB gene structures) are not universal. In contrast, the elongation event that led to the extant hisA gene from two homologous ancestral modules, as well as the subsequent paralogous duplication that originated hisF, appear to be irreversible and are conserved in all known organisms. The available evidence supports the hypothesis that histidine biosynthesis was assembled by a gene recruitment process.

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

PubMed Central

Senderek, Jan; Müller, Juliane S.; Dusl, Marina; Strom, Tim M.; Guergueltcheva, Velina; Diepolder, Irmgard; Laval, Steven H.; Maxwell, Susan; Cossins, Judy; Krause, Sabine; Muelas, Nuria; Vilchez, Juan J.; Colomer, Jaume; Mallebrera, Cecilia Jimenez; Nascimento, Andres; Nafissi, Shahriar; Kariminejad, Ariana; Nilipour, Yalda; Bozorgmehr, Bita; Najmabadi, Hossein; Rodolico, Carmelo; Sieb, Jörn P.; Steinlein, Ortrud K.; Schlotter, Beate; Schoser, Benedikt; Kirschner, Janbernd; Herrmann, Ralf; Voit, Thomas; Oldfors, Anders; Lindbergh, Christopher; Urtizberea, Andoni; von der Hagen, Maja; Hübner, Angela; Palace, Jacqueline; Bushby, Kate; Straub, Volker; Beeson, David; Abicht, Angela; Lochmüller, Hanns

2011-01-01

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general. PMID:21310273

Two-dimensional isobutyl acetate production pathways to improve carbon yield

PubMed Central

Tashiro, Yohei; Desai, Shuchi H.; Atsumi, Shota

2015-01-01

For an economically competitive biological process, achieving high carbon yield of a target chemical is crucial. In biochemical production, pyruvate and acetyl-CoA are primary building blocks. When sugar is used as the sole biosynthetic substrate, acetyl-CoA is commonly generated by pyruvate decarboxylation. However, pyruvate decarboxylation during acetyl-CoA formation limits the theoretical maximum carbon yield (TMCY) by releasing carbon, and in some cases also leads to redox imbalance. To avoid these problems, we describe here the construction of a metabolic pathway that simultaneously utilizes glucose and acetate. Acetate is utilized to produce acetyl-CoA without carbon loss or redox imbalance. We demonstrate the utility of this approach for isobutyl acetate (IBA) production, wherein IBA production with glucose and acetate achieves a higher carbon yield than with either sole carbon source. These results highlight the potential for this multiple carbon source approach to improve the TMCY and balance redox in biosynthetic pathways. PMID:26108471

A stilbenoid-specific prenyltransferase utilizes dimethylallyl pyrophosphate from the plastidic terpenoid pathway

USDA-ARS?s Scientific Manuscript database

Prenylated stilbenoids found preferentially in a few legume plants exhibit phytoalexin properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy r...

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties.

PubMed

Rama Reddy, Nagaraja Reddy; Mehta, Rucha Harishbhai; Soni, Palak Harendrabhai; Makasana, Jayanti; Gajbhiye, Narendra Athamaram; Ponnuchamy, Manivel; Kumar, Jitendra

2015-01-01

Senna (Cassia angustifolia Vahl.) is a world's natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with 'green plant database (txid 33090)', Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various

Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (Cassia angustifolia Vahl.), a Non-Model Plant with Potent Laxative Properties

PubMed Central

Rama Reddy, Nagaraja Reddy; Mehta, Rucha Harishbhai; Soni, Palak Harendrabhai; Makasana, Jayanti; Gajbhiye, Narendra Athamaram; Ponnuchamy, Manivel; Kumar, Jitendra

2015-01-01

Senna (Cassia angustifolia Vahl.) is a world’s natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with ‘green plant database (txid 33090)’, Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various

Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440

PubMed Central

Molina‐Henares, M. Antonia; García‐Salamanca, Adela; Molina‐Henares, A. Jesús; De La Torre, Jesús; Herrera, M. Carmen; Ramos, Juan L.; Duque, Estrella

2009-01-01

Summary Pseudomonas putida KT2440 is a non‐pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini‐Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene‐encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB‐like genes are present in the host chromosome. PMID:21261884

A novel MVA-mediated pathway for isoprene production in engineered E. coli.

PubMed

Yang, Jianming; Nie, Qingjuan; Liu, Hui; Xian, Mo; Liu, Huizhou

2016-01-20

To deal with the increasingly severe energy crisis and environmental consequences, biofuels and biochemicals generated from renewable resources could serve as a promising alternative for replacing petroleum as a source of fuel and chemicals, among which isoprene (2-methyl-1,3-butadiene) in particular is of great significance in that it is an important platform chemical, which has been used in industrial production of synthetic rubber for tires and coatings or aviation fuel. We firstly introduced fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species into E. coli to directly convert MVA(mevalonate) into 3-methy-3-buten-1-ol. And then to transform 3-methy-3-buten-1-ol to isoprene, oleate hydratase (OhyAEM) from Elizabethkingia meningoseptica was overexpressed in E. coli. A novel biosynthetic pathway of isoprene in E. coli was established by co-expressing the heterologous mvaE gene encoding acetyl-CoA acetyltransferase/HMG-CoA reductase and mvaS gene encoding HMG-CoA synthase from Enterococcus faecalis, fatty acid decarboxylase (OleTJE) and oleate hydratase (OhyAEM). Furthermore, to enhance isoprene production, a further optimization of expression level of OleTJE, OhyAEM was carried out by using different promoters and copy numbers of plasmids. Thereafter, the fermentation process was also optimized to improve the production of isoprene. The final engineered strain, YJM33, bearing the innovative biosynthetic pathway of isoprene, was found to produce isoprene up to 2.2 mg/L and 620 mg/L under flask and fed-batch fermentation conditions, respectively. In this study, by using metabolic engineering techniques, the novel MVA-mediated biosynthetic pathway of isoprene was successfully assembled in E. coli BL21(DE3) with the heterologous MVA upper pathway, OleTJE from Jeotgalicoccus species and OhyAEM from Elizabethkingia meningoseptica. Compared with traditional MVA pathway, the novel pathway is shortened by 3 steps. In addition, this is the first report on the

Chapter 7. Cloning and analysis of natural product pathways.

PubMed

Gust, Bertolt

2009-01-01

The identification of gene clusters of natural products has lead to an enormous wealth of information about their biosynthesis and its regulation, and about self-resistance mechanisms. Well-established routine techniques are now available for the cloning and sequencing of gene clusters. The subsequent functional analysis of the complex biosynthetic machinery requires efficient genetic tools for manipulation. Until recently, techniques for the introduction of defined changes into Streptomyces chromosomes were very time-consuming. In particular, manipulation of large DNA fragments has been challenging due to the absence of suitable restriction sites for restriction- and ligation-based techniques. The homologous recombination approach called recombineering (referred to as Red/ET-mediated recombination in this chapter) has greatly facilitated targeted genetic modifications of complex biosynthetic pathways from actinomycetes by eliminating many of the time-consuming and labor-intensive steps. This chapter describes techniques for the cloning and identification of biosynthetic gene clusters, for the generation of gene replacements within such clusters, for the construction of integrative library clones and their expression in heterologous hosts, and for the assembly of entire biosynthetic gene clusters from the inserts of individual library clones. A systematic approach toward insertional mutation of a complete Streptomyces genome is shown by the use of an in vitro transposon mutagenesis procedure.

Towards first-principles calculation of electronic excitations in the ring of the protein-bound bacteriochlorophylls

NASA Astrophysics Data System (ADS)

Polyakov, Igor V.; Khrenova, Maria G.; Moskovsky, Alexander A.; Shabanov, Boris M.; Nemukhin, Alexander V.

2018-04-01

Modeling electronic excitation of bacteriochlorophyll (BChl) molecules in light-harvesting (LH) antennae from photosynthetic centers presents a challenge for the quantum theory. We report on a quantum chemical study of the ring of 32 BChl molecules from the bacterial core complex LH1-RC. Diagonal and off-diagonal elements of the excitonic Hamiltonian matrices are estimated in quantum chemical calculations of relevant fragments using the TD-DFT and CIS approaches. The deviation of the computed excitation energy of this BChl system from the experimental data related to the Qy band maximum of this LH1-RC complex is about 0.2 eV. We demonstrate that corrections due to improvement in modeling of an individual BChl molecule and due to contributions from the protein environment are in the range of the obtained discrepancy between theory and experiment. Differences between results of the excitonic model and direct quantum chemical calculations of BChl aggregates fall in the same range.

Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.

PubMed

Rodriguez, Alberto; Martínez, Juan A; Millard, Pierre; Gosset, Guillermo; Portais, Jean-Charles; Létisse, Fabien; Bolivar, Francisco

2017-06-01

Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Recent advances in biosynthetic modeling of nitric oxide reductases and insights gained from nuclear resonance vibrational and other spectroscopic studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborty, Saumen; Reed, Julian; Sage, Timothy

This Forum Article focuses on recent advances in structural and spectroscopic studies of biosynthetic models of nitric oxide reductases (NORs). NORs are complex metalloenzymes found in the denitrification pathway of Earth's nitrogen cycle where they catalyze the proton-dependent twoelectron reduction of nitric oxide (NO) to nitrous oxide (N 2O). While much progress has been made in biochemical and biophysical studies of native NORs and their variants, a. clear mechanistic understanding of this important metalloenzyme related to its function is still elusive. We report herein UV vis and nuclear resonance vibrational spectroscopy (NRVS) studies of mononitrosylated intermediates of the NOR reactionmore » of a biosynthetic model. The ability to selectively substitute metals at either heme or nonheme metal sites allows the introduction of independent 57Fe probe atoms at either site, as well as allowing the preparation of analogues of stable reaction intermediates by replacing either metal with a redox inactive metal. Together with previous structural and spectroscopic results, we summarize insights gained from studying these biosynthetic models toward understanding structural features responsible for the NOR activity and its mechanism. As a result, the outlook on NOR modeling is also discussed, with an emphasis on the design of models capable of catalytic turnovers designed based on close mimics of the secondary coordination sphere of native NORs.« less

Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism

PubMed Central

Xie, Zhengzhi; Gang, David R.

2009-01-01

Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules—groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants. PMID:19073964

Gene transcript profiles of the TIA biosynthetic pathway in response to ethylene and copper reveal their interactive role in modulating TIA biosynthesis in Catharanthus roseus.

PubMed

Pan, Ya-Jie; Liu, Jia; Guo, Xiao-Rui; Zu, Yuan-Gang; Tang, Zhong-Hua

2015-05-01

Research on transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis of the medicinal plant, Catharanthus roseus, has largely been focused on gene function and not clustering analysis of multiple genes at the transcript level. Here, more than ten key genes encoding key enzyme of alkaloid synthesis in TIA biosynthetic pathways were chosen to investigate the integrative responses to exogenous elicitor ethylene and copper (Cu) at both transcriptional and metabolic levels. The ethylene-induced gene transcripts in leaves and roots, respectively, were subjected to principal component analysis (PCA) and the results showed the overall expression of TIA pathway genes indicated as the Q value followed a standard normal distribution after ethylene treatments. Peak gene expression was at 15-30 μM of ethephon, and the pre-mature leaf had a higher Q value than the immature or mature leaf and root. Treatment with elicitor Cu found that Cu up-regulated overall TIA gene expression more in roots than in leaves. The combined effects of Cu and ethephon on TIA gene expression were stronger than their separate effects. It has been documented that TIA gene expression is tightly regulated by the transcriptional factor (TF) ethylene responsive factor (ERF) and mitogen-activated protein kinase (MAPK) cascade. The loading plot combination with correlation analysis for the genes of C. roseus showed that expression of the MPK gene correlated with strictosidine synthase (STR) and strictosidine b-D-glucosidase(SGD). In addition, ERF expression correlated with expression of secologanin synthase (SLS) and tryptophan decarboxylase (TDC), specifically in roots, whereas MPK and myelocytomatosis oncogene (MYC) correlated with STR and SGD genes. In conclusion, the ERF regulates the upstream pathway genes in response to heavy metal Cu mainly in C. roseus roots, while the MPK mainly participates in regulating the STR gene in response to ethylene in pre-mature leaf. Interestingly, the

Structure, function and regulation of the enzymes in the starch biosynthetic pathway.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geiger, Jim

structure of ADP- Glucose pyrophosphorylase from potato in its inhibited conformation, and bound to both ATP and ADP-glucose. In addition, we have determined the first structure of glycogen synthase in its "closed", catalytically active conformation bound to ADP-glucose. We also determined the structure of glycogen synthase bound to malto-oligosaccharides, showing for the first time that an enzyme in the starch biosynthetic pathway recognizes glucans not just in its active site but on binding sites on the surface of the enzyme ten’s of Angstroms from the active site. In addition our structure of a glycogen branching enzyme bound to malto-oligosaccharides identified seven distinct binding sites distributed about the surface of the enzyme. We will now determine the function of these sites to get a molecular-level picture of exactly how these enzymes interact with their polymeric substrates and confer specificity leading to the complex structure of the starch granule. We will extend our studies to other isoforms of the enzymes, to understand how their structures give rise to their distinct function. Our goal is to understand what accounts for the various functional differences between SS and SBE isoforms at a molecular level.« less

Integrative genomic mining for enzyme function to enable engineering of a non-natural biosynthetic pathway.

PubMed

Mak, Wai Shun; Tran, Stephen; Marcheschi, Ryan; Bertolani, Steve; Thompson, James; Baker, David; Liao, James C; Siegel, Justin B

2015-11-24

The ability to biosynthetically produce chemicals beyond what is commonly found in Nature requires the discovery of novel enzyme function. Here we utilize two approaches to discover enzymes that enable specific production of longer-chain (C5-C8) alcohols from sugar. The first approach combines bioinformatics and molecular modelling to mine sequence databases, resulting in a diverse panel of enzymes capable of catalysing the targeted reaction. The median catalytic efficiency of the computationally selected enzymes is 75-fold greater than a panel of naively selected homologues. This integrative genomic mining approach establishes a unique avenue for enzyme function discovery in the rapidly expanding sequence databases. The second approach uses computational enzyme design to reprogramme specificity. Both approaches result in enzymes with >100-fold increase in specificity for the targeted reaction. When enzymes from either approach are integrated in vivo, longer-chain alcohol production increases over 10-fold and represents >95% of the total alcohol products.

Discrepancy between experimental and theoretical excitation transfer rates in LH2 bacteriochlorophyll-protein complexes of purple bacteria.

PubMed

Borisov, A Y

2008-02-01

Discrepancy is revealed between the values of excitation transfer times measured experimentally, and those calculated, for the atomic structures of B800 --> B850 bacteriochlorophylls within the LH2 light-harvesting pigment-protein complex of the purple bacterium Rhodopseudomonas acidophila. The value 2.9-3.2 ps for the B800 --> B850 excitation transfer, calculated on the basis of atomic structure of LH2, is about 4-times longer than that measured for this bacterium (0.7 ps). This discrepancy appears common in at least two purple bacteria. Possible sources responsible for this discrepancy are discussed. It may either signify some drawback/s/ in our notions about the precise in vivo structure of LH2 complexes, for example, possible changes of LH2 structure during crystallization, or it may reflect our ignorance of some mechanisms involved in excitation migration.

Protein Design for Pathway Engineering

PubMed Central

Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

2013-01-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

Protein design for pathway engineering.

PubMed

Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

2014-02-01

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria.

PubMed

Dittmann, Elke; Gugger, Muriel; Sivonen, Kaarina; Fewer, David P

2015-10-01

Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with intricate chemical structures and potent biological activities. The bulk of these natural products are known from just a handful of genera. Recent efforts have elucidated the mechanisms underpinning the biosynthesis of a diverse array of natural products from cyanobacteria. Many of the biosynthetic mechanisms are unique to cyanobacteria or rarely described from other organisms. Advances in genome sequence technology have precipitated a deluge of genome sequences for cyanobacteria. This makes it possible to link known natural products to biosynthetic gene clusters but also accelerates the discovery of new natural products through genome mining. These studies demonstrate that cyanobacteria encode a huge variety of cryptic gene clusters for the production of natural products, and the known chemical diversity is likely to be just a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms. Copyright © 2015. Published by Elsevier Ltd.

Integrated Interactive Chart as a Tool for Teaching Metabolic Pathways

ERIC Educational Resources Information Center

Kalogiannis, Stavros; Pagkalos, Ioannis; Koufoudakis, Panagiotis; Dashi, Ino; Pontikeri, Kyriaki; Christodoulou, Constantina

2014-01-01

An interactive chart of energy metabolism with didactic function, complementary to the already existing metabolic maps, located at the URL www.metpath.teithe.gr is being presented. The chart illustrates the major catabolic and biosynthetic pathways of glucose, fatty acids, and aminoacids, individually as well as in an integrated view. For every…

Key roles of Arf small G proteins and biosynthetic trafficking for animal development.

PubMed

Rodrigues, Francisco F; Harris, Tony J C

2017-04-14

Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.

A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway1[OPEN

PubMed Central

2016-01-01

Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases. PMID:27356974

Giant Virus Megavirus chilensis Encodes the Biosynthetic Pathway for Uncommon Acetamido Sugars*

PubMed Central

Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G.

2014-01-01

Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways. PMID:25035429

Giant virus Megavirus chilensis encodes the biosynthetic pathway for uncommon acetamido sugars.

PubMed

Piacente, Francesco; De Castro, Cristina; Jeudy, Sandra; Molinaro, Antonio; Salis, Annalisa; Damonte, Gianluca; Bernardi, Cinzia; Abergel, Chantal; Tonetti, Michela G

2014-08-29

Giant viruses mimicking microbes, by the sizes of their particles and the heavily glycosylated fibrils surrounding their capsids, infect Acanthamoeba sp., which are ubiquitous unicellular eukaryotes. The glycans on fibrils are produced by virally encoded enzymes, organized in gene clusters. Like Mimivirus, Megavirus glycans are mainly composed of virally synthesized N-acetylglucosamine (GlcNAc). They also contain N-acetylrhamnosamine (RhaNAc), a rare sugar; the enzymes involved in its synthesis are encoded by a gene cluster specific to Megavirus close relatives. We combined activity assays on two enzymes of the pathway with mass spectrometry and NMR studies to characterize their specificities. Mg534 is a 4,6-dehydratase 5-epimerase; its three-dimensional structure suggests that it belongs to a third subfamily of inverting dehydratases. Mg535, next in the pathway, is a bifunctional 3-epimerase 4-reductase. The sequential activity of the two enzymes leads to the formation of UDP-l-RhaNAc. This study is another example of giant viruses performing their glycan synthesis using enzymes different from their cellular counterparts, raising again the question of the origin of these pathways. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant Escherichia coli

PubMed Central

Lacmata, Stephen Tamekou; Kuiate, Jules-Roger; Ding, Yamei; Xian, Mo; Liu, Huizhou; Boudjeko, Thaddée; Feng, Xinjun; Zhao, Guang

2017-01-01

Poly(3-hydroxypropionate) (P3HP) is a thermoplastic with great compostability and biocompatibility, and can be produced through several biosynthetic pathways, in which the glycerol pathway achieved the highest P3HP production. However, exogenous supply of vitamin B12 was required to maintain the activity of glycerol dehydratase, resulting in high production cost. To avoid the addition of VB12, we have previously constructed a P3HP biosynthetic route with β-alanine as intermediate, and the present study aimed to improve the P3HP production of this pathway. L-aspartate decarboxylase PanD was found to be the rate-limiting enzyme in the β-alanine pathway firstly. To improve the pathway efficiency, PanD was screened from four different sources (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Corynebacterium glutamicum). And PanD from C. glutamicum was found to have the highest activity, the P3HP production was improved in flask cultivation with this enzyme. To further improve the production, the host strain was screened and the culture condition was optimized. Under optimal conditions, production and content of P3HP reached to 10.2 g/L and 39.1% (wt/wt [cell dry weight]) in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without VB12. PMID:28253372

Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol-anchored proteins in biosynthetic and recycling routes in Madin-Darby canine kidney cells.

PubMed

Castillon, Guillaume A; Burriat-Couleru, Patricia; Abegg, Daniel; Criado Santos, Nina; Watanabe, Reika

2018-03-01

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal-to-apical transcytosis routes. The apical distribution of the t-SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical-destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI-APs in polarized MDCK cells. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Insights into a divergent phenazine biosynthetic pathway governed by a plasmid-born esmeraldin gene cluster.

PubMed

Rui, Zhe; Ye, Min; Wang, Shuoguo; Fujikawa, Kaori; Akerele, Bankole; Aung, May; Floss, Heinz G; Zhang, Wenjun; Yu, Tin-Wein

2012-09-21

Phenazine-type metabolites arise from either phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). Although the biosynthesis of PCA has been studied extensively, PDC assembly remains unclear. Esmeraldins and saphenamycin, the PDC originated products, are antimicrobial and antitumor metabolites isolated from Streptomyces antibioticus Tü 2706. Herein, the esmeraldin biosynthetic gene cluster was identified on a dispensable giant plasmid. Twenty-four putative esm genes were characterized by bioinformatics, mutagenesis, genetic complementation, and functional protein expressions. Unlike enzymes involved in PCA biosynthesis, EsmA1 and EsmA2 together decisively promoted the PDC yield. The resulting PDC underwent a series of conversions to give 6-acetylphenazine-1-carboxylic acid, saphenic acid, and saphenamycin through a unique one-carbon extension by EsmB1-B5, a keto reduction by EsmC, and an esterification by EsmD1-D3, the atypical polyketide sythases, respectively. Two transcriptional regulators, EsmT1 and EsmT2, are required for esmeraldin production. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243.

PubMed

Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu

2003-01-01

The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

Structure and function of polyketide biosynthetic enzymes: various strategies for production of structurally diverse polyketides.

PubMed

Miyanaga, Akimasa

2017-12-01

Polyketides constitute a large family of natural products that display various biological activities. Polyketides exhibit a high degree of structural diversity, although they are synthesized from simple acyl building blocks. Recent biochemical and structural studies provide a better understanding of the biosynthetic logic of polyketide diversity. This review highlights the biosynthetic mechanisms of structurally unique polyketides, β-amino acid-containing macrolactams, enterocin, and phenolic lipids. Functional and structural studies of macrolactam biosynthetic enzymes have revealed the unique biosynthetic machinery used for selective incorporation of a rare β-amino acid starter unit into the polyketide skeleton. Biochemical and structural studies of cyclization enzymes involved in the biosynthesis of enterocin and phenolic lipids provide mechanistic insights into how these enzymes diversify the carbon skeletons of their products.

Analysis of the Expression of Anthocyanin Pathway Genes in Developing Vitis vinifera L. cv Shiraz Grape Berries and the Implications for Pathway Regulation.

PubMed Central

Boss, P. K.; Davies, C.; Robinson, S. P.

1996-01-01

Anthocyanin synthesis in Vitis vinifera L. cv Shiraz grape berries began 10 weeks postflowering and continued throughout berry ripening. Expression of seven genes of the anthocyanin biosynthetic pathway (phenylalanine ammonia lyase [PAL], chalcone synthase [CHS], chalcone isomerase [CHI], flavanone-3-hydroxylase [F3H], dihydroflavonol 4-reductase [DFR], leucoanthocyanidin dioxygen-ase [LDOX], and UDP glucose-flavonoid 3-o-glucosyl transferase [UFGT]) was determined. In flowers and grape berry skins, expression of all of the genes, except UFGT, was detected up to 4 weeks postflowering, followed by a reduction in this expression 6 to 8 weeks postflowering. Expression of CHS, CHI, F3H, DFR, LDOX, and UFGT then increased 10 weeks postflowering, coinciding with the onset of anthocyanin synthesis. In grape berry flesh, no PAL or UFGT expression was detected at any stage of development, but CHS, CHI, F3H, DFR, and LDOX were expressed up to 4 weeks postflowering. These results indicate that the onset of anthocyanin synthesis in ripening grape berry skins coincides with a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway, suggesting the involvement of regulatory genes. UFGT is regulated independently of the other genes, suggesting that in grapes the major control point in this pathway is later than that observed in maize, petunia, and snapdragon. PMID:12226348

Promzea: a pipeline for discovery of co-regulatory motifs in maize and other plant species and its application to the anthocyanin and phlobaphene biosynthetic pathways and the Maize Development Atlas.

PubMed

Liseron-Monfils, Christophe; Lewis, Tim; Ashlock, Daniel; McNicholas, Paul D; Fauteux, François; Strömvik, Martina; Raizada, Manish N

2013-03-15

The discovery of genetic networks and cis-acting DNA motifs underlying their regulation is a major objective of transcriptome studies. The recent release of the maize genome (Zea mays L.) has facilitated in silico searches for regulatory motifs. Several algorithms exist to predict cis-acting elements, but none have been adapted for maize. A benchmark data set was used to evaluate the accuracy of three motif discovery programs: BioProspector, Weeder and MEME. Analysis showed that each motif discovery tool had limited accuracy and appeared to retrieve a distinct set of motifs. Therefore, using the benchmark, statistical filters were optimized to reduce the false discovery ratio, and then remaining motifs from all programs were combined to improve motif prediction. These principles were integrated into a user-friendly pipeline for motif discovery in maize called Promzea, available at http://www.promzea.org and on the Discovery Environment of the iPlant Collaborative website. Promzea was subsequently expanded to include rice and Arabidopsis. Within Promzea, a user enters cDNA sequences or gene IDs; corresponding upstream sequences are retrieved from the maize genome. Predicted motifs are filtered, combined and ranked. Promzea searches the chosen plant genome for genes containing each candidate motif, providing the user with the gene list and corresponding gene annotations. Promzea was validated in silico using a benchmark data set: the Promzea pipeline showed a 22% increase in nucleotide sensitivity compared to the best standalone program tool, Weeder, with equivalent nucleotide specificity. Promzea was also validated by its ability to retrieve the experimentally defined binding sites of transcription factors that regulate the maize anthocyanin and phlobaphene biosynthetic pathways. Promzea predicted additional promoter motifs, and genome-wide motif searches by Promzea identified 127 non-anthocyanin/phlobaphene genes that each contained all five predicted promoter

Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes.

PubMed

Zhong, Xingyu; Tian, Yuqing; Niu, Guoqing; Tan, Huarong

2013-07-01

A draft genome sequence of Streptomyces ansochromogenes 7100 was generated using 454 sequencing technology. In combination with local BLAST searches and gap filling techniques, a comprehensive antiSMASH-based method was adopted to assemble the secondary metabolite biosynthetic gene clusters in the draft genome of S. ansochromogenes. A total of at least 35 putative gene clusters were identified and assembled. Transcriptional analysis showed that 20 of the 35 gene clusters were expressed in either or all of the three different media tested, whereas the other 15 gene clusters were silent in all three different media. This study provides a comprehensive method to identify and assemble secondary metabolite biosynthetic gene clusters in draft genomes of Streptomyces, and will significantly promote functional studies of these secondary metabolite biosynthetic gene clusters.

Genetic and Metabolomic Dissection of the Ergothioneine and Selenoneine Biosynthetic Pathway in the Fission Yeast, S. pombe, and Construction of an Overproduction System

PubMed Central

Pluskal, Tomáš; Ueno, Masaru; Yanagida, Mitsuhiro

2014-01-01

Ergothioneine is a small, sulfur-containing metabolite (229 Da) synthesized by various species of bacteria and fungi, which can accumulate to millimolar levels in tissues or cells (e.g. erythrocytes) of higher eukaryotes. It is commonly marketed as a dietary supplement due to its proposed protective and antioxidative functions. In this study we report the genes forming the two-step ergothioneine biosynthetic pathway in the fission yeast, Schizosaccharomyces pombe. We identified the first gene, egt1+ (SPBC1604.01), by sequence homology to previously published genes from Neurospora crassa and Mycobacterium smegmatis. We showed, using metabolomic analysis, that the Δegt1 deletion mutant completely lacked ergothioneine and its precursors (trimethyl histidine/hercynine and hercynylcysteine sulfoxide). Since the second step of ergothioneine biosynthesis has not been characterized in eukaryotes, we examined four putative homologs (Nfs1/SPBC21D10.11c, SPAC11D3.10, SPCC777.03c, and SPBC660.12c) of the corresponding mycobacterial enzyme EgtE. Among deletion mutants of these genes, only one (ΔSPBC660.12c, designated Δegt2) showed a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibited no phenotypic defects during vegetative growth or quiescence. To effectively study the role of ergothioneine, we constructed an egt1+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. We employed three versions of the nmt1 promoter with increasing strength of expression and confirmed corresponding accumulations of ergothioneine. We quantified the intracellular concentration of ergothioneine in S. pombe (0.3, 157.4, 41.6, and up to 1606.3 µM in vegetative, nitrogen-starved, glucose-starved, and egt1+-overexpressing cells, respectively) and described its gradual accumulation under long-term quiescence

Microbial synthesis of propane by engineering valine pathway and aldehyde-deformylating oxygenase.

PubMed

Zhang, Lei; Liang, Yajing; Wu, Wei; Tan, Xiaoming; Lu, Xuefeng

2016-01-01

Propane, a major component of liquid petroleum gas (LPG) derived from fossil fuels, has widespread applications in vehicles, cooking, and ambient heating. Given the concerns about fossil fuel depletion and carbon emission, exploiting alternative and renewable source of propane have become attractive. In this study, we report the construction of a novel propane biosynthetic pathway in Escherichia coli. We constructed an aldehyde reductases (ALR)-deprived E. coli strain BW25113(DE3) Δ13 via genetic engineering, which produced sufficient isobutyraldehyde precursors and finally achieved de novo synthesis of propane (91 μg/L) by assembling the engineered valine pathway and cyanobacterial aldehyde-deformylating oxygenase (ADO). Additionally, after extensive screening of ADO mutants generated by engineering the active center to accommodate branched-chain isobutyraldehyde, we identified two ADO mutants (I127G, I127G/A48G) which exhibited higher catalytic activity for isobutyraldehyde and improved propane productivity by three times (267 μg/L). The propane biosynthetic pathway constructed here through the engineered valine pathway can produce abundant isobutyraldehyde for ADO and overcome the low availability of precursors in propane production. Furthermore, the rational design aiming at the ADO active center illustrates the plasticity and catalytic potential of ADO. These results together highlight the potential for developing a microbial biomanufacturing platform for propane.

A Global Coexpression Network Approach for Connecting Genes to Specialized Metabolic Pathways in Plants

PubMed Central

Borowsky, Alexander T.

2017-01-01

Plants produce diverse specialized metabolites (SMs), but the genes responsible for their production and regulation remain largely unknown, hindering efforts to tap plant pharmacopeia. Given that genes comprising SM pathways exhibit environmentally dependent coregulation, we hypothesized that genes within a SM pathway would form tight associations (modules) with each other in coexpression networks, facilitating their identification. To evaluate this hypothesis, we used 10 global coexpression data sets, each a meta-analysis of hundreds to thousands of experiments, across eight plant species to identify hundreds of coexpressed gene modules per data set. In support of our hypothesis, 15.3 to 52.6% of modules contained two or more known SM biosynthetic genes, and module genes were enriched in SM functions. Moreover, modules recovered many experimentally validated SM pathways, including all six known to form biosynthetic gene clusters (BGCs). In contrast, bioinformatically predicted BGCs (i.e., those lacking an associated metabolite) were no more coexpressed than the null distribution for neighboring genes. These results suggest that most predicted plant BGCs are not genuine SM pathways and argue that BGCs are not a hallmark of plant specialized metabolism. We submit that global gene coexpression is a rich, largely untapped resource for discovering the genetic basis and architecture of plant natural products. PMID:28408660

The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

PubMed Central

Dugrand-Judek, Audray; Olry, Alexandre; Hehn, Alain; Costantino, Gilles; Ollitrault, Patrick; Froelicher, Yann; Bourgaud, Frédéric

2015-01-01

Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in

Identification of biosynthetic intermediates of teaghrelins and teaghrelin-like compounds in oolong teas, and their molecular docking to the ghrelin receptor.

PubMed

Hsieh, Sheng-Kuo; Lo, Yuan-Hao; Wu, Chia-Chang; Chung, Tse-Yu; Tzen, Jason T C

2015-12-01

Teaghrelins are unique acylated flavonoid tetraglycosides found in Chin-shin oolong tea, and have been demonstrated to be promising oral ghrelin analogues. The biosynthetic pathway of teaghrelins from quercetin-3-O-rutinoside (rutin) or kaempferol-3-O-rutinoside (nicotiflorin) was proposed to comprise three enzymatic steps according to the identification of putative intermediates in Chin-shin oolong tea. In addition to the two known teaghrelins in Chin-shin oolong tea, four teaghrelin-like compounds with different attachments of glycosides were identified in various oolong teas. Molecular modeling and docking were used to evaluate theoretically whether the putative biosynthetic intermediates of teaghrelins and the four teaghrelin-like compounds could be potential candidates of ghrelin analogues. The results showed that the attachment of a coumaroyl group was crucial for these tea compounds to bind to the ghrelin receptor. However, the additional attachment of a rhamnosyl glycoside to the flavonoid backbone of teaghrelin-like compounds at C-7 significantly reduced their binding affinity with the ghrelin receptor. Copyright © 2015. Published by Elsevier B.V.

Genetic tool development and systemic regulation in biosynthetic technology.

PubMed

Dai, Zhongxue; Zhang, Shangjie; Yang, Qiao; Zhang, Wenming; Qian, Xiujuan; Dong, Weiliang; Jiang, Min; Xin, Fengxue

2018-01-01

With the increased development in research, innovation, and policy interest in recent years, biosynthetic technology has developed rapidly, which combines engineering, electronics, computer science, mathematics, and other disciplines based on classical genetic engineering and metabolic engineering. It gives a wider perspective and a deeper level to perceive the nature of life via cell mechanism, regulatory networks, or biological evolution. Currently, synthetic biology has made great breakthrough in energy, chemical industry, and medicine industries, particularly in the programmable genetic control at multiple levels of regulation to perform designed goals. In this review, the most advanced and comprehensive developments achieved in biosynthetic technology were represented, including genetic engineering as well as synthetic genomics. In addition, the superiority together with the limitations of the current genome-editing tools were summarized.

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

PubMed Central

Horsman, Geoff P.; Chen, Yihua; Thorson, Jon S.; Shen, Ben

2010-01-01

Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence. PMID:20534556

Structure of the Bacillus anthracis dTDP- L -rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baumgartner, Jackson; Lee, Jesi; Halavaty, Andrei S.

L-Rhamnose is a ubiquitous bacterial cell-wall component. The biosynthetic pathway for its precursor dTDP-L-rhamnose is not present in humans, which makes the enzymes of the pathway potential drug targets. In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), fromBacillus anthraciswas determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP-α-D-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs.more » However, RfbA exhibits a shorter sequence at its C-terminus, which results in the absence of three α-helices involved in allosteric site formation. Consequently, RfbA was observed to exhibit a quaternary structure that is unique among currently reported glucose-1-phosphate thymidylyltransferase bacterial homologs. These structural analyses suggest that RfbA may not be allosterically regulated in some organisms and is structurally distinct from other RmlA homologs.« less

Crosstalk of Autophagy and the Secretory Pathway and Its Role in Diseases.

PubMed

Zahoor, Muhammad; Farhan, Hesso

2018-01-01

The secretory and autophagic pathways are two fundamental, evolutionary highly conserved endomembrane processes. Typically, secretion is associated with biosynthesis and delivery of proteins. In contrast, autophagy is usually considered as a degradative pathway. Thus, an analogy to metabolic pathways is evident. Anabolic (biosynthetic) and catabolic (degradative) pathways are usually intimately linked and intertwined, and likewise, the secretory and autophagy pathways are intertwined. Investigation of this link is an emerging area of research, and we will provide an overview of some of the major advances that have been made to contribute to understanding of how secretion regulates autophagy and vice versa. Finally, we will highlight evidence that supports a potential involvement of the autophagy-secretion crosstalk in human diseases. © 2018 Elsevier Inc. All rights reserved.

Production of dehydrogingerdione derivatives in Escherichia coli by exploiting a curcuminoid synthase from Oryza sativa and a β-oxidation pathway from Saccharomyces cerevisiae.

PubMed

Katsuyama, Yohei; Ohnishi, Yasuo; Horinouchi, Sueharu

2010-09-24

Gingerol derivatives are bioactive compounds isolated from the rhizome of ginger. They possess various beneficial activities, such as anticancer and hepatoprotective activities, and are therefore attractive targets of bioengineering. However, the microbial production of gingerol derivatives has not yet been established, primarily because the biosynthetic pathway of gingerol is unknown. Here, we report the production of several dehydrogingerdione (a gingerol derivative) analogues from a recombinant Escherichia coli strain that has an "artificial" biosynthesis pathway for dehydrogingerdione that was not based on the original biosynthesis pathway of gingerol derivatives in plants. The system consists of a 4-coumarate:CoA ligase from Lithospermum erythrorhizon, a fatty acid CoA ligase from Oryza sativa, a β-oxidation system from Saccharomyces cerevisiae, and a curcuminoid synthase from O. sativa. To our knowledge, this is the first report of the microbial production of a plant metabolite the biosynthetic pathway of which has not yet been identified.

Ascorbate as a Biosynthetic Precursor in Plants

PubMed Central

Debolt, Seth; Melino, Vanessa; Ford, Christopher M.

2007-01-01

Background and Aims l-Ascorbate (vitamin C) has well-documented roles in many aspects of redox control and anti-oxidant activity in plant cells. This Botanical Briefing highlights recent developments in another aspect of l-ascorbate metabolism: its function as a precursor for specific processes in the biosynthesis of organic acids. Scope The Briefing provides a summary of recent advances in our understanding of l-ascorbate metabolism, covering biosynthesis, translocation and functional aspects. The role of l-ascorbate as a biosynthetic precursor in the formation of oxalic acid, l-threonic acid and l-tartaric acid is described, and progress in elaborating the mechanisms of the formation of these acids is reviewed. The potential conflict between the two roles of l-ascorbate in plant cells, functional and biosynthetic, is highlighted. Conclusions Recent advances in the understanding of l-ascorbate catabolism and the formation of oxalic and l-tartaric acids provide compelling evidence for a major role of l-ascorbate in plant metabolism. Combined experimental approaches, using classic biochemical and emerging ‘omics’ technologies, have provided recent insight to previously under-investigated areas. PMID:17098753

Sugars as the Optimal Biosynthetic Carbon Substrate of Aqueous Life throughout the Universe

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1999-01-01

Our previous analysis of the energetics of metabolism showed that both the biosynthesis of amino acids and lipids from sugars, and the fermentation of organic substrates, were energetically driven by electron transfer reactions resulting in carbon redox disproportionation (Weber 1997). Redox disproportionation -- the spontaneous (energetically favorable) direction of carbon group transformation in biosynthesis -- is brought about and driven by the energetically downhill transfer of electron pairs from more oxidized carbon groups (with lower half-cell reduction potentials) to more reduced carbon groups (with higher half-cell reduction potentials). In this report, we compare the redox and kinetic properties of carbon groups in order to evaluate the relative biosynthetic capability of organic substrates, and to identify the optimal biosubstrate. This analysis revealed that sugars (monocarbonyl alditols) are the optimal biosynthetic substrate because they contain the maximum number of biosynthetically useful .high energy electrons/carbon atom , while still containing a single carbonyl group needed to kinetically facilitate their conversion to useful biosynthetic intermediates. This conclusion applies to aqueous life throughout the Universe because it is based on invariant aqueous carbon chemistry -- primarily, the universal reduction potentials of carbon groups.

Sugars as the optimal biosynthetic carbon substrate of aqueous life throughout the universe

NASA Technical Reports Server (NTRS)

Weber, A. L.

2000-01-01

Our previous analysis of the energetics of metabolism showed that both the biosynthesis of amino acids and lipids from sugars, and the fermentation of organic substrates, were energetically driven by electron transfer reactions resulting in carbon redox disproportionation (Weber, 1997). Redox disproportionation--the spontaneous (energetically favorable) direction of carbon group transformation in biosynthesis--is brought about and driven by the energetically downhill transfer of electron pairs from more oxidized carbon groups (with lower half-cell reduction potentials) to more reduced carbon groups (with higher half-cell reduction potentials). In this report, we compare the redox and kinetic properties of carbon groups in order to evaluate the relative biosynthetic capability of organic substrates, and to identify the optimal biosubstrate. This analysis revealed that sugars (monocarbonyl alditols) are the optimal biosynthetic substrate because they contain the maximum number of biosynthetically useful high energy electrons/carbon atom while still containing a single carbonyl group needed to kinetically facilitate their conversion to useful biosynthetic intermediates. This conclusion applies to aqueous life throughout the Universe because it is based on invariant aqueous carbon chemistry--primarily, the universal reduction potentials of carbon groups.

Quantification of two forms of green sulfur bacteria in their natural habitat using bacteriochlorophyll fluorescence spectra

NASA Astrophysics Data System (ADS)

Kharcheva, Anastasia V.; Zhiltsova, Anna A.; Lunina, Olga N.; Savvichev, Alexander S.; Patsaeva, Svetlana V.

2016-04-01

Detection of phototropic organisms in their natural habitat using optical instruments operating under water is urgently needed for many tasks of ecological monitoring. While fluorescence methods are widely applied nowadays to detect and characterize phytoplankton communities, the techniques for detection and recognition of anoxygenic phototrophs are considered challenging. Differentiation of the forms of anoxygenic green sulfur bacteria in natural water using spectral techniques remains problematic. Green sulfur bacteria could be found in two forms, green-colored (containing BChl d in pigment compound) and brown-colored (containing BChl e), have the special ecological niche in such reservoirs. Separate determination of these microorganisms by spectral methods is complicated because of similarity of spectral characteristics of their pigments. We describe the novel technique of quantification of two forms of green sulfur bacteria directly in water using bacteriochlorophyll fluorescence without pigment extraction. This technique is noninvasive and could be applied in remote mode in the water bodies with restricted water circulation to determine simultaneously concentrations of two forms of green sulfur bacteria in their natural habitat.

Intermolecular Modes between LH2 Bacteriochlorophylls and Protein Residues: The Effect on the Excitation Energies.

PubMed

Anda, André; De Vico, Luca; Hansen, Thorsten

2017-06-08

Light-harvesting system 2 (LH2) executes the primary processes of photosynthesis in purple bacteria; photon absorption, and energy transportation to the reaction center. A detailed mechanistic insight into these operations is obscured by the complexity of the light-harvesting systems, particularly by the chromophore-environment interaction. In this work, we focus on the effects of the protein residues that are ligated to the bacteriochlorophylls (BChls) and construct potential energy surfaces of the ground and first optically excited state for the various BChl-residue systems where we in each case consider two degrees of freedom in the intermolecular region. We find that the excitation energies are only slightly affected by the considered modes. In addition, we see that axial ligands and hydrogen-bonded residues have opposite effects on both excitation energies and oscillator strengths by comparing to the isolated BChls. Our results indicate that only a small part of the chromophore-environment interaction can be associated with the intermolecular region between a BChl and an adjacent residue, but that it may be possible to selectively raise or lower the excitation energy at the axial and planar residue positions, respectively.

Biosynthetic porphyrins and the origin of photosynthesis

NASA Technical Reports Server (NTRS)

Mauzerall, D.; Ley, A.; Mercer-Smith, J. A.

1986-01-01

Since the prebiotic atmosphere was anaerobic, if not reducing, a useful function of primordial photosynthesis would have been to photooxidize reduced substrates such as Fe(+2), S(-2) or reduced organic molecules and to emit hydrogen. Experiments have shown that the early biogenic pigments uroporphyrin and coproporphyrin do photooxidize organic compounds and emit hydrogen in the presence of a platinum catalyst. These experiments were carried out in dilute aqueous solution near neutral pH under anaerobic atmosphere, and quantum yields near 10-2 were obtained. Thus relevant prebiotic conditions were maintained. Rather then to further optimize conditions, attempts were made to replace the platinum catalyst by a more prebiotically suitable catalyst. Trials with an Fe4S4(SR)4 cluster, in analogy to the present hydrogenase and nitrogenase, were not successful. However, experiments using cobalt complexes to catalyze the formation of hydrogen are promising. In analogy with biological photosynthetic systems which group pigments, electron transfer molecules and enzymes in clusters for efficiency, it was found that binding the biogenic porphyrins to the polyvinyl alcohol used to support the platinum catalyst did increase the quantum yield of the reaction. It was also found that ultraviolet light can serve to photo-oxidize porphyrinogens to porphyrins under anaerobic conditions. Thus the formation of the colorless porphyriogens by the extraordinarily simple biosynthetic pathway would not be a problem because of the prevalence of UV light in the prebiotic, anoxic atmosphere.

A Global Coexpression Network Approach for Connecting Genes to Specialized Metabolic Pathways in Plants.

PubMed

Wisecaver, Jennifer H; Borowsky, Alexander T; Tzin, Vered; Jander, Georg; Kliebenstein, Daniel J; Rokas, Antonis

2017-05-01

Plants produce diverse specialized metabolites (SMs), but the genes responsible for their production and regulation remain largely unknown, hindering efforts to tap plant pharmacopeia. Given that genes comprising SM pathways exhibit environmentally dependent coregulation, we hypothesized that genes within a SM pathway would form tight associations (modules) with each other in coexpression networks, facilitating their identification. To evaluate this hypothesis, we used 10 global coexpression data sets, each a meta-analysis of hundreds to thousands of experiments, across eight plant species to identify hundreds of coexpressed gene modules per data set. In support of our hypothesis, 15.3 to 52.6% of modules contained two or more known SM biosynthetic genes, and module genes were enriched in SM functions. Moreover, modules recovered many experimentally validated SM pathways, including all six known to form biosynthetic gene clusters (BGCs). In contrast, bioinformatically predicted BGCs (i.e., those lacking an associated metabolite) were no more coexpressed than the null distribution for neighboring genes. These results suggest that most predicted plant BGCs are not genuine SM pathways and argue that BGCs are not a hallmark of plant specialized metabolism. We submit that global gene coexpression is a rich, largely untapped resource for discovering the genetic basis and architecture of plant natural products. © 2017 American Society of Plant Biologists. All rights reserved.

Identification of the putrescine biosynthetic genes in Pseudomonas aeruginosa and characterization of agmatine deiminase and N-carbamoylputrescine amidohydrolase of the arginine decarboxylase pathway.

PubMed

Nakada, Yuji; Itoh, Yoshifumi

2003-03-01

Putrescine can be synthesized either directly from ornithine by ornithine decarboxylase (ODC; the speC product) or indirectly from arginine via arginine decarboxylase (ADC; the speA product). The authors identified the speA and speC genes in Pseudomonas aeruginosa PAO1. The activities of the two decarboxylases were similar and each enzyme alone appeared to direct sufficient formation of the polyamine for normal growth. A mutant defective in both speA and speC was a putrescine auxotroph. In this strain, agmatine deiminase (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), which were initially identified as the catabolic enzymes of agmatine, biosynthetically convert agmatine to putrescine in the ADC pathway: a double mutant of aguAB and speC was a putrescine auxotroph. AguA was purified as a homodimer of 43 kDa subunits and AguB as a homohexamer of 33 kDa subunits. AguA specifically deiminated agmatine with K(m) and K(cat) values of 0.6 mM and 4.2 s(-1), respectively. AguB was specific to N-carbamoylputrescine and the K(m) and K(cat) values of the enzyme for the substrate were 0.5 mM and 3.3 s(-1), respectively. Whereas AguA has no structural relationship to any known C-N hydrolases, AguB is a protein of the nitrilase family that performs thiol-assisted catalysis. Inhibition by SH reagents and the conserved cysteine residue in AguA and its homologues suggested that this enzyme is also involved in thiol-mediated catalysis.

Specific inhibition of antenna bacteriochlorophyll synthesis in Chlorobium vibrioforme by anesthetic gases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ormerod, J.G.; Nesbakken, T.; Beale, S.I.

1990-03-01

The green sulfur bacterium Chlorobium vibrioforme contains two types of bacteriochlorophll (Bchl). The minor pigment, Bchl a, is associated primarily with the cell membrane and its reaction centers; and the major light-harvesting antenna pigment, Bchl d, is found primarily in the chlorosomes, which are attached to the inner surface of the cell membrane. Anesthetic gases, such as N{sub 2}O, ethylene, and acetylene, were found to inhibit the synthesis of Bchl d, but not of Bchl a, thus allowing the cells to grow at high light intensities with a greatly diminished content of antenna pgiment. Chlorosomes were absent or sparse inmore » inhibited cells. Porphyrins accumulated in the inhibited cells. Result suggest that the gases act at a step in pigment biosynthesis that affect the utilization of the major one was identified as the Bchl precursor magnesium-protoporphyrin IX monomethyl ester (Mg-PPME) for isocyclic ring formation. Synthesis of Bchl d and Bchl a may be differentially affected by the gases because of compartmentation of their biosynthetic apparatus or because competition for precursors favors Bchl a synthesis. An ethephon-resistant mutant strain was isolated by selection for growth in dim, long-wavelength light. The mutant cells were also resistant to acetylene, but not to N{sub 2}O. The ability to reversibly generate viable Chlorobium cells that lack antenna pigments may be useful in photosynthesis research. The ethephon-and acetylene-resistant strain may be useful in the study of the enzymes and genes that are involved in the biosynthetic step that the gases affect.« less

BciD Is a Radical S-Adenosyl-l-methionine (SAM) Enzyme That Completes Bacteriochlorophyllide e Biosynthesis by Oxidizing a Methyl Group into a Formyl Group at C-7.

PubMed

Thweatt, Jennifer L; Ferlez, Bryan H; Golbeck, John H; Bryant, Donald A

2017-01-27

Green bacteria are chlorophotorophs that synthesize bacteriochlorophyll (BChl) c, d, or e, which assemble into supramolecular, nanotubular structures in large light-harvesting structures called chlorosomes. The biosynthetic pathways of these chlorophylls are known except for one reaction. Null mutants of bciD, which encodes a putative radical S-adenosyl-l-methionine (SAM) protein, are unable to synthesize BChl e but accumulate BChl c; however, it is unknown whether BciD is sufficient to convert BChl c (or its precursor, bacteriochlorophyllide (BChlide) c) into BChl e (or BChlide e). To determine the function of BciD, we expressed the bciD gene of Chlorobaculum limnaeum strain DSMZ 1677 T in Escherichia coli and purified the enzyme under anoxic conditions. Electron paramagnetic resonance spectroscopy of BciD indicated that it contains a single [4Fe-4S] cluster. In assays containing SAM, BChlide c or d, and sodium dithionite, BciD catalyzed the conversion of SAM into 5'-deoxyadenosine and BChlide c or d into BChlide e or f, respectively. Our analyses also identified intermediates that are proposed to be 7 1 -OH-BChlide c and d Thus, BciD is a radical SAM enzyme that converts the methyl group of BChlide c or d into the formyl group of BChlide e or f This probably occurs by a mechanism involving consecutive hydroxylation reactions of the C-7 methyl group to form a geminal diol intermediate, which spontaneously dehydrates to produce the final products, BChlide e or BChlide f The demonstration that BciD is sufficient to catalyze the conversion of BChlide c into BChlide e completes the biosynthetic pathways for all "Chlorobium chlorophylls." © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

PubMed

Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

2016-08-03

Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols

PubMed Central

Avalos, José L.; Fink, Gerald R.; Stephanopoulos, Gregory

2013-01-01

Efforts to improve the production of a compound of interest in Saccharomyces cerevisiae have mainly involved engineering or overexpression of cytoplasmic enzymes. We show that targeted expression of metabolic pathways to mitochondria can increase production levels compared with expression of the same pathways in the cytoplasm. Compartmentalisation of the Ehrlich pathway into mitochondria increased isobutanol production by 260%, whereas overexpression of the same pathway in the cytoplasm only improved yields by 10%, compared with a strain overexpressing only the first three steps of the biosynthetic pathway. Subcellular fractionation of engineered strains reveals that targeting the enzymes of the Ehrlich pathway to the mitochondria achieves higher local enzyme concentrations. Other benefits of compartmentalization may include increased availability of intermediates, removing the need to transport intermediates out of the mitochondrion, and reducing the loss of intermediates to competing pathways. PMID:23417095

Molecular cloning and heterologous expression of a biosynthetic gene cluster for the antitubercular agent D-cycloserine produced by Streptomyces lavendulae.

PubMed

Kumagai, Takanori; Koyama, Yusuke; Oda, Kosuke; Noda, Masafumi; Matoba, Yasuyuki; Sugiyama, Masanori

2010-03-01

In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding D-alanyl-D-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, L-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-L-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-L-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the L-arginine derivative, N(G)-hydroxy-L-arginine. The resulting O-ureido-L-serine is then racemized to O-ureido-D-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-D-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.

Identification of the Pr1 gene product completes the anthocyanin biosynthesis pathway of maize

USDA-ARS?s Scientific Manuscript database

In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3'H encoding gene (Zmf3'h1), and showed by segrega...

What is the evidence for the use of biologic or biosynthetic meshes in abdominal wall reconstruction?

PubMed

Köckerling, F; Alam, N N; Antoniou, S A; Daniels, I R; Famiglietti, F; Fortelny, R H; Heiss, M M; Kallinowski, F; Kyle-Leinhase, I; Mayer, F; Miserez, M; Montgomery, A; Morales-Conde, S; Muysoms, F; Narang, S K; Petter-Puchner, A; Reinpold, W; Scheuerlein, H; Smietanski, M; Stechemesser, B; Strey, C; Woeste, G; Smart, N J

2018-04-01

Although many surgeons have adopted the use of biologic and biosynthetic meshes in complex abdominal wall hernia repair, others have questioned the use of these products. Criticism is addressed in several review articles on the poor standard of studies reporting on the use of biologic meshes for different abdominal wall repairs. The aim of this consensus review is to conduct an evidence-based analysis of the efficacy of biologic and biosynthetic meshes in predefined clinical situations. A European working group, "BioMesh Study Group", composed of invited surgeons with a special interest in surgical meshes, formulated key questions, and forwarded them for processing in subgroups. In January 2016, a workshop was held in Berlin where the findings were presented, discussed, and voted on for consensus. Findings were set out in writing by the subgroups followed by consensus being reached. For the review, 114 studies and background analyses were used. The cumulative data regarding biologic mesh under contaminated conditions do not support the claim that it is better than synthetic mesh. Biologic mesh use should be avoided when bridging is needed. In inguinal hernia repair biologic and biosynthetic meshes do not have a clear advantage over the synthetic meshes. For prevention of incisional or parastomal hernias, there is no evidence to support the use of biologic/biosynthetic meshes. In complex abdominal wall hernia repairs (incarcerated hernia, parastomal hernia, infected mesh, open abdomen, enterocutaneous fistula, and component separation technique), biologic and biosynthetic meshes do not provide a superior alternative to synthetic meshes. The routine use of biologic and biosynthetic meshes cannot be recommended.

Integrating nitric oxide into salicylic acid and jasmonic acid/ ethylene plant defense pathways.

PubMed

Mur, Luis A J; Prats, Elena; Pierre, Sandra; Hall, Michael A; Hebelstrup, Kim H

2013-01-01

Plant defense against pests and pathogens is known to be conferred by either salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) pathways, depending on infection or herbivore-grazing strategy. It is well attested that SA and JA/ET pathways are mutually antagonistic allowing defense responses to be tailored to particular biotic stresses. Nitric oxide (NO) has emerged as a major signal influencing resistance mediated by both signaling pathways but no attempt has been made to integrate NO into established SA/JA/ET interactions. NO has been shown to act as an inducer or suppressor of signaling along each pathway. NO will initiate SA biosynthesis and nitrosylate key cysteines on TGA-class transcription factors to aid in the initiation of SA-dependent gene expression. Against this, S-nitrosylation of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) will promote the NPR1 oligomerization within the cytoplasm to reduce TGA activation. In JA biosynthesis, NO will initiate the expression of JA biosynthetic enzymes, presumably to over-come any antagonistic effects of SA on JA-mediated transcription. NO will also initiate the expression of ET biosynthetic genes but a suppressive role is also observed in the S-nitrosylation and inhibition of S-adenosylmethionine transferases which provides methyl groups for ET production. Based on these data a model for NO action is proposed but we have also highlighted the need to understand when and how inductive and suppressive steps are used.

Integrating nitric oxide into salicylic acid and jasmonic acid/ ethylene plant defense pathways

PubMed Central

Mur, Luis A. J.; Prats, Elena; Pierre, Sandra; Hall, Michael A.; Hebelstrup, Kim H.

2013-01-01

Plant defense against pests and pathogens is known to be conferred by either salicylic acid (SA) or jasmonic acid (JA)/ethylene (ET) pathways, depending on infection or herbivore-grazing strategy. It is well attested that SA and JA/ET pathways are mutually antagonistic allowing defense responses to be tailored to particular biotic stresses. Nitric oxide (NO) has emerged as a major signal influencing resistance mediated by both signaling pathways but no attempt has been made to integrate NO into established SA/JA/ET interactions. NO has been shown to act as an inducer or suppressor of signaling along each pathway. NO will initiate SA biosynthesis and nitrosylate key cysteines on TGA-class transcription factors to aid in the initiation of SA-dependent gene expression. Against this, S-nitrosylation of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) will promote the NPR1 oligomerization within the cytoplasm to reduce TGA activation. In JA biosynthesis, NO will initiate the expression of JA biosynthetic enzymes, presumably to over-come any antagonistic effects of SA on JA-mediated transcription. NO will also initiate the expression of ET biosynthetic genes but a suppressive role is also observed in the S-nitrosylation and inhibition of S-adenosylmethionine transferases which provides methyl groups for ET production. Based on these data a model for NO action is proposed but we have also highlighted the need to understand when and how inductive and suppressive steps are used. PMID:23818890

An antagonist treatment in combination with tracer experiments revealed isocitrate pathway dominant to oxalate biosynthesis in Rumex obtusifolius L

USDA-ARS?s Scientific Manuscript database

Oxalate accumulates in leaves of certain plants such as Rumex species (Polygonaceae). Oxalate plays important roles in defense to predator, detoxification of metallic ions, and in hydroxyl peroxide formation upon wounding/senescence. However, biosynthetic pathways of soluble oxalate are largely unkn...

Lifetimes of bacteriochlorophyll fluorescence in Rhodopseudomonas viridis and Heliobacterium chlorum at low temperatures

NASA Technical Reports Server (NTRS)

Kleinherenbrink, F. A.; Cheng, P.; Amesz, J.; Blankenship, R. E.

1993-01-01

Fluorescence lifetimes of isolated membranes of Rhodopseudomonas viridis were measured in the temperature range of 77 K to 25 K. At room temperature, the main component of the fluorescence decay of bacteriochlorophyll (BChl) b had a time constant of 50 ps. In contrast to other purple bacteria, the emission at low temperature was spectrally homogeneous and showed essentially single lifetimes of 140 ps at 77 K and 180 ps at 25 K, with the primary electron donor in the oxidized state. Taking into account the relative fluorescence yields with open and closed reaction centers, we arrive at numbers of 125 ps and 215 ps, respectively, for open reaction centers. These numbers are significantly smaller than expected on the basis of measurements of the efficiency of charge separation, perhaps suggesting that the excitation decay in the absence of reaction centers is considerably faster at low temperature than at room temperature. At least four different spectral components with different lifetimes were observed at 25 K in the emission of Heliobacterium chlorum, a short-wavelength component of about 30 ps and three longer-wavelength components of about 100 ps, 300 ps, and 900 ps. This indicates a strong heterogeneity in the emitting pigment, BChl g-808. The component with the shortest lifetime does not appear to be affected by the redox state of the reaction center and might reflect energy transfer to BChl g species which are connected to the reaction center.

Carotenoid-to-bacteriochlorophyll energy transfer through vibronic coupling in LH2 from Phaeosprillum molischianum.

PubMed

Thyrhaug, Erling; Lincoln, Craig N; Branchi, Federico; Cerullo, Giulio; Perlík, Václav; Šanda, František; Lokstein, Heiko; Hauer, Jürgen

2018-03-01

The peripheral light-harvesting antenna complex (LH2) of purple photosynthetic bacteria is an ideal testing ground for models of structure-function relationships due to its well-determined molecular structure and ultrafast energy deactivation. It has been the target for numerous studies in both theory and ultrafast spectroscopy; nevertheless, certain aspects of the convoluted relaxation network of LH2 lack a satisfactory explanation by conventional theories. For example, the initial carotenoid-to-bacteriochlorophyll energy transfer step necessary on visible light excitation was long considered to follow the Förster mechanism, even though transfer times as short as 40 femtoseconds (fs) have been observed. Such transfer times are hard to accommodate by Förster theory, as the moderate coupling strengths found in LH2 suggest much slower transfer within this framework. In this study, we investigate LH2 from Phaeospirillum (Ph.) molischianum in two types of transient absorption experiments-with narrowband pump and white-light probe resulting in 100 fs time resolution, and with degenerate broadband 10 fs pump and probe pulses. With regard to the split Q x band in this system, we show that vibronically mediated transfer explains both the ultrafast carotenoid-to-B850 transfer, and the almost complete lack of transfer to B800. These results are beyond Förster theory, which predicts an almost equal partition between the two channels.

A novel pathway for the biosynthesis of heme in Archaea: genome-based bioinformatic predictions and experimental evidence.

PubMed

Storbeck, Sonja; Rolfes, Sarah; Raux-Deery, Evelyne; Warren, Martin J; Jahn, Dieter; Layer, Gunhild

2010-12-13

Heme is an essential prosthetic group for many proteins involved in fundamental biological processes in all three domains of life. In Eukaryota and Bacteria heme is formed via a conserved and well-studied biosynthetic pathway. Surprisingly, in Archaea heme biosynthesis proceeds via an alternative route which is poorly understood. In order to formulate a working hypothesis for this novel pathway, we searched 59 completely sequenced archaeal genomes for the presence of gene clusters consisting of established heme biosynthetic genes and colocalized conserved candidate genes. Within the majority of archaeal genomes it was possible to identify such heme biosynthesis gene clusters. From this analysis we have been able to identify several novel heme biosynthesis genes that are restricted to archaea. Intriguingly, several of the encoded proteins display similarity to enzymes involved in heme d(1) biosynthesis. To initiate an experimental verification of our proposals two Methanosarcina barkeri proteins predicted to catalyze the initial steps of archaeal heme biosynthesis were recombinantly produced, purified, and their predicted enzymatic functions verified.

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway

PubMed Central

Moghe, Gaurav D; Leong, Bryan J; Hurney, Steven M; Daniel Jones, A

2017-01-01

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored. PMID:28853706

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway.

PubMed

Moghe, Gaurav D; Leong, Bryan J; Hurney, Steven M; Daniel Jones, A; Last, Robert L

2017-08-30

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored.

Cloning and heterologous expression of blasticidin S biosynthetic genes from Streptomyces griseochromogenes.

PubMed

Cone, M C; Petrich, A K; Gould, S J; Zabriskie, T M

1998-06-01

Two small chromosomal DNA fragments (2.6 and 4.8 kb) from the blasticidin S producer Streptomyces griseochromogenes were cloned in the high copy number vector pIJ702 and shown to confer increased resistance to blasticidin S upon S. lividans TK24. These fragments were used to screen a library of S. griseochromogenes DNA prepared in the cosmid shuttle vector pOJ446. Cosmids containing DNA inserts of at least 23 kb were identified which hybridized to one or the other resistance fragment, but not to both. Transformation of S. lividans TK24 with several cosmids hybridizing with the 4.8 kb resistance fragment resulted in clones that produced cytosylglucuronic acid, the first intermediate of the blasticidin S biosynthetic pathway, and other blasticidin-related metabolites. A strain of S. lividans TK24 harboring both the 4.8 kb-hybridizing cosmid and the 2.6 kb resistance fragment cloned in pIJ702 produced 12.5 times as much demethylblasticidin S as the transformant harboring the cosmid alone.

Engineering dynamic pathway regulation using stress-response promoters.

PubMed

Dahl, Robert H; Zhang, Fuzhong; Alonso-Gutierrez, Jorge; Baidoo, Edward; Batth, Tanveer S; Redding-Johanson, Alyssa M; Petzold, Christopher J; Mukhopadhyay, Aindrila; Lee, Taek Soon; Adams, Paul D; Keasling, Jay D

2013-11-01

Heterologous pathways used in metabolic engineering may produce intermediates toxic to the cell. Dynamic control of pathway enzymes could prevent the accumulation of these metabolites, but such a strategy requires sensors, which are largely unknown, that can detect and respond to the metabolite. Here we applied whole-genome transcript arrays to identify promoters that respond to the accumulation of toxic intermediates, and then used these promoters to control accumulation of the intermediate and improve the final titers of a desired product. We apply this approach to regulate farnesyl pyrophosphate (FPP) production in the isoprenoid biosynthetic pathway in Escherichia coli. This strategy improved production of amorphadiene, the final product, by twofold over that from inducible or constitutive promoters, eliminated the need for expensive inducers, reduced acetate accumulation and improved growth. We extended this approach to another toxic intermediate to demonstrate the broad utility of identifying novel sensor-regulator systems for dynamic regulation.

Bacterial natural product biosynthetic domain composition in soil correlates with changes in latitude on a continent-wide scale.

PubMed

Lemetre, Christophe; Maniko, Jeffrey; Charlop-Powers, Zachary; Sparrow, Ben; Lowe, Andrew J; Brady, Sean F

2017-10-31

Although bacterial bioactive metabolites have been one of the most prolific sources of lead structures for the development of small-molecule therapeutics, very little is known about the environmental factors associated with changes in secondary metabolism across natural environments. Large-scale sequencing of environmental microbiomes has the potential to shed light on the richness of bacterial biosynthetic diversity hidden in the environment, how it varies from one environment to the next, and what environmental factors correlate with changes in biosynthetic diversity. In this study, the sequencing of PCR amplicons generated using primers targeting either ketosynthase domains from polyketide biosynthesis or adenylation domains from nonribosomal peptide biosynthesis was used to assess biosynthetic domain composition and richness in soils collected across the Australian continent. Using environmental variables collected at each soil site, we looked for environmental factors that correlated with either high overall domain richness or changes in the domain composition. Among the environmental variables we measured, changes in biosynthetic domain composition correlate most closely with changes in latitude and to a lesser extent changes in pH. Although it is unclear at this time the exact mix of factors that may drive the relationship between biosynthetic domain composition and latitude, from a practical perspective the identification of a latitudinal basis for differences in soil metagenome biosynthetic domain compositions should help guide future natural product discovery efforts. Published under the PNAS license.

Dynamics of energy transfer from lycopene to bacteriochlorophyll in genetically-modified LH2 complexes of Rhodobacter sphaeroides.

PubMed

Hörvin Billsten, H; Herek, J L; Garcia-Asua, G; Hashøj, L; Polívka, T; Hunter, C N; Sundström, V

2002-03-26

LH2 complexes from Rb. sphaeroides were modified genetically so that lycopene, with 11 saturated double bonds, replaced the native carotenoids which contain 10 saturated double bonds. Tuning the S1 level of the carotenoid in LH2 in this way affected the dynamics of energy transfer within LH2, which were investigated using both steady-state and time-resolved techniques. The S1 energy of lycopene in n-hexane was determined to be approximately 12 500 +/- 150 cm(-1), by direct measurement of the S1-S2 transient absorption spectrum using a femtosecond IR-probing technique, thus placing an upper limit on the S1 energy of lycopene in the LH2 complex. Fluorescence emission and excitation spectra demonstrated that energy can be transferred from lycopene to the bacteriochlorophyll molecules within this LH2 complex. The energy-transfer dynamics within the mutant complex were compared to wild-type LH2 from Rb. sphaeroides containing the carotenoid spheroidene and from Rs. molischianum, in which lycopene is the native carotenoid. The results show that the overall efficiency for Crt --> B850 energy transfer is approximately 80% in lyco-LH2 and approximately 95% in WT-LH2 of Rb. sphaeroides. The difference in overall Crt --> BChl transfer efficiency of lyco-LH2 and WT-LH2 mainly relates to the low efficiency of the Crt S(1) --> BChl pathway for complexes containing lycopene, which was 20% in lyco-LH2. These results show that in an LH2 complex where the Crt S1 energy is sufficiently high to provide efficient spectral overlap with both B800 and B850 Q(y) states, energy transfer via the Crt S1 state occurs to both pigments. However, the introduction of lycopene into the Rb. sphaeroides LH2 complex lowers the S1 level of the carotenoid sufficiently to prevent efficient transfer of energy to the B800 Q(y) state, leaving only the Crt S1 --> B850 channel, strongly suggesting that Crt S1 --> BChl energy transfer is controlled by the relative Crt S1 and BChl Q(y) energies.

Salicylic acid induces vanillin synthesis through the phospholipid signaling pathway in Capsicum chinense cell cultures

PubMed Central

Rodas-Junco, Beatriz A; Cab-Guillen, Yahaira; Muñoz-Sanchez, J Armando; Vázquez-Flota, Felipe; Monforte-Gonzalez, Miriam; Hérnandez-Sotomayor, S M Teresa

2013-01-01

Signal transduction via phospholipids is mediated by phospholipases such as phospholipase C (PLC) and D (PLD), which catalyze hydrolysis of plasma membrane structural phospholipids. Phospholipid signaling is also involved in plant responses to phytohormones such as salicylic acid (SA). The relationships between phospholipid signaling, SA, and secondary metabolism are not fully understood. Using a Capsicum chinense cell suspension as a model, we evaluated whether phospholipid signaling modulates SA-induced vanillin production through the activation of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthetic pathway. Salicylic acid was found to elicit PAL activity and consequently vanillin production, which was diminished or reversed upon exposure to the phosphoinositide-phospholipase C (PI-PLC) signaling inhibitors neomycin and U73122. Exposure to the phosphatidic acid inhibitor 1-butanol altered PLD activity and prevented SA-induced vanillin production. Our results suggest that PLC and PLD-generated secondary messengers may be modulating SA-induced vanillin production through the activation of key biosynthetic pathway enzymes.

Evidence for a Saponin Biosynthesis Pathway in the Body Wall of the Commercially Significant Sea Cucumber Holothuria scabra.

PubMed

Mitu, Shahida Akter; Bose, Utpal; Suwansa-Ard, Saowaros; Turner, Luke H; Zhao, Min; Elizur, Abigail; Ogbourne, Steven M; Shaw, Paul Nicholas; Cummins, Scott F

2017-11-07

The sea cucumber (phylum Echinodermata) body wall is the first line of defense and is well known for its production of secondary metabolites; including vitamins and triterpenoid glycoside saponins that have important ecological functions and potential benefits to human health. The genes involved in the various biosynthetic pathways are unknown. To gain insight into these pathways in an echinoderm, we performed a comparative transcriptome analysis and functional annotation of the body wall and the radial nerve of the sea cucumber Holothuria scabra ; to define genes associated with body wall metabolic functioning and secondary metabolite biosynthesis. We show that genes related to signal transduction mechanisms were more highly represented in the H. scabra body wall, including genes encoding enzymes involved in energy production. Eight of the core triterpenoid biosynthesis enzymes were found, however, the identity of the saponin specific biosynthetic pathway enzymes remains unknown. We confirm the body wall release of at least three different triterpenoid saponins using solid phase extraction followed by ultra-high-pressure liquid chromatography-quadrupole time of flight-mass spectrometry. The resource we have established will help to guide future research to explore secondary metabolite biosynthesis in the sea cucumber.

Extending shikimate pathway for the production of muconic acid and its precursor salicylic acid in Escherichia coli.

PubMed

Lin, Yuheng; Sun, Xinxiao; Yuan, Qipeng; Yan, Yajun

2014-05-01

cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered strain is able to produce 1.2g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of up to 1.5g/L MA within 48h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novo biosynthesis of valuable degradation metabolites. Copyright © 2014. Published by Elsevier Inc.

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway

PubMed Central

Hsiao, Yu-Yun; Tsai, Wen-Chieh; Kuoh, Chang-Sheng; Huang, Tian-Hsiang; Wang, Hei-Chia; Wu, Tian-Shung; Leu, Yann-Lii; Chen, Wen-Huei; Chen, Hong-Hwa

2006-01-01

Background Floral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis. Results The main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives. Conclusion This systems

Comparison of transcripts in Phalaenopsis bellina and Phalaenopsis equestris (Orchidaceae) flowers to deduce monoterpene biosynthesis pathway.

PubMed

Hsiao, Yu-Yun; Tsai, Wen-Chieh; Kuoh, Chang-Sheng; Huang, Tian-Hsiang; Wang, Hei-Chia; Wu, Tian-Shung; Leu, Yann-Lii; Chen, Wen-Huei; Chen, Hong-Hwa

2006-07-13

Floral scent is one of the important strategies for ensuring fertilization and for determining seed or fruit set. Research on plant scents has hampered mainly by the invisibility of this character, its dynamic nature, and complex mixtures of components that are present in very small quantities. Most progress in scent research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. Although volatile components have been identified in several orchid species, the biosynthetic pathways of orchid flower fragrance are far from understood. We investigated how flower fragrance was generated in certain Phalaenopsis orchids by determining the chemical components of the floral scent, identifying floral expressed-sequence-tags (ESTs), and deducing the pathways of floral scent biosynthesis in Phalaneopsis bellina by bioinformatics analysis. The main chemical components in the P. bellina flower were shown by gas chromatography-mass spectrometry to be monoterpenoids, benzenoids and phenylpropanoids. The set of floral scent producing enzymes in the biosynthetic pathway from glyceraldehyde-3-phosphate (G3P) to geraniol and linalool were recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina were distinguished by comparing the scent floral dbEST to that of a scentless species, P. equestris, and included those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase. In addition, EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, were detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpointed 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives. This systems biology program combined

A Catharanthus roseus BPF-1 homologue interacts with an elicitor-responsive region of the secondary metabolite biosynthetic gene Str and is induced by elicitor via a JA-independent signal transduction pathway.

PubMed

van der Fits, L; Zhang, H; Menke, F L; Deneka, M; Memelink, J

2000-11-01

Plants respond to pathogen attack by induction of various defence responses, including the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the elicitor-induced expression of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str) is mediated via the plant stress hormonejasmonate. In the promoters of several defence-related genes, cis-acting elements have been identified that are important for transcriptional regulation upon stress signals. Here we show that an upstream region in the Str promoter confers responsiveness to partially purified yeast elicitor and jasmonate. Yeast one-hybrid screening with this element as a bait identified a MYB-like protein, which shows high homology to parsley box P-binding factor-1 (PcBPF-1). In vitro analyses showed that the Str promoter fragment contained a novel binding site for BPF-1-like proteins with higher binding affinity than the previously described box P. CrBPF-1 mRNA accumulated rapidly in elicitor-treated C. roseus suspension cells, whereas no induction was observed with jasmonate. Inhibitor studies indicated that CrBPF-1 plays a role in an elicitor-responsive but jasmonate-independent signal transduction pathway, acting downstream of protein phosphorylation and calcium influx.

Biosynthetic Polymers as Functional Materials

PubMed Central

2016-01-01

The synthesis of functional polymers encoded with biomolecules has been an extensive area of research for decades. As such, a diverse toolbox of polymerization techniques and bioconjugation methods has been developed. The greatest impact of this work has been in biomedicine and biotechnology, where fully synthetic and naturally derived biomolecules are used cooperatively. Despite significant improvements in biocompatible and functionally diverse polymers, our success in the field is constrained by recognized limitations in polymer architecture control, structural dynamics, and biostabilization. This Perspective discusses the current status of functional biosynthetic polymers and highlights innovative strategies reported within the past five years that have made great strides in overcoming the aforementioned barriers. PMID:27375299

In Planta Variation of Volatile Biosynthesis: An Alternative Biosynthetic Route to the Formation of the Pathogen-Induced Volatile Homoterpene DMNT via Triterpene Degradation in Arabidopsis Roots

PubMed Central

Sohrabi, Reza; Huh, Jung-Hyun; Badieyan, Somayesadat; Rakotondraibe, Liva Harinantenaina; Kliebenstein, Daniel J.; Sobrado, Pablo; Tholl, Dorothea

2015-01-01

Plant-derived volatile compounds such as terpenes exhibit substantial structural variation and serve multiple ecological functions. Despite their structural diversity, volatile terpenes are generally produced from a small number of core 5- to 20-carbon intermediates. Here, we present unexpected plasticity in volatile terpene biosynthesis by showing that irregular homo/norterpenes can arise from different biosynthetic routes in a tissue specific manner. While Arabidopsis thaliana and other angiosperms are known to produce the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) or its C16-analog (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene by the breakdown of sesquiterpene and diterpene tertiary alcohols in aboveground tissues, we demonstrate that Arabidopsis roots biosynthesize DMNT by the degradation of the C30 triterpene diol, arabidiol. The reaction is catalyzed by the Brassicaceae-specific cytochrome P450 monooxygenase CYP705A1 and is transiently induced in a jasmonate-dependent manner by infection with the root-rot pathogen Pythium irregulare. CYP705A1 clusters with the arabidiol synthase gene ABDS, and both genes are coexpressed constitutively in the root stele and meristematic tissue. We further provide in vitro and in vivo evidence for the role of the DMNT biosynthetic pathway in resistance against P. irregulare. Our results show biosynthetic plasticity in DMNT biosynthesis in land plants via the assembly of triterpene gene clusters and present biochemical and genetic evidence for volatile compound formation via triterpene degradation in plants. PMID:25724638

A trehalose biosynthetic enzyme doubles as an osmotic stress sensor to regulate bacterial morphogenesis.

PubMed

Chen, Ximing; An, Lizhe; Fan, Xiaochuan; Ju, Furong; Zhang, Binglin; Sun, Haili; Xiao, Jianxi; Hu, Wei; Qu, Tao; Guan, Liping; Tang, Shukun; Chen, Tuo; Liu, Guangxiu; Dyson, Paul

2017-10-01

The dissacharide trehalose is an important intracellular osmoprotectant and the OtsA/B pathway is the principal pathway for trehalose biosynthesis in a wide range of bacterial species. Scaffolding proteins and other cytoskeletal elements play an essential role in morphogenetic processes in bacteria. Here we describe how OtsA, in addition to its role in trehalose biosynthesis, functions as an osmotic stress sensor to regulate cell morphology in Arthrobacter strain A3. In response to osmotic stress, this and other Arthrobacter species undergo a transition from bacillary to myceloid growth. An otsA null mutant exhibits constitutive myceloid growth. Osmotic stress leads to a depletion of trehalose-6-phosphate, the product of the OtsA enzyme, and experimental depletion of this metabolite also leads to constitutive myceloid growth independent of OtsA function. In vitro analyses indicate that OtsA can self-assemble into protein networks, promoted by trehalose-6-phosphate, a property that is not shared by the equivalent enzyme from E. coli, despite the latter's enzymatic activity when expressed in Arthrobacter. This, and the localization of the protein in non-stressed cells at the mid-cell and poles, indicates that OtsA from Arthrobacter likely functions as a cytoskeletal element regulating cell morphology. Recruiting a biosynthetic enzyme for this morphogenetic function represents an intriguing adaptation in bacteria that can survive in extreme environments.

Different sensitivities to oxygen between two strains of the photosynthetic green sulfur bacterium Chlorobium vibrioforme NCIB 8327 with bacteriochlorophyll c and d.

PubMed

Harada, Jiro; Saga, Yoshitaka; Oh-oka, Hirozo; Tamiaki, Hitoshi

2005-11-01

Two sub-strains of the anoxygenic photosynthetic green sulfur bacterium Chlorobium vibrioforme NCIB 8327 were derived from the same clone and could be discriminated only by their possession of either bacteriochlorophyll (BChl) c or d as the major pigment in the peripheral light-harvesting antenna system, chlorosome (Saga Y et al. (2003) Anal Sci 19: 1575-1579). In the presence of a proper amount of oxygen in the initial culture medium, the BChl d strain showed longer retardation on its growth initiation than the BChl c strain, indicating that the latter was advantageous for survival under aerobic light conditions which produced reactive oxygen species in vivo. The result would be ascribable to the difference of the midpoint potentials between two kinds of chlorosomes formed by self-aggregates of BChl c and d as measured by their fluorescence quenching.

Structural and Kinetic Characterization of the LPS Biosynthetic Enzyme D-alpha,beta-D-heptose-1,7-bisphosphate Phosphatase (GmhB) from Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, P.; Sugiman-Marangos, S; Zhang, K

2010-01-01

Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-{alpha},{beta}-D-Heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis.more » This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting Gram-negative bacterial infection.« less

TTG2 controls the developmental regulation of seed coat tannins in Arabidopsis by regulating vacuolar transport steps in the proanthocyanidin pathway.

PubMed

Gonzalez, Antonio; Brown, Matthew; Hatlestad, Greg; Akhavan, Neda; Smith, Tyler; Hembd, Austin; Moore, Joshua; Montes, David; Mosley, Trenell; Resendez, Juan; Nguyen, Huy; Wilson, Lyndsey; Campbell, Annabelle; Sudarshan, Duncan; Lloyd, Alan

2016-11-01

The brown color of Arabidopsis seeds is caused by the deposition of proanthocyanidins (PAs or condensed tannins) in their inner testa layer. A transcription factor complex consisting of TT2, TT8 and TTG1 controls expression of PA biosynthetic genes, just as similar TTG1-dependent complexes have been shown to control flavonoid pigment pathway gene expression in general. However, PA synthesis is controlled by at least one other gene. TTG2 mutants lack the pigmentation found in wild-type seeds, but produce other flavonoid compounds, such as anthocyanins in the shoot, suggesting that TTG2 regulates genes in the PA biosynthetic branch of the flavonoid pathway. We analyzed the expression of PA biosynthetic genes within the developing seeds of ttg2-1 and wild-type plants for potential TTG2 regulatory targets. We found that expression of TT12, encoding a MATE type transporter, is dependent on TTG2 and that TTG2 can bind to the upstream regulatory region of TT12 suggesting that TTG2 directly regulates TT12. Ectopic expression of TT12 in ttg2-1 plants partially restores seed coat pigmentation. Moreover, we show that TTG2 regulation of TT12 is dependent on TTG1 and that TTG1 and TTG2 physically interact. The observation that TTG1 interacts with TTG2, a WRKY type transcription factor, proposes the existence of a novel TTG1-containing complex, and an addendum to the existing paradigm of flavonoid pathway regulation. Copyright © 2016 Elsevier Inc. All rights reserved.

Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria.

PubMed

Ross, Avena C; Xu, Ying; Lu, Liang; Kersten, Roland D; Shao, Zongze; Al-Suwailem, Abdulaziz M; Dorrestein, Pieter C; Qian, Pei-Yuan; Moore, Bradley S

2013-01-23

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus.

Biosynthetic Multitasking Facilitates Thalassospiramide Structural Diversity in Marine Bacteria

PubMed Central

Ross, Avena C.; Xu, Ying; Lu, Liang; Kersten, Roland D.; Shao, Zongze; Al-Suwailem, Abdulaziz M.; Dorrestein, Pieter C.; Qian, Pei-Yuan; Moore, Bradley S.

2013-01-01

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multi-module skipping and iteration. Preliminary biochemical analysis of the N-terminal NRPS module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N-terminus. PMID:23270364

Ultrafast Energy Flow and Equilibration Dynamics in Photosynthetic Light-Harvesting Complexes

NASA Astrophysics Data System (ADS)

Maiuri, Margherita; Lüer, Larry; Henry, Sarah; Carey, Anne-Marie; Cogdell, Richard J.; Cerullo, Giulio; Polli, Dario

We disentangle various energy transfer pathways in the bacterio-chlorophyll excitation cascade from LH2 to LH1 in Chromatium vinosum grown under high-light or low-light illumination using tunable narrowband selective excitation and broadband infrared probing.

Central Role of the Trehalose Biosynthesis Pathway in the Pathogenesis of Human Fungal Infections: Opportunities and Challenges for Therapeutic Development.

PubMed

Thammahong, Arsa; Puttikamonkul, Srisombat; Perfect, John R; Brennan, Richard G; Cramer, Robert A

2017-06-01

Invasive fungal infections cause significant morbidity and mortality in part due to a limited antifungal drug arsenal. One therapeutic challenge faced by clinicians is the significant host toxicity associated with antifungal drugs. Another challenge is the fungistatic mechanism of action of some drugs. Consequently, the identification of fungus-specific drug targets essential for fitness in vivo remains a significant goal of medical mycology research. The trehalose biosynthetic pathway is found in a wide variety of organisms, including human-pathogenic fungi, but not in humans. Genes encoding proteins involved in trehalose biosynthesis are mechanistically linked to the metabolism, cell wall homeostasis, stress responses, and virulence of Candida albicans , Cryptococcus neoformans , and Aspergillus fumigatus . While there are a number of pathways for trehalose production across the tree of life, the TPS/TPP (trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase) pathway is the canonical pathway found in human-pathogenic fungi. Importantly, data suggest that proteins involved in trehalose biosynthesis play other critical roles in fungal metabolism and in vivo fitness that remain to be fully elucidated. By further defining the biology and functions of trehalose and its biosynthetic pathway components in pathogenic fungi, an opportunity exists to leverage this pathway as a potent antifungal drug target. The goal of this review is to cover the known roles of this important molecule and its associated biosynthesis-encoding genes in the human-pathogenic fungi studied to date and to employ these data to critically assess the opportunities and challenges facing development of this pathway as a therapeutic target. Copyright © 2017 American Society for Microbiology.

Central Role of the Trehalose Biosynthesis Pathway in the Pathogenesis of Human Fungal Infections: Opportunities and Challenges for Therapeutic Development

PubMed Central

Thammahong, Arsa; Puttikamonkul, Srisombat; Perfect, John R.; Brennan, Richard G.

2017-01-01

SUMMARY Invasive fungal infections cause significant morbidity and mortality in part due to a limited antifungal drug arsenal. One therapeutic challenge faced by clinicians is the significant host toxicity associated with antifungal drugs. Another challenge is the fungistatic mechanism of action of some drugs. Consequently, the identification of fungus-specific drug targets essential for fitness in vivo remains a significant goal of medical mycology research. The trehalose biosynthetic pathway is found in a wide variety of organisms, including human-pathogenic fungi, but not in humans. Genes encoding proteins involved in trehalose biosynthesis are mechanistically linked to the metabolism, cell wall homeostasis, stress responses, and virulence of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. While there are a number of pathways for trehalose production across the tree of life, the TPS/TPP (trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase) pathway is the canonical pathway found in human-pathogenic fungi. Importantly, data suggest that proteins involved in trehalose biosynthesis play other critical roles in fungal metabolism and in vivo fitness that remain to be fully elucidated. By further defining the biology and functions of trehalose and its biosynthetic pathway components in pathogenic fungi, an opportunity exists to leverage this pathway as a potent antifungal drug target. The goal of this review is to cover the known roles of this important molecule and its associated biosynthesis-encoding genes in the human-pathogenic fungi studied to date and to employ these data to critically assess the opportunities and challenges facing development of this pathway as a therapeutic target. PMID:28298477

Recent advances in the metabolic engineering of lignan biosynthesis pathways for the production of transgenic plant-based foods and supplements.

PubMed

Satake, Honoo; Ono, Eiichiro; Murata, Jun

2013-12-04

Plant physiological, epidemiological, and food science studies have shed light on lignans as healthy diets for the reduction of the risk of lifestyle-related noncommunicable diseases and, thus, the demand for lignans has been rapidly increasing. However, the low efficiency and instability of lignan production via extraction from plant resources remain to be resolved, indicating the requirement for the development of new procedures for lignan production. The metabolic engineering of lignan-biosynthesizing plants is expected to be most promising for efficient, sustainable, and stable lignan production. This is supported by the recent verification of biosynthetic pathways of major dietary lignans and the exploration of lignan production via metabolic engineering using transiently gene-transfected or transgenic plants. The aim of this review is to present an overview of the biosynthetic pathways, biological activities, and metabolic engineering of lignans and also perspectives in metabolic engineering-based lignan production using transgenic plants for practical application.

A Novel Pathway for the Biosynthesis of Heme in Archaea: Genome-Based Bioinformatic Predictions and Experimental Evidence

PubMed Central

Storbeck, Sonja; Rolfes, Sarah; Raux-Deery, Evelyne; Warren, Martin J.; Jahn, Dieter; Layer, Gunhild

2010-01-01

Heme is an essential prosthetic group for many proteins involved in fundamental biological processes in all three domains of life. In Eukaryota and Bacteria heme is formed via a conserved and well-studied biosynthetic pathway. Surprisingly, in Archaea heme biosynthesis proceeds via an alternative route which is poorly understood. In order to formulate a working hypothesis for this novel pathway, we searched 59 completely sequenced archaeal genomes for the presence of gene clusters consisting of established heme biosynthetic genes and colocalized conserved candidate genes. Within the majority of archaeal genomes it was possible to identify such heme biosynthesis gene clusters. From this analysis we have been able to identify several novel heme biosynthesis genes that are restricted to archaea. Intriguingly, several of the encoded proteins display similarity to enzymes involved in heme d 1 biosynthesis. To initiate an experimental verification of our proposals two Methanosarcina barkeri proteins predicted to catalyze the initial steps of archaeal heme biosynthesis were recombinantly produced, purified, and their predicted enzymatic functions verified. PMID:21197080

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

PubMed

Unal, Meral; Palavan-Unsal, Narcin; Tufekci, M A

2008-03-01

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

An Integrated workflow for phenazine biosynthetic gene cluster discovery and characterization

USDA-ARS?s Scientific Manuscript database

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limi...

Tailoring the Oxidative Stress Tolerance of Clostridium tyrobutyricum CCTCC W428 by Introducing Trehalose Biosynthetic Capability.

PubMed

Wu, Qian; Zhu, Liying; Xu, Qing; Huang, He; Jiang, Ling; Yang, Shang-Tian

2017-10-11

Fermentations employing anaerobes always suffer from the restriction of stringent anaerobic conditions during the production of bulk and fine chemicals. This work aims to improve the oxidative stress tolerance of C. tyrobutyricum CCTCC W428, an ideal butyric-acid-producing anaerobe, via the introduction of trehalose biosynthesis capability. Compared with the wild type, the engineered strain showed a wider substrate spectrum, an improved metabolic profile, and a significantly increased specific growth rate upon aeration and acid challenge. Molecular simulation experiments indicated that CoA transferase maintained its native folded state when protected by the trehalose system. Furthermore, qRT-PCR was combined assays for acid-related enzyme activities under various conditions to verify the effects of trehalose. These results demonstrate that introducing a trehalose biosynthetic pathway, which is redundant for the metabolism of C. tyrobutyricum, can increase the robustness of the host to achieve a better oxidative resistance.

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator

PubMed Central

Chang, Xiu-bao; Mengos, April; Hou, Yue-xian; Cui, Liying; Jensen, Timothy J.; Aleksandrov, Andrei; Riordan, John R.; Gentzsch, Martina

2009-01-01

Summary The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway. PMID:18682497

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

PubMed

Chang, Xiu-Bao; Mengos, April; Hou, Yue-Xian; Cui, Liying; Jensen, Timothy J; Aleksandrov, Andrei; Riordan, John R; Gentzsch, Martina

2008-09-01

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and DeltaF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated DeltaF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges.

PubMed

Wei, Xu; Chen, Chunxian; Yu, Qibin; Gady, Antoine; Yu, Yuan; Liang, Guolu; Gmitter, Frederick G

2014-10-01

Carotenoid accumulation and biosynthetic gene expression levels during fruit maturation were compared between ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV). The two cultivars exhibited different carotenoid profiles and regulatory mechanisms in flavedo and juice sacs, respectively. In flavedo, there was uncoordinated carotenoid accumulation and gene expression in RRV during green stages, which might be related to the expression of certain gene(s) in the MEP (methylerythritol phosphate) pathway. The carotenoid biosynthesis pathway shifting from α,β-xanthophylls to β,β-xanthophylls synthesis occurred in RRV earlier than VAL during orange stages. In juice sacs, the low carotenoid content in both cultivars coincided with low expression of LCYE-Contig03 and LCYE-Contig24 during green stages, suggesting LCYE might be a limiting step for carotenoid accumulation. VAL mainly accumulated violaxanthin, but RRV accumulated β-cryptoxanthin and violaxanthin during orange stages, which corresponded to differences in juice color. Several upstream genes (PDS-Contig17, LCYB-Contig19, and ZDS members) and a downstream gene (ZEP) were expressed at higher levels in RRV than VAL, which might be responsible for greater accumulation of β-cryptoxanthin and violaxanthin in RRV, respectively. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Distribution of secondary metabolite biosynthetic gene clusters in 343 Fusarium genomes

USDA-ARS?s Scientific Manuscript database

Fusarium consists of over 200 phylogenetically distinct species, many of which cause important crop diseases and/or produce mycotoxins and other secondary metabolites (SMs). Some fusaria also cause opportunistic infections in humans and other animals. To investigate the distribution of biosynthetic ...

Induction of DREB2A pathway with repression of E2F, jasmonic acid biosynthetic and photosynthesis pathways in cold acclimation-specific freeze-resistant wheat crown.

PubMed

Karki, Amrit; Horvath, David P; Sutton, Fedora

2013-03-01

Winter wheat lines can achieve cold acclimation (development of tolerance to freezing temperatures) and vernalization (delay in transition from vegetative to reproductive phase) in response to low non-freezing temperatures. To describe cold-acclimation-specific processes and pathways, we utilized cold acclimation transcriptomic data from two lines varying in freeze survival but not vernalization. These lines, designated freeze-resistant (FR) and freeze-susceptible (FS), were the source of crown tissue RNA. Well-annotated differentially expressed genes (p ≤ 0.005 and fold change ≥ 2 in response to 4 weeks cold acclimation) were used for gene ontology and pathway analysis. "Abiotic stimuli" was identified as the most enriched and unique for FR. Unique to FS was "cytoplasmic components." Pathway analysis revealed the "triacylglycerol degradation" pathway as significantly downregulated and common to both FR and FS. The most enriched of FR pathways was "neighbors of DREB2A," with the highest positive median fold change. The "13-LOX and 13-HPL" and the "E2F" pathways were enriched in FR only with a negative median fold change. The "jasmonic acid biosynthesis" pathway and four "photosynthetic-associated" pathways were enriched in both FR and FS but with a more negative median fold change in FR than in FS. A pathway unique to FS was "binding partners of LHCA1," which was enriched only in FS with a significant negative median fold change. We propose that the DREB2A, E2F, jasmonic acid biosynthesis, and photosynthetic pathways are critical for discrimination between cold-acclimated lines varying in freeze survival.

A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, J.; Xu, C.; Andre, C.

2011-06-23

Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to producemore » TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.« less

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Aram; George, Kevin W.; Wang, George

Branched C 5 alcohols are promising biofuels with excellent combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C 5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C 5 alcohols and initial precursor to longermore » chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C 5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.« less

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

DOE PAGES

Kang, Aram; George, Kevin W.; Wang, George; ...

2015-12-17

Branched C 5 alcohols are promising biofuels with excellent combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C 5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C 5 alcohols and initial precursor to longermore » chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C 5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.« less

Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway

PubMed Central

Ruiz-Sola, M. Águila; Rodríguez-Concepción, Manuel

2012-01-01

Plant carotenoids are a family of pigments that participate in light harvesting and are essential for photoprotection against excess light. Furthermore, they act as precursors for the production of apocarotenoid hormones such as abscisic acid and strigolactones. In this review, we summarize the current knowledge on the genes and enzymes of the carotenoid biosynthetic pathway (which is now almost completely elucidated) and on the regulation of carotenoid biosynthesis at both transcriptional and post-transcriptional levels. We also discuss the relevance of Arabidopsis as a model system for the study of carotenogenesis and how metabolic engineering approaches in this plant have taught important lessons for carotenoid biotechnology. PMID:22582030

Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways.

PubMed

Caveney, Nathanael A; Li, Franco Kk; Strynadka, Natalie Cj

2018-06-06

The bacterial cell wall is a complex polymeric structure with essential roles in defence, survival and pathogenesis. Common to both Gram-positive and Gram-negative bacteria is the mesh-like peptidoglycan sacculus that surrounds the outer leaflet of the cytoplasmic membrane. Recent crystallographic studies of enzymes that comprise the peptidoglycan biosynthetic pathway have led to significant new understanding of all stages. These include initial multi-step cytosolic formation of sugar-pentapeptide precursors, transfer of the precursors to activated polyprenyl lipids at the membrane inner leaflet and flippase mediated relocalization of the resulting lipid II precursors to the outer leaflet where glycopolymerization and subsequent peptide crosslinking are finalized. Additional, species-specific enzymes allow customized peptidoglycan modifications and biosynthetic regulation that are important to bacterial virulence and survival. These studies have reinforced the unique and specific catalytic mechanisms at play in cell wall biogenesis and expanded the atomic foundation to develop novel, structure guided, antibacterial agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis of covalently linked dimeric derivatives of chlorophyll a, pyrochlorophyll a, chlorophyll b, and bacteriochlorophyll a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wasielewski, M.R.; Svec, W.A.

1980-05-09

Bis(chlorophyllide) ethylene glycol diesters were prepared for each of the title compounds. Pheophytins a and b isolated from alfalfa and bacteriochlorophyll a isolated from R. sphaeroides were treated with 80% aqueous trifluoroacetic acid to yield the corresponding pheophorbides. Pyropheophorbide was prepared by a literature procedure. Carbonic anhydride and benzotriazole-1-methanesulfonate activation methods were used in the esterification of the pheophorbides with ethylene glycol at ambient temperature. Each method yielded 75%+ of the pheophorbide ethylene glycol monoester. These monoesters were treated with equimolar amounts of the corresponding pheophorbide by using benzotriazol-1-methanesulfonate/4-(dimethylamino)pyridine in CH/sub 2/Cl/sub 2/ or dicyclohexylcarbodiimide/4-(dimethylamino)pyridine in CH/sub 2/Cl/sub 2/ atmore » ambient temperature. Yields of bis(phenophorbide) ethylene glycol diesters averaged about 50% for the former method and 70% for the latter method. Insertion of the magnesium atoms into the a series macrocycles was accomplished with iodomagnesium 2,6-di-tert-butyl-4-methylphenolate, IMgBHT, in CH/sub 2/Cl/sub 2/, while the metalation of the b and bacterial series macrocycles was carried out with a mixture of IMgBHT and lithium 2,2,6,6-tetramethylpiperidide in thiophen, all at ambient temperature. Both mono- and dimetalated derivatives were isolated and characterized in each case.« less

Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity.

PubMed

Ochocki, Joshua D; Khare, Sanika; Hess, Markus; Ackerman, Daniel; Qiu, Bo; Daisak, Jennie I; Worth, Andrew J; Lin, Nan; Lee, Pearl; Xie, Hong; Li, Bo; Wubbenhorst, Bradley; Maguire, Tobi G; Nathanson, Katherine L; Alwine, James C; Blair, Ian A; Nissim, Itzhak; Keith, Brian; Simon, M Celeste

2018-05-04

Kidney cancer, one of the ten most prevalent malignancies in the world, has exhibited increased incidence over the last decade. The most common subtype is "clear cell" renal cell carcinoma (ccRCC), which features consistent metabolic abnormalities, such as highly elevated glycogen and lipid deposition. By integrating metabolomics, genomic, and transcriptomic data, we determined that enzymes in multiple metabolic pathways are universally depleted in human ccRCC tumors, which are otherwise genetically heterogeneous. Notably, the expression of key urea cycle enzymes, including arginase 2 (ARG2) and argininosuccinate synthase 1 (ASS1), is strongly repressed in ccRCC. Reduced ARG2 activity promotes ccRCC tumor growth through at least two distinct mechanisms: conserving the critical biosynthetic cofactor pyridoxal phosphate and avoiding toxic polyamine accumulation. Pharmacological approaches to restore urea cycle enzyme expression would greatly expand treatment strategies for ccRCC patients, where current therapies only benefit a subset of those afflicted with renal cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Involvement of 2-C-methyl-D-erythritol-4-phosphate pathway in biosynthesis of aphidicolin-like tetracyclic diterpene of Scoparia dulcis.

PubMed

Nkembo, Marguerite Kasidimoko; Lee, Jung-Bum; Nakagiri, Takeshi; Hayashi, Toshimitsu

2006-05-01

Specific inhibitors of the MVA pathway (pravastatin) and the MEP pathway (fosmidomycin) were used to interfere with the biosynthetic flux which leads to the production of aphidicolin-like diterpene in leaf organ cultures of Scoparia dulcis. Treatment of leaf organs with fosmidomycin resulted in dose dependent inhibition of chlorophylls, carotenoids, scopadulcic acid B (SDB) and phytol production, and no effect on sterol production was observed. In response to the pravastatin treatment, a significant decrease in sterol and perturbation of SDB production was observed.

Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallagher, Kelley A.; Jensen, Paul R.

Background: Considerable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemicalmore » scaffolds and include many potent antibiotic and cytotoxic agents. Results: One hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the ‘MAR4’ clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites

Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade

DOE PAGES

Gallagher, Kelley A.; Jensen, Paul R.

2015-11-17

Background: Considerable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemicalmore » scaffolds and include many potent antibiotic and cytotoxic agents. Results: One hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the ‘MAR4’ clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites

Rational synthetic pathway refactoring of natural products biosynthesis in actinobacteria.

PubMed

Tan, Gao-Yi; Liu, Tiangang

2017-01-01

Natural products (NPs) and their derivatives are widely used as frontline treatments for many diseases. Actinobacteria spp. are used to produce most of NP antibiotics and have also been intensively investigated for NP production, derivatization, and discovery. However, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in Actinobacteria, especially in the cases of genome mining and heterologous expression, it is often difficult to rationally and systematically engineer synthetic pathways to maximize biosynthetic efficiency. With the emergence of new tools and methods in metabolic engineering, the synthetic pathways of many chemicals, such as fatty acids and biofuels, in model organisms (e.g. Escherichia coli ), have been refactored to realize precise and flexible control of production. These studies also offer a promising approach for synthetic pathway refactoring in Actinobacteria. In this review, the great potential of Actinobacteria as a microbial cell factory for biosynthesis of NPs is discussed. To this end, recent progress in metabolic engineering of NP synthetic pathways in Actinobacteria are summarized and strategies and perspectives to rationally and systematically refactor synthetic pathways in Actinobacteria are highlighted. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Mammalian O-Mannosylation Pathway: Glycan Structures, Enzymes, and Protein Substrates

PubMed Central

2015-01-01

The mammalian O-mannosylation pathway for protein post-translational modification is intricately involved in modulating cell–matrix interactions in the musculature and nervous system. Defects in enzymes of this biosynthetic pathway are causative for multiple forms of congenital muscular dystophy. The application of advanced genetic and biochemical technologies has resulted in remarkable progress in this field over the past few years, culminating with the publication of three landmark papers in 2013 alone. In this review, we will highlight recent progress focusing on the dramatic expansion of the set of genes known to be involved in O-mannosylation and disease processes, the concurrent acceleration of the rate of O-mannosylation pathway protein functional assignments, the tremendous increase in the number of proteins now known to be modified by O-mannosylation, and the recent progress in protein O-mannose glycan quantification and site assignment. Also, we attempt to highlight key outstanding questions raised by this abundance of new information. PMID:24786756

Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus.

PubMed

Yanai, Koji; Murakami, Takeshi; Bibb, Mervyn

2006-06-20

Streptomyces kanamyceticus 12-6 is a derivative of the wild-type strain developed for industrial kanamycin (Km) production. Southern analysis and DNA sequencing revealed amplification of a large genomic segment including the entire Km biosynthetic gene cluster in the chromosome of strain 12-6. At 145 kb, the amplifiable unit of DNA (AUD) is the largest AUD reported in Streptomyces. Striking repetitive DNA sequences belonging to the clustered regularly interspaced short palindromic repeats family were found in the AUD and may play a role in its amplification. Strain 12-6 contains a mixture of different chromosomes with varying numbers of AUDs, sometimes exceeding 36 copies and producing an amplified region >5.7 Mb. The level of Km production depended on the copy number of the Km biosynthetic gene cluster, suggesting that DNA amplification occurred during strain improvement as a consequence of selection for increased Km resistance. Amplification of DNA segments including entire antibiotic biosynthetic gene clusters might be a common mechanism leading to increased antibiotic production in industrial strains.

A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects.

PubMed

Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L; Busby, Scott A; Griffin, Patrick R; Pathak, Manish C; Ortlund, Eric A; Moore, David D

2011-05-25

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

Revealing the first uridyl peptide antibiotic biosynthetic gene cluster and probing pacidamycin biosynthesis.

PubMed

Rackham, Emma J; Grüschow, Sabine; Goss, Rebecca J M

2011-01-01

There is an urgent need for new antibiotics with resistance continuing to emerge toward existing classes. The pacidamycin antibiotics possess a novel scaffold and exhibit unexploited bioactivity rendering them attractive research targets. We recently reported the first identification of a biosynthetic cluster encoding uridyl peptide antibiotic assembly and the engineering of pacidamycin biosynthesis into a heterologous host. We report here our methods toward identifying the biosynthetic cluster. Our initial experiments employed conventional methods of probing a cosmid library using PCR and Southern blotting, however it became necessary to adopt a state-of-the-art genome scanning  and in silico hybridization approach  to pin point the cluster. Here we describe our "real" and "virtual" probing methods and contrast the benefits and pitfalls of each approach. 

Yellow flowers generated by expression of the aurone biosynthetic pathway

PubMed Central

Ono, Eiichiro; Fukuchi-Mizutani, Masako; Nakamura, Noriko; Fukui, Yuko; Yonekura-Sakakibara, Keiko; Yamaguchi, Masaatsu; Nakayama, Toru; Tanaka, Takaharu; Kusumi, Takaaki; Tanaka, Yoshikazu

2006-01-01

Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4′-O-glucosyltransferase (4′CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4′CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4′CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4′-O-glucosylated in the cytoplasm, their 4′-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant. PMID:16832053

A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis

PubMed Central

Zhao, Qing; Zhang, Yang; Wang, Gang; Hill, Lionel; Weng, Jing-Ke; Chen, Xiao-Ya; Xue, Hongwei; Martin, Cathie

2016-01-01

Wogonin and baicalein are bioactive flavones in the popular Chinese herbal remedy Huang-Qin (Scutellaria baicalensis Georgi). These specialized flavones lack a 4′-hydroxyl group on the B ring (4′-deoxyflavones) and induce apoptosis in a wide spectrum of human tumor cells in vitro and inhibit tumor growth in vivo in different mouse tumor models. Root-specific flavones (RSFs) from Scutellaria have a variety of reported additional beneficial effects including antioxidant and antiviral properties. We describe the characterization of a new pathway for the synthesis of these compounds, in which pinocembrin (a 4′-deoxyflavanone) serves as a key intermediate. Although two genes encoding flavone synthase II (FNSII) are expressed in the roots of S. baicalensis, FNSII-1 has broad specificity for flavanones as substrates, whereas FNSII-2 is specific for pinocembrin. FNSII-2 is responsible for the synthesis of 4′-deoxyRSFs, such as chrysin and wogonin, wogonoside, baicalein, and baicalin, which are synthesized from chrysin. A gene encoding a cinnamic acid–specific coenzyme A ligase (SbCLL-7), which is highly expressed in roots, is required for the synthesis of RSFs by FNSII-2, as demonstrated by gene silencing. A specific isoform of chalcone synthase (SbCHS-2) that is highly expressed in roots producing RSFs is also required for the synthesis of chrysin. Our studies reveal a recently evolved pathway for biosynthesis of specific, bioactive 4′-deoxyflavones in the roots of S. baicalensis. PMID:27152350

Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts1[C][W][OA

PubMed Central

Hennen-Bierwagen, Tracie A.; Lin, Qiaohui; Grimaud, Florent; Planchot, Véronique; Keeling, Peter L.; James, Martha G.; Myers, Alan M.

2009-01-01

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds. PMID:19168640

Forster energy transfer in chlorosomes of green photosynthetic bacteria

NASA Technical Reports Server (NTRS)

Causgrove, T. P.; Brune, D. C.; Blankenship, R. E.

1992-01-01

Energy transfer properties of whole cells and chlorosome antenna complexes isolated from the green sulfur bacteria Chlorobium limicola (containing bacteriochlorophyll c), Chlorobium vibrioforme (containing bacteriochlorophyll d) and Pelodictyon phaeoclathratiforme (containing bacteriochlorophyll e) were measured. The spectral overlap of the major chlorosome pigment (bacteriochlorophyll c, d or, e) with the bacteriochlorophyll a B795 chlorosome baseplate pigment is greatest for bacteriochlorophyll c and smallest for bacteriochlorophyll e. The absorbance and fluorescence spectra of isolated chlorosomes were measured, fitted to gaussian curves and the overlap factors with B795 calculated. Energy transfer times from the bacteriochlorophyll c, d or e to B795 were measured in whole cells and the results interpreted in terms of the Forster theory of energy transfer.

Mutation of a Rice Gene Encoding a Phenylalanine Biosynthetic Enzyme Results in Accumulation of Phenylalanine and Tryptophan[W

PubMed Central

Yamada, Tetsuya; Matsuda, Fumio; Kasai, Koji; Fukuoka, Shuichi; Kitamura, Keisuke; Tozawa, Yuzuru; Miyagawa, Hisashi; Wakasa, Kyo

2008-01-01

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size. PMID:18487352

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity▿

PubMed Central

Withers, Sydnor T.; Gottlieb, Shayin S.; Lieu, Bonny; Newman, Jack D.; Keasling, Jay D.

2007-01-01

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol. PMID:17693564

Nicotinamide riboside kinase structures reveal new pathways to NAD+.

PubMed

Tempel, Wolfram; Rabeh, Wael M; Bogan, Katrina L; Belenky, Peter; Wojcik, Marzena; Seidle, Heather F; Nedyalkova, Lyudmila; Yang, Tianle; Sauve, Anthony A; Park, Hee-Won; Brenner, Charles

2007-10-02

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

Arabidopsis ERG28 Tethers the Sterol C4-Demethylation Complex to Prevent Accumulation of a Biosynthetic Intermediate That Interferes with Polar Auxin Transport[C][W

PubMed Central

Mialoundama, Alexis Samba; Jadid, Nurul; Brunel, Julien; Di Pascoli, Thomas; Heintz, Dimitri; Erhardt, Mathieu; Mutterer, Jérôme; Bergdoll, Marc; Ayoub, Daniel; Van Dorsselaer, Alain; Rahier, Alain; Nkeng, Paul; Geoffroy, Philippe; Miesch, Michel; Camara, Bilal; Bouvier, Florence

2013-01-01

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development. PMID:24326590

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

PubMed Central

Dailey, Tamara A.; Gerdes, Svetlana; Jahn, Dieter; O'Brian, Mark R.; Warren, Martin J.

2017-01-01

SUMMARY The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized. PMID:28123057

Manipulation of ubiquitin/SUMO pathways in human herpesviruses infection.

PubMed

Gan, Jin; Qiao, Niu; Strahan, Roxanne; Zhu, Caixia; Liu, Lei; Verma, Subhash C; Wei, Fang; Cai, Qiliang

2016-11-01

Post-translational modification of proteins with ubiquitin/small ubiquitin-like modifier (SUMO) molecules triggers multiple signaling pathways that are critical for many aspects of cellular physiology. Given that viruses hijack the biosynthetic and degradative systems of their host, it is not surprising that viruses encode proteins to manipulate the host's cellular machinery for ubiquitin/SUMO modification at multiple levels. Infection with a herpesvirus, among the most ubiquitous human DNA viruses, has been linked to many human diseases, including cancers. The interplay between human herpesviruses and the ubiquitylation/SUMOylation modification system has been extensively investigated in the past decade. In this review, we present an overview of recent advances to address how the ubiquitin/SUMO-modified system alters the latency and lytic replication of herpesvirus and how herpesviruses usurp the ubiquitin/SUMO pathways against the host's intrinsic and innate immune response to favor their pathogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Induction and Repression in the S-Adenosylmethionine and Methionine Biosynthetic Systems of Saccharomyces cerevisiae

PubMed Central

Ferro, A. J.; Spence, K. D.

1973-01-01

Two methionine biosynthetic enzymes and the methionine adenosyltransferase are repressed in Saccharomyces cerevisiae when grown under conditions where the intracellular levels of S-adenosylmethionine are high. The nature of the co-repressor molecule of this repression was investigated by following the intracellular levels of methionine, S-adenosylmethionine, and S-adenosylhomocysteine, as well as enzyme activities, after growth under various conditions. Under all of the conditions found to repress these enzymes, there is an accompanying induction of the S-adenosylmethionine-homocysteine methyltransferase which suggests that this enzyme may play a key role in the regulation of S-adenosylmethionine and methionine balance and synthesis. S-methylmethionine also induces the methyltransferase, but unlike S-adenosylmethionine, it does not repress the methionine adenosyltransferase or other methionine biosynthetic enzymes tested. PMID:4583251

Identification of flavonoids and expression of flavonoid biosynthetic genes in two coloured tree peony flowers.

PubMed

Zhao, Daqiu; Tang, Wenhui; Hao, Zhaojun; Tao, Jun

2015-04-10

Tree peony (Paeonia suffruticosa Andr.) has been named the "king of flowers" because of its elegant and gorgeous flower colour. Among these colours, the molecular mechanisms of white formation and how white turned to red in P. suffruticosa is little known. In this study, flower colour variables, flavonoid accumulation and expression of flavonoid biosynthetic genes of white ('Xueta') and red ('Caihui') P. suffruticosa were investigated. The results showed that the flower colours of both cultivars were gradually deepened with the development of flowers. Moreover, two anthoxanthin compositions apigenin 7-O-glucoside together with apigenin deoxyheso-hexoside were identified in 'Xueta' and 'Caihui', but one main anthocyanin composition peonidin 3,5-di-O-glucoside (Pn3G5G) was only found in 'Caihui'. Total contents of anthocyanins in 'Caihui' was increased during flower development, and the same trend was presented in anthoxanthins and flavonoids of these two cultivars, but the contents of these two category flavonoid in 'Caihui' were always higher than those in 'Xueta'. Furthermore, nine structural genes in flavonoid biosynthetic pathway were isolated including the full-length cDNAs of phenylalanine ammonialyase gene (PAL), chalcone synthase gene (CHS) and chalcone isomerase gene (CHI), together with the partial-length cDNAs of flavanone 3-hydroxylase gene (F3H), flavonoid 3'-hydroxylase gene (F3'H), dihydroflavonol 4-reductase gene (DFR), anthocyanidin synthase gene (ANS), UDP-glucose: flavonoid 3-O-glucosyltransferase gene (UF3GT) and UDP-glucose: flavonoid 5-O-glucosyltransferase gene (UF5GT), and PAL, UF3GT and UF5GT were reported in P. suffruticosa for the first time. Their expression patterns showed that transcription levels of downstream genes in 'Caihui' were basically higher than those in 'Xueta', especially PsDFR and PsANS, suggesting that these two genes may play a key role in the anthocyanin biosynthesis which resulted in the shift from white to red in

Diversity of Culturable Thermophilic Actinobacteria in Hot Springs in Tengchong, China and Studies of their Biosynthetic Gene Profiles.

PubMed

Liu, Lan; Salam, Nimaichand; Jiao, Jian-Yu; Jiang, Hong-Chen; Zhou, En-Min; Yin, Yi-Rui; Ming, Hong; Li, Wen-Jun

2016-07-01

The class Actinobacteria has been a goldmine for the discovery of antibiotics and has attracted interest from both academics and industries. However, an absence of novel approaches during the last few decades has limited the discovery of new microbial natural products useful for industries. Scientists are now focusing on the ecological aspects of diverse environments including unexplored or underexplored habitats and extreme environments in the search for new metabolites. This paper reports on the diversity of culturable actinobacteria associated with hot springs located in Tengchong County, Yunnan Province, southwestern China. A total of 58 thermophilic actinobacterial strains were isolated from the samples collected from ten hot springs distributed over three geothermal fields (e.g., Hehua, Rehai, and Ruidian). Phylogenetic positions and their biosynthetic profiles were analyzed by sequencing 16S rRNA gene and three biosynthetic gene clusters (KS domain of PKS-I, KSα domain of PKS-II and A domain of NRPS). On the basis of 16S rRNA gene phylogenetic analysis, the 58 strains were affiliated with 12 actinobacterial genera: Actinomadura Micromonospora, Microbispora, Micrococcus, Nocardiopsis, Nonomuraea, Promicromonospora, Pseudonocardia, Streptomyces, Thermoactinospora, Thermocatellispora, and Verrucosispora, of which the two novel genera Thermoactinospora and Thermocatellisopora were recently described from among these strains. Considering the biosynthetic potential of these actinobacterial strains, 22 were positive for PCR amplification of at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, and NRPS). These actinobacteria were further subjected to antimicrobial assay against five opportunistic human pathogens (Acinetobacter baumannii, Escherichia coli, Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis). All of the 22 strains that were positive for PCR amplification of at least one of the biosynthetic gene domains exhibited

MiYA, an efficient machine-learning workflow in conjunction with the YeastFab assembly strategy for combinatorial optimization of heterologous metabolic pathways in Saccharomyces cerevisiae.

PubMed

Zhou, Yikang; Li, Gang; Dong, Junkai; Xing, Xin-Hui; Dai, Junbiao; Zhang, Chong

2018-05-01

Facing boosting ability to construct combinatorial metabolic pathways, how to search the metabolic sweet spot has become the rate-limiting step. We here reported an efficient Machine-learning workflow in conjunction with YeastFab Assembly strategy (MiYA) for combinatorial optimizing the large biosynthetic genotypic space of heterologous metabolic pathways in Saccharomyces cerevisiae. Using β-carotene biosynthetic pathway as example, we first demonstrated that MiYA has the power to search only a small fraction (2-5%) of combinatorial space to precisely tune the expression level of each gene with a machine-learning algorithm of an artificial neural network (ANN) ensemble to avoid over-fitting problem when dealing with a small number of training samples. We then applied MiYA to improve the biosynthesis of violacein. Feed with initial data from a colorimetric plate-based, pre-screened pool of 24 strains producing violacein, MiYA successfully predicted, and verified experimentally, the existence of a strain that showed a 2.42-fold titer improvement in violacein production among 3125 possible designs. Furthermore, MiYA was able to largely avoid the branch pathway of violacein biosynthesis that makes deoxyviolacein, and produces very pure violacein. Together, MiYA combines the advantages of standardized building blocks and machine learning to accelerate the Design-Build-Test-Learn (DBTL) cycle for combinatorial optimization of metabolic pathways, which could significantly accelerate the development of microbial cell factories. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Biosynthetic Investigations of Lactonamycin and Lactonamycin Z: Cloning of the Biosynthetic Gene Clusters and Discovery of an Unusual Starter Unit▿ †

PubMed Central

Zhang, Xiujun; Alemany, Lawrence B.; Fiedler, Hans-Peter; Goodfellow, Michael; Parry, Ronald J.

2008-01-01

The antibiotics lactonamycin and lactonamycin Z provide attractive leads for antibacterial drug development. Both antibiotics contain a novel aglycone core called lactonamycinone. To gain insight into lactonamycinone biosynthesis, cloning and precursor incorporation experiments were undertaken. The lactonamycin gene cluster was initially cloned from Streptomyces rishiriensis. Sequencing of ca. 61 kb of S. rishiriensis DNA revealed the presence of 57 open reading frames. These included genes coding for the biosynthesis of l-rhodinose, the sugar found in lactonamycin, and genes similar to those in the tetracenomycin biosynthetic gene cluster. Since lactonamycin production by S. rishiriensis could not be sustained, additional proof for the identity of the S. rishiriensis cluster was obtained by cloning the lactonamycin Z gene cluster from Streptomyces sanglieri. Partial sequencing of the S. sanglieri cluster revealed 15 genes that exhibited a very high degree of similarity to genes within the lactonamycin cluster, as well as an identical organization. Double-crossover disruption of one gene in the S. sanglieri cluster abolished lactonamycin Z production, and production was restored by complementation. These results confirm the identity of the genetic locus cloned from S. sanglieri and indicate that the highly similar locus in S. rishiriensis encodes lactonamycin biosynthetic genes. Precursor incorporation experiments with S. sanglieri revealed that lactonamycinone is biosynthesized in an unusual manner whereby glycine or a glycine derivative serves as a starter unit that is extended by nine acetate units. Analysis of the gene clusters and of the precursor incorporation data suggested a hypothetical scheme for lactonamycinone biosynthesis. PMID:18070976

Identification and characterization of L-lysine decarboxylase from Huperzia serrata and its role in the metabolic pathway of lycopodium alkaloid.

PubMed

Xu, Baofu; Lei, Lei; Zhu, Xiaocen; Zhou, Yiqing; Xiao, Youli

2017-04-01

Lysine decarboxylation is the first biosynthetic step of Huperzine A (HupA). Six cDNAs encoding lysine decarboxylases (LDCs) were cloned from Huperzia serrata by degenerate PCR and rapid amplification of cDNA ends (RACE). One HsLDC isoform was functionally characterized as lysine decarboxylase. The HsLDC exhibited greatest catalytic efficiency (k cat /K m , 2.11 s -1  mM -1 ) toward L-lysine in vitro among all reported plant-LDCs. Moreover, transient expression of the HsLDC in tobacco leaves specifically increased cadaverine content from zero to 0.75 mg per gram of dry mass. Additionally, a convenient and reliable method used to detect the two catalytic products was developed. With the novel method, the enzymatic products of HsLDC and HsCAO, namely cadaverine and 5-aminopentanal, respectively, were detected simultaneously both in assay with purified enzymes and in transgenic tobacco leaves. This work not only provides direct evidence of the first two-step in biosynthetic pathway of HupA in Huperzia serrata and paves the way for further elucidation of the pathway, but also enables engineering heterologous production of HupA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters.

PubMed

Schorn, Michelle A; Alanjary, Mohammad M; Aguinaldo, Kristen; Korobeynikov, Anton; Podell, Sheila; Patin, Nastassia; Lincecum, Tommie; Jensen, Paul R; Ziemert, Nadine; Moore, Bradley S

2016-12-01

Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites.

Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters

PubMed Central

Schorn, Michelle A.; Alanjary, Mohammad M.; Aguinaldo, Kristen; Korobeynikov, Anton; Podell, Sheila; Patin, Nastassia; Lincecum, Tommie; Jensen, Paul R.; Ziemert, Nadine

2016-01-01

Traditional natural product discovery methods have nearly exhausted the accessible diversity of microbial chemicals, making new sources and techniques paramount in the search for new molecules. Marine actinomycete bacteria have recently come into the spotlight as fruitful producers of structurally diverse secondary metabolites, and remain relatively untapped. In this study, we sequenced 21 marine-derived actinomycete strains, rarely studied for their secondary metabolite potential and under-represented in current genomic databases. We found that genome size and phylogeny were good predictors of biosynthetic gene cluster diversity, with larger genomes rivalling the well-known marine producers in the Streptomyces and Salinispora genera. Genomes in the Micrococcineae suborder, however, had consistently the lowest number of biosynthetic gene clusters. By networking individual gene clusters into gene cluster families, we were able to computationally estimate the degree of novelty each genus contributed to the current sequence databases. Based on the similarity measures between all actinobacteria in the Joint Genome Institute's Atlas of Biosynthetic gene Clusters database, rare marine genera show a high degree of novelty and diversity, with Corynebacterium, Gordonia, Nocardiopsis, Saccharomonospora and Pseudonocardia genera representing the highest gene cluster diversity. This research validates that rare marine actinomycetes are important candidates for exploration, as they are relatively unstudied, and their relatives are historically rich in secondary metabolites. PMID:27902408

Lipidomics Characterization of Biosynthetic and Remodeling Pathways of Cardiolipins in Genetically and Nutritionally Manipulated Yeast Cells.

PubMed

Tyurina, Yulia Y; Lou, Wenjia; Qu, Feng; Tyurin, Vladimir A; Mohammadyani, Dariush; Liu, Jenney; Hüttemann, Maik; Frasso, Michael A; Wipf, Peter; Bayir, Hülya; Greenberg, Miriam L; Kagan, Valerian E

2017-01-20

Cardioipins (CLs) are unique tetra-acylated phospholipids of mitochondria and define the bioenergetics and regulatory functions of these organelles. An unresolved paradox is the high uniformity of CL molecular species (tetra-linoleoyl-CL) in the heart, liver, and skeletal muscles-in contrast to their high diversification in the brain. Here, we combined liquid chromatography-mass-spectrometry-based phospholipidomics with genetic and nutritional manipulations to explore CLs' biosynthetic vs postsynthetic remodeling processes in S. cerevisiae yeast cells. By applying the differential phospholipidomics analysis, we evaluated the contribution of Cld1 (CL-specific phospholipase A) and Taz1 (acyl-transferase) as the major regulatory mechanisms of the remodeling process. We further established that nutritional "pressure" by high levels of free fatty acids triggered a massive synthesis of homoacylated molecular species in all classes of phospholipids, resulting in the preponderance of the respective homoacylated CLs. We found that changes in molecular speciation of CLs induced by exogenous C18-fatty acids (C18:1 and C18:2) in wild-type (wt) cells did not occur in any of the remodeling mutant cells, including cld1Δ, taz1Δ, and cld1Δtaz1Δ. Interestingly, molecular speciation of CLs in wt and double mutant cells cld1Δtaz1Δ was markedly different. Given that the bioenergetics functions are preserved in the double mutant, this suggests that the accumulated MLCL-rather than the changed CL speciation-are the likely major contributors to the mitochondrial dysfunction in taz1Δ mutant cells (also characteristic of Barth syndrome). Biochemical studies of Cld1 specificity and computer modeling confirmed the hydrolytic selectivity of the enzyme toward C16-CL substrates and the preservation of C18:1-containing CL species.

New insights into the organization and regulation of trichothecene biosynthetic genes in Trichoderma

USDA-ARS?s Scientific Manuscript database

Collectively, species of the genus Trichoderma can produce numerous structurally diverse secondary metabolites (SM). This ability is conferred by the presence of SM biosynthetic gene clusters in their genomes. Species of Trichoderma in the Brevicompactum clade are able to produce trichothecenes, a f...

Nanolipoprotein particles comprising a natural rubber biosynthetic enzyme complex and related products, methods and systems

DOEpatents

Hoeprich, Paul D.; Whalen, Maureen

2016-04-05

Provided herein are nanolipoprotein particles that comprise a biosynthetic enzyme more particularly an enzyme capable of catalyzing rubber or other rubbers polymerization, and related assemblies, devices, methods and systems.

Heterologous expression and transcript analysis of gibberellin biosynthetic genes of grasses reveals novel functionality in the GA3ox family.

PubMed

Pearce, Stephen; Huttly, Alison K; Prosser, Ian M; Li, Yi-dan; Vaughan, Simon P; Gallova, Barbora; Patil, Archana; Coghill, Jane A; Dubcovsky, Jorge; Hedden, Peter; Phillips, Andrew L

2015-06-05

The gibberellin (GA) pathway plays a central role in the regulation of plant development, with the 2-oxoglutarate-dependent dioxygenases (2-ODDs: GA20ox, GA3ox, GA2ox) that catalyse the later steps in the biosynthetic pathway of particularly importance in regulating bioactive GA levels. Although GA has important impacts on crop yield and quality, our understanding of the regulation of GA biosynthesis during wheat and barley development remains limited. In this study we identified or assembled genes encoding the GA 2-ODDs of wheat, barley and Brachypodium distachyon and characterised the wheat genes by heterologous expression and transcript analysis. The wheat, barley and Brachypodium genomes each contain orthologous copies of the GA20ox, GA3ox and GA2ox genes identified in rice, with the exception of OsGA3ox1 and OsGA2ox5 which are absent in these species. Some additional paralogs of 2-ODD genes were identified: notably, a novel gene in the wheat B genome related to GA3ox2 was shown to encode a GA 1-oxidase, named as TaGA1ox-B1. This enzyme is likely to be responsible for the abundant 1β-hydroxylated GAs present in developing wheat grains. We also identified a related gene in barley, located in a syntenic position to TaGA1ox-B1, that encodes a GA 3,18-dihydroxylase which similarly accounts for the accumulation of unusual GAs in barley grains. Transcript analysis showed that some paralogs of the different classes of 2-ODD were expressed mainly in a single tissue or at specific developmental stages. In particular, TaGA20ox3, TaGA1ox1, TaGA3ox3 and TaGA2ox7 were predominantly expressed in developing grain. More detailed analysis of grain-specific gene expression showed that while the transcripts of biosynthetic genes were most abundant in the endosperm, genes encoding inactivation and signalling components were more highly expressed in the seed coat and pericarp. The comprehensive expression and functional characterisation of the multigene families encoding the 2-ODD

Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy.

PubMed

Escalera-Fanjul, Ximena; Campero-Basaldua, Carlos; Colón, Maritrini; González, James; Márquez, Dariel; González, Alicia

2017-01-01

Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, Sc Alt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1 , alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display Lk Alt1 and Kl Alt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s). Furthermore, phenotypic analysis of null mutants uncovered the fact that Kl Alt1 and Lk Alt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that Sc Alt2 conserves 64% identity with

Evolutionary Diversification of Alanine Transaminases in Yeast: Catabolic Specialization and Biosynthetic Redundancy

PubMed Central

Escalera-Fanjul, Ximena; Campero-Basaldua, Carlos; Colón, Maritrini; González, James; Márquez, Dariel; González, Alicia

2017-01-01

Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s). Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64% identity with LkAlt1

Descriptions of Roseiarcus fermentans gen. nov., sp. nov., a bacteriochlorophyll a-containing fermentative bacterium related phylogenetically to alphaproteobacterial methanotrophs, and of the family Roseiarcaceae fam. nov.

PubMed

Kulichevskaya, Irina S; Danilova, Olga V; Tereshina, Vera M; Kevbrin, Vadim V; Dedysh, Svetlana N

2014-08-01

A light-pink-pigmented, microaerophilic bacterium was obtained from a methanotrophic consortium enriched from acidic Sphagnum peat and designated strain Pf56(T). Cells of this bacterium were Gram-negative, non-motile, thick curved rods that contained a vesicular intracytoplasmic membrane system characteristic of some purple non-sulfur alphaproteobacteria. The absorption spectrum of acetone/methanol extracts of cells grown in the light showed maxima at 363, 475, 505, 601 and 770 nm; the peaks at 363 and 770 nm are characteristic of bacteriochlorophyll a. However, in contrast to purple non-sulfur bacteria, strain Pf56(T) was unable to grow phototrophically under anoxic conditions in the light. Best growth occurred on some sugars and organic acids under micro-oxic conditions by means of fermentation. The fermentation products were propionate, acetate and hydrogen. Slow chemo-organotrophic growth was also observed under fully oxic conditions. Light stimulated growth. C1 substrates were not utilized. Strain Pf56(T) grew at pH 4.0-7.0 (optimum pH 5.5-6.5) and at 15-30 °C (optimum 22-28 °C). The major cellular fatty acids were 19 : 0 cyclo ω8c and 18 : 1ω7c; quinones were represented by ubiquinone Q-10. The G+C content of the DNA was 70.0 mol%. Strain Pf56 displays 93.6-94.7 and 92.7-93.7% 16S rRNA gene sequence similarity to members of the families Methylocystaceae and Beijerinckiaceae, respectively, and belongs to a large cluster of environmental sequences retrieved from various wetlands and forest soils in cultivation-independent studies. Phenotypic, genotypic and chemotaxonomic characteristics of strain Pf56(T) suggest that it represents a novel genus and species of bacteriochlorophyll a-containing fermentative bacteria, for which the name Roseiarcus fermentans gen. nov., sp. nov. is proposed. Strain Pf56(T) ( = DSM 24875(T) = VKM B-2876(T)) is the type strain of Roseiarcus fermentans, and is also the first characterized member of a novel family

Comparative genomic analysis of secondary metabolite biosynthetic gene clusters in 207 isolates of Fusarium

USDA-ARS?s Scientific Manuscript database

Fusarium species are known for their ability to produce secondary metabolites (SMs), including plant hormones, pigments, mycotoxins, and other compounds with potential agricultural, pharmaceutical, and biotechnological impact. Understanding the distribution of SM biosynthetic gene clusters across th...

Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties.

PubMed

Hoang, Van L T; Innes, David J; Shaw, P Nicholas; Monteith, Gregory R; Gidley, Michael J; Dietzgen, Ralf G

2015-07-30

Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and

Step of Dichlorvos Inhibition in the Pathway of Aflatoxin Biosynthesis

PubMed Central

Yao, Raymond C.; Hsieh, Dennis P. H.

1974-01-01

Dichlorvos (dimethyl 2,2-dichlorovinyl phosphate) inhibits the biosynthesis of aflatoxin by Aspergillus parasiticus. Cultures treated with dichlorvos excrete an orange pigment which can be converted into aflatoxin B1 by the untreated mycelia. The orange pigment was partially identified as an acetyl derivative of versiconol-type compound. In the presence of dichlorvos, sterigmatocystin is converted into aflatoxin B1 without being interfered, but averufin is converted into the orange pigment instead of aflatoxin B1. Therefore, dichlorvos appears to block an enzymatic step in the aflatoxin biosynthetic pathway, which lies beyond averufin but before sterigmatocystin, at the formation of the orange pigment. PMID:4844267

Description of an orthologous cluster of ochratoxin A biosynthetic genes in Aspergillus and Penicillium species. A comparative analysis.

PubMed

Gil-Serna, Jessica; García-Díaz, Marta; González-Jaén, María Teresa; Vázquez, Covadonga; Patiño, Belén

2018-03-02

Ochratoxin A (OTA) is one of the most important mycotoxins due to its toxic properties and worldwide distribution which is produced by several Aspergillus and Penicillium species. The knowledge of OTA biosynthetic genes and understanding of the mechanisms involved in their regulation are essential. In this work, we obtained a clear picture of biosynthetic genes organization in the main OTA-producing Aspergillus and Penicillium species (A. steynii, A. westerdijkiae, A. niger, A. carbonarius and P. nordicum) using complete genome sequences obtained in this work or previously available on databases. The results revealed a region containing five ORFs which predicted five proteins: halogenase, bZIP transcription factor, cytochrome P450 monooxygenase, non-ribosomal peptide synthetase and polyketide synthase in all the five species. Genetic synteny was conserved in both Penicillium and Aspergillus species although genomic location seemed to be different since the clusters presented different flanking regions (except for A. steynii and A. westerdijkiae); these observations support the hypothesis of the orthology of this genomic region and that it might have been acquired by horizontal transfer. New real-time RT-PCR assays for quantification of the expression of these OTA biosynthetic genes were developed. In all species, the five genes were consistently expressed in OTA-producing strains in permissive conditions. These protocols might favour futures studies on the regulation of biosynthetic genes in order to develop new efficient control methods to avoid OTA entering the food chain. Copyright © 2018 Elsevier B.V. All rights reserved.

A plug-and-play pathway refactoring workflow for natural product research in Escherichia coli and Saccharomyces cerevisiae.

PubMed

Ren, Hengqian; Hu, Pingfan; Zhao, Huimin

2017-08-01

Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization, and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Here we report a plug-and-play pathway refactoring workflow for high-throughput, flexible pathway construction, and expression in both Escherichia coli and Saccharomyces cerevisiae. Biosynthetic genes were firstly cloned into pre-assembled helper plasmids with promoters and terminators, resulting in a series of expression cassettes. These expression cassettes were further assembled using Golden Gate reaction to generate fully refactored pathways. The inclusion of spacer plasmids in this system would not only increase the flexibility for refactoring pathways with different number of genes, but also facilitate gene deletion and replacement. As proof of concept, a total of 96 pathways for combinatorial carotenoid biosynthesis were built successfully. This workflow should be generally applicable to different classes of natural products produced by various organisms. Biotechnol. Bioeng. 2017;114: 1847-1854. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Mutasynthesis of pyrrole spiroketal compound using calcimycin 3-hydroxy anthranilic acid biosynthetic mutant.

PubMed

Gou, Lixia; Wu, Qiulin; Lin, Shuangjun; Li, Xiangmei; Liang, Jingdan; Zhou, Xiufen; An, Derong; Deng, Zixin; Wang, Zhijun

2013-09-01

The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies.

PubMed

Kirkton, Robert D; Bursac, Nenad

2011-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair.

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies

PubMed Central

Kirkton, Robert D.; Bursac, Nenad

2012-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair. PMID:21556054

Accumulation of kaempferitrin and expression of phenyl-propanoid biosynthetic genes in kenaf (Hibiscus cannabinus).

PubMed

Zhao, Shicheng; Li, Xiaohua; Cho, Dong Ha; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Sang Un

2014-10-23

Kenaf (Hibiscus cannabinus) is cultivated worldwide for its fiber; however, the medicinal properties of this plant are currently attracting increasing attention. In this study, we investigated the expression levels of genes involved in the biosynthesis of kaempferitrin, a compound with many biological functions, in different kenaf organs. We found that phenylalanine ammonia lyase (HcPAL) was more highly expressed in stems than in other organs. Expression levels of cinnamate 4-hydroxylase (HcC4H) and 4-coumarate-CoA ligase (Hc4CL) were highest in mature leaves, followed by stems and young leaves, and lowest in roots and mature flowers. The expression of chalcone synthase (HcCHS), chalcone isomerase (HcCHI), and flavone 3-hydroxylase (HcF3H) was highest in young flowers, whereas that of flavone synthase (HcFLS) was highest in leaves. An analysis of kaempferitrin accumulation in the different organs of kenaf revealed that the accumulation of this compound was considerably higher (>10-fold) in leaves than in other organs. On the basis of a comparison of kaempferitrin contents with the expression levels of different genes in different organs, we speculate that HcFLS plays an important regulatory role in the kaempferitrin biosynthetic pathway in kenaf.

A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIα is important for protein trafficking from the endoplasmic reticulum to the plasma membrane.

PubMed

Bryant, Kirsten L; Baird, Barbara; Holowka, David

2015-02-27

Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking. To investigate the source of PI4P that orchestrates trafficking events, we developed a novel flow cytometry based method to monitor biosynthetic trafficking of transiently transfected proteins. We demonstrated that our method can be used to assess the trafficking of both type-1 transmembrane and GPI-linked proteins, and that it can accurately monitor the pharmacological disruption of biosynthetic trafficking with brefeldin A, a well-documented inhibitor of early biosynthetic trafficking. Furthermore, utilizing our newly developed method, we applied pharmacological inhibition of different isoforms of PI 4-kinase to reveal a role for a distinct pool of PI4P, synthesized by PI4KIIIα, in ER-to-PM trafficking. Taken together, these findings provide evidence that a specific pool of PI4P plays a role in biosynthetic trafficking of two different classes of proteins from the ER to the Golgi complex. Furthermore, our simple, flow cytometry-based biosynthetic trafficking assay can be widely applied to the study of multiple classes of proteins and varied pharmacological and genetic perturbations.

Altered expression of polyketide biosynthetic gene clusters in fumonisin-deficient mutants of Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

Fusarium verticillioides is a pathogen of maize and produces fumonisins, a group of polyketide derived secondary metabolites. Fumonisins cause diseases in animals, and they have been correlated epidemiologically with esophageal cancer and birth defects in humans. Fumonisin biosynthetic genes are c...

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase

PubMed Central

Faehnle, Christopher R.; Liu, Xuying; Pavlovsky, Alexander; Viola, Ronald E.

2006-01-01

The activation of the β-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate l-­aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups. PMID:17012784

Viral exploitation of the MEK/ERK pathway - A tale of vaccinia virus and other viruses.

PubMed

Bonjardim, Cláudio A

2017-07-01

The VACV replication cycle is remarkable in the sense that it is performed entirely in the cytoplasmic compartment of vertebrate cells, due to its capability to encode enzymes required either for regulating the macromolecular precursor pool or the biosynthetic processes. Although remarkable, this gene repertoire is not sufficient to confer the status of a free-living microorganism to the virus, and, consequently, the virus relies heavily on the host to successfully generate its progeny. During the complex virus-host interaction, viruses must deal not only with the host pathways to accomplish their temporal demands but also with pathways that counteract viral infection, including the inflammatory, innate and acquired immune responses. This review focuses on VACV and other DNA or RNA viruses that stimulate the MEK (MAPK - Mitogen Activated Protein Kinase)/ERK- Extracellular signal-Regulated Kinase) pathway as part of their replication cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

Improved L-ornithine production in Corynebacterium crenatum by introducing an artificial linear transacetylation pathway.

PubMed

Shu, Qunfeng; Xu, Meijuan; Li, Jing; Yang, Taowei; Zhang, Xian; Xu, Zhenghong; Rao, Zhiming

2018-05-04

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis.

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

PubMed

Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

2009-03-04

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in Acinetobacter baumannii

PubMed Central

Wang, Lisha; Chan, Helen; De Pascale, Gianfranco; Six, David A.; Wei, Jun-Rong; Dean, Charles R.

2018-01-01

Acinetobacter baumannii ATCC 19606 can grow without lipooligosaccharide (LOS). Lack of LOS can result from disruption of the early lipid A biosynthetic pathway genes lpxA, lpxC or lpxD. Although LOS itself is not essential for growth of A. baumannii ATCC 19606, it was previously shown that depletion of the lipid A biosynthetic enzyme LpxK in cells inhibited growth due to the toxic accumulation of lipid A pathway intermediates. Growth of LpxK-depleted cells was restored by chemical inhibition of LOS biosynthesis using CHIR-090 (LpxC) and fatty acid biosynthesis using cerulenin (FabB/F) and pyridopyrimidine (acetyl-CoA-carboxylase). Here, we expand on this by showing that inhibition of enoyl-acyl carrier protein reductase (FabI), responsible for converting trans-2-enoyl-ACP into acyl-ACP during the fatty acid elongation cycle also restored growth during LpxK depletion. Inhibition of fatty acid biosynthesis during LpxK depletion rescued growth at 37°C, but not at 30°C, whereas rescue by LpxC inhibition was temperature independent. We exploited these observations to demonstrate proof of concept for a targeted medium-throughput growth restoration screening assay to identify small molecule inhibitors of LOS and fatty acid biosynthesis. The differential temperature dependence of fatty acid and LpxC inhibition provides a simple means by which to separate growth stimulating compounds by pathway. Targeted cell-based screening platforms such as this are important for faster identification of compounds inhibiting pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens. PMID:29505586

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways.

PubMed

Wingler, Laura M; Cornish, Virginia W

2011-09-13

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

PubMed Central

Wingler, Laura M.; Cornish, Virginia W.

2011-01-01

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 104 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo. PMID:21876185

Ketol-acid reductoisomerase enzymes and methods of use

DOEpatents

Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

2015-10-27

Provided herein are polypeptides having ketol-aid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

The oxalic acid biosynthetic activity of Burkholderia mallei is encoded by a single locus

USDA-ARS?s Scientific Manuscript database

Although it is known that oxalic acid provides a selective advantage to the secreting microbe, our understanding of how this acid is biosynthesized remains incomplete. This study reports the identification, cloning, and partial characterization of the oxalic acid biosynthetic enzyme from the animal ...

Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains1

PubMed Central

Gandía-Herrero, Fernando; Escribano, Josefa; García-Carmona, Francisco

2005-01-01

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis and in the enzymatic browning of fruits and vegetables. The role of tyrosinase in the secondary metabolism of plants still remains unclear, but its implication in betalain biosynthesis has been proposed. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this article, the betaxanthins, tyrosine-betaxanthin (portulacaxanthin II) and dopaxanthin, are reported to be physiological substrates for tyrosinase. The direct activity of tyrosinase on selected betaxanthins is characterized in depth, and conversion of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are described. Identity of the reaction products was studied by high-performance liquid chromatography and electrospray ionization-mass spectrometry. Masses determined for the reaction products were the same in all cases, 389 m/z ([M + H]+) and equal to that determined for betanidin. Data indicate that dopaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. Kinetic parameters for tyrosinase acting on dopaxanthin were evaluated, showing a high affinity for this substrate (Km = 84.3 μm). The biosynthetic scheme of betalains is reviewed and a branch is proposed based on the description of physiological substrates for tyrosinase. Lampranthus productus, Glottiphylum oligocarpum, and Glottiphylum pigmaeum are described as sources of stereopure (2S/S)-dopaxanthin. PMID:15805475

Identification of the Coumermycin A1 Biosynthetic Gene Cluster of Streptomyces rishiriensis DSM 40489

PubMed Central

Wang, Zhao-Xin; Li, Shu-Ming; Heide, Lutz

2000-01-01

The biosynthetic gene cluster of the aminocoumarin antibiotic coumermycin A1 was cloned by screening of a cosmid library of Streptomyces rishiriensis DSM 40489 with heterologous probes from a dTDP-glucose 4,6-dehydratase gene, involved in deoxysugar biosynthesis, and from the aminocoumarin resistance gyrase gene gyrBr. Sequence analysis of a 30.8-kb region upstream of gyrBr revealed the presence of 28 complete open reading frames (ORFs). Fifteen of the identified ORFs showed, on average, 84% identity to corresponding ORFs in the biosynthetic gene cluster of novobiocin, another aminocoumarin antibiotic. Possible functions of 17 ORFs in the biosynthesis of coumermycin A1 could be assigned by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by an insertional gene inactivation experiment, which resulted in an abolishment of coumermycin A1 production. PMID:11036020

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

PubMed

Rabatel, Andréane; Febvay, Gérard; Gaget, Karen; Duport, Gabrielle; Baa-Puyoulet, Patrice; Sapountzis, Panagiotis; Bendridi, Nadia; Rey, Marjolaine; Rahbé, Yvan; Charles, Hubert; Calevro, Federica; Colella, Stefano

2013-04-10

Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch

The synthesis of chlorophyll-a biosynthetic precursors and methyl substituted iron porphyrins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matera, K.M.

1988-01-01

The biosynthetic intermediates were incubated in a plant system. The activity levels calculated show that magnesium 6-acrylate porphyrins and one of the magnesium 6-{beta}-hydroxypropionate porphyrins are not intermediates. In addition, plant systems incubated with {sup 18}O{sub 2} were found to synthesize magnesium 2,4-divinyl pheoporphyrin-a{sub 5} incorporated with {sup 18}O at the 9-carbonyl oxygen. Mass spectroscopy confirmed the presence of the oxygen label, thus eliminating one of two hypothesized pathways to chlorophyll-a. An overall description is given of iron porphyrins and iron porphyrin containing proteins. The function of the propionic side chains of the heme prosthetic group during electron transport reactionsmore » will be investigated. The synthesis of a series of iron(III) hexamethyl porphyrins with increasingly longer substituents in the remaining two peripheral positions of the porphyrin is described. Models for NMR studies of iron chlorin containing enzymes are discussed. Iron(III) pyropheophorbide-a and methyl pyropheophorbide-a were synthesized in addition to 5-CD{sub 3}, 10-CD{sub 2} iron(III) pyropheophorbide-a and methyl pyropheophorbide-a. Together, these pyropheophorbides were used to assign NMR resonances and ultimately provide a model for other iron chlorins. The synthesis of nickel(II) anhydro-mesorhodoporphyrin from zinc(III) anhydromesorhodochlorin is described; this nickel porphyrin was used as a standard for ring current calculations of reduced nickel analogs of anhydromesorhodoporphyrin.« less

Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis

PubMed Central

Qu, Xudong

2014-01-01

Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5’-fluoro-5’-deoxyadenosine (5’-FDA) from inorganic fluoride and S-adenosyl-l-methionine (SAM). The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of the fl pathway. Kinetic parameters for fluoride ( K m 4153 μM, k cat 0.073 min -1) and SAM ( K m 416 μM, k cat 0.139 min -1) have been determined, revealing that NobA is slightly (2.3 fold) slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity. PMID:24795808

De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-alzheimer's properties.

PubMed

Guo, Qianqian; Ma, Xiaojun; Wei, Shugen; Qiu, Deyou; Wilson, Iain W; Wu, Peng; Tang, Qi; Liu, Lijun; Dong, Shoukun; Zu, Wei

2014-08-12

The major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid β protein (a pathological marker of Alzheimer's disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown. In this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis. A combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria, and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.

Microorganism genomics, compositions and methods related thereto

DOEpatents

Handelsman, Jo; Goodman, Robert M.; Rondon, Michelle R.

2001-01-01

The present invention provides methods and compositions for accessing, in a generally unbaised manner, a diverse genetic pool for genes involved in biosynthetic pathways. The invention also provides compounds which can be identified by cloning biosynthetic pathways.

Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology.

PubMed

Pandey, Ramesh Prasad; Parajuli, Prakash; Koffas, Mattheos A G; Sohng, Jae Kyung

2016-01-01

In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described. Copyright © 2016 Elsevier Inc. All rights reserved.

Complete genome sequence of the bacteriochlorophyll a-containing Roseibacterium elongatum type strain (DSM 19469(T)), a representative of the Roseobacter group isolated from Australian coast sand.

PubMed

Riedel, Thomas; Fiebig, Anne; Göker, Markus; Klenk, Hans-Peter

2014-06-15

Roseibacterium elongatum Suzuki et al. 2006 is a pink-pigmented and bacteriochlorophyll a-producing representative of the Roseobacter group within the alphaproteobacterial family Rhodobacteraceae. Representatives of the marine 'Roseobacter group' were found to be abundant in the ocean and play an important role in global and biogeochemical processes. In the present study we describe the features of R. elongatum strain OCh 323(T) together with its genome sequence and annotation. The 3,555,102 bp long genome consists of one circular chromosome with no extrachromosomal elements and is one of the smallest known Roseobacter genomes. It contains 3,540 protein-coding genes and 59 RNA genes. Genome analysis revealed the presence of a photosynthetic gene cluster, which putatively enables a photoheterotrophic lifestyle. Gene sequences associated with quorum sensing, motility, surface attachment, and thiosulfate and carbon monoxide oxidation could be detected. The genome was sequenced as part of the activities of the Transregional Collaborative Research Centre 51 (TRR51) funded by the German Research Foundation (DFG).

IMG-ABC: An Atlas of Biosynthetic Gene Clusters to Fuel the Discovery of Novel Secondary Metabolites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, I-Min; Chu, Ken; Ratner, Anna

2014-10-28

In the discovery of secondary metabolites (SMs), large-scale analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of relevant computational resources. We present IMG-ABC (https://img.jgi.doe.gov/abc/) -- An Atlas of Biosynthetic gene Clusters within the Integrated Microbial Genomes (IMG) system1. IMG-ABC is a rich repository of both validated and predicted biosynthetic clusters (BCs) in cultured isolates, single-cells and metagenomes linked with the SM chemicals they produce and enhanced with focused analysis tools within IMG. The underlying scalable framework enables traversal of phylogenetic dark matter and chemical structure space -- serving as a doorwaymore » to a new era in the discovery of novel molecules.« less

Enhancement of L-valine production in Bacillus licheniformis by blocking three branched pathways.

PubMed

Liang, Chengwen; Huo, Yanli; Qi, Gaofu; Wei, Xuetuan; Wang, Qin; Chen, Shouwen

2015-06-01

Bacillus licheniformis WX-02 is used for the production of many valuable chemicals. Here, we have sought to improve L-valine production by blocking the metabolic pathways related to branched-chain amino acids. The synthesis genes of L-leucine (leuA) and L-isoleucine (ilvA) were deleted to obtain mutant strains. L-Valine yields of WX-02ΔleuA and WX-02ΔilvA reached 33.2 and 21.1 mmol/l, respectively, which are 22 and 14 times higher than the wild-type WX-02 (1.53 mmol/l). After further deletion of L-lactate dehydrogenase gene (ldh) from WX-02ΔleuA, the productivity reached 0.47 mmol/l h, an increase of 19 %. We provide a possibility to over-produce L-valine using genetically-modified B. licheniformis using remodeling of the biosynthetic pathway to L-valine.

In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli.

PubMed

Zhu, Fayin; Zhong, Xiaofang; Hu, Mengzhu; Lu, Lei; Deng, Zixin; Liu, Tiangang

2014-07-01

Approaches using metabolic engineering and synthetic biology to overproduce terpenoids, such as the precursors of taxol and artemisinin, in microbial systems have achieved initial success. However, due to the lack of steady-state kinetic information and incomplete understanding of the terpenoid biosynthetic pathway, it has been difficult to build a highly efficient, universal system. Here, we reconstituted the mevalonate pathway to produce farnesene (a precursor of new jet fuel) in vitro using purified protein components. The information from this in vitro reconstituted system guided us to rationally optimize farnesene production in E. coli by quantitatively overexpressing each component. Targeted proteomic assays and intermediate assays were used to determine the metabolic status of each mutant. Through targeted engineering, farnesene production could be increased predictably step by step, up to 1.1 g/L (∼ 2,000 fold) 96 h after induction at the shake-flask scale. The strategy developed to release the potential of the mevalonate pathway for terpenoid overproduction should also work in other multistep synthetic pathways. © 2014 Wiley Periodicals, Inc.

Tools of pathway reconstruction and production of economically relevant plant secondary metabolites in recombinant microorganisms.

PubMed

Dziggel, Clarissa; Schäfer, Holger; Wink, Michael

2017-01-01

Plant secondary metabolites exhibit a variety of biological activities and therefore serve as valuable therapeutics or flavoring compounds. However, the small amounts isolated from plants often cannot meet market demands. This led to the exploration of other, more profitable methods for their production, including plant cell culture systems, chemical synthesis and biotechnological production in microbial hosts. The biotechnological production can be pursued by reconstructing metabolic pathways in selected microbial systems. But due to their complexity, most of these pathways are not completely understood and require the expression of a multitude of genes in a foreign organism. Recently, next generation sequencing data and advances in gene silencing in plants allowed the elucidation of some biosynthetic pathways in more detail. Thus, the de novo production of some natural products, including morphine, strictosidine, artemisinin, taxol ® and resveratrol, in extensively engineered microbial hosts has become feasible. This review highlights the reconstruction of these pathways, missing pieces and novel techniques employed. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Review on Abyssomicins: Inhibitors of the Chorismate Pathway and Folate Biosynthesis.

PubMed

Sadaka, Carmen; Ellsworth, Edmund; Hansen, Paul Robert; Ewin, Richard; Damborg, Peter; Watts, Jeffrey L

2018-06-06

Antifolates targeting folate biosynthesis within the shikimate-chorismate-folate metabolic pathway are ideal and selective antimicrobials, since higher eukaryotes lack this pathway and rely on an exogenous source of folate. Resistance to the available antifolates, inhibiting the folate pathway, underlines the need for novel antibiotic scaffolds and molecular targets. While para-aminobenzoic acid synthesis within the chorismate pathway constitutes a novel molecular target for antifolates, abyssomicins are its first known natural inhibitors. This review describes the abyssomicin family, a novel spirotetronate polyketide Class I antimicrobial. It summarizes synthetic and biological studies, structural, biosynthetic, and biological properties of the abyssomicin family members. This paper aims to explain their molecular target, mechanism of action, structure⁻activity relationship, and to explore their biological and pharmacological potential. Thirty-two natural abyssomicins and numerous synthetic analogues have been reported. The biological activity of abyssomicins includes their antimicrobial activity against Gram-positive bacteria and mycobacteria, antitumor properties, latent human immunodeficiency virus (HIV) reactivator, anti-HIV and HIV replication inducer properties. Their antimalarial properties have not been explored yet. Future analoging programs using the structure⁻activity relationship data and synthetic approaches may provide a novel abyssomicin structure that is active and devoid of cytotoxicity. Abyssomicin J and atrop- o -benzyl-desmethylabyssomicin C constitute promising candidates for such programs.

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI

PubMed Central

Wang, Cheng; Zeng, Jian; Li, Yin; Yang, Guangxiao; He, Guangyuan

2014-01-01

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 μg g–1 of seed dry weight, a β-carotene increase of 65-fold to 3.21 μg g–1 of seed dry weight, and a provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold to 3.82 μg g–1 of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm. PMID:24692648

Enrichment of provitamin A content in wheat (Triticum aestivum L.) by introduction of the bacterial carotenoid biosynthetic genes CrtB and CrtI.

PubMed

Wang, Cheng; Zeng, Jian; Li, Yin; Hu, Wei; Chen, Ling; Miao, Yingjie; Deng, Pengyi; Yuan, Cuihong; Ma, Cheng; Chen, Xi; Zang, Mingli; Wang, Qiong; Li, Kexiu; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2014-06-01

Carotenoid content is a primary determinant of wheat nutritional value and affects its end-use quality. Wheat grains contain very low carotenoid levels and trace amounts of provitamin A content. In order to enrich the carotenoid content in wheat grains, the bacterial phytoene synthase gene (CrtB) and carotene desaturase gene (CrtI) were transformed into the common wheat cultivar Bobwhite. Expression of CrtB or CrtI alone slightly increased the carotenoid content in the grains of transgenic wheat, while co-expression of both genes resulted in a darker red/yellow grain phenotype, accompanied by a total carotenoid content increase of approximately 8-fold achieving 4.76 μg g(-1) of seed dry weight, a β-carotene increase of 65-fold to 3.21 μg g(-1) of seed dry weight, and a provitamin A content (sum of α-carotene, β-carotene, and β-cryptoxanthin) increase of 76-fold to 3.82 μg g(-1) of seed dry weight. The high provitamin A content in the transgenic wheat was stably inherited over four generations. Quantitative PCR analysis revealed that enhancement of provitamin A content in transgenic wheat was also a result of the highly coordinated regulation of endogenous carotenoid biosynthetic genes, suggesting a metabolic feedback regulation in the wheat carotenoid biosynthetic pathway. These transgenic wheat lines are not only valuable for breeding wheat varieties with nutritional benefits for human health but also for understanding the mechanism regulating carotenoid biosynthesis in wheat endosperm. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Impairment of chondrocyte biosynthetic activity by exposure to 3-tesla high-field magnetic resonance imaging is temporary

PubMed Central

Sunk, Ilse-Gerlinde; Trattnig, Siegfried; Graninger, Winfried B; Amoyo, Love; Tuerk, Birgit; Steiner, Carl-Walter; Smolen, Josef S; Bobacz, Klaus

2006-01-01

The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissues is unknown. We investigated the effects of a 3-tesla electromagnetic field (EMF) on the biosynthetic activity of bovine articular cartilage. Bovine articular cartilage was obtained from juvenile and adult animals. Whole joints or cartilage explants were subjected to a pulsed 3-tesla EMF; controls were left unexposed. Synthesis of sulfated glycosaminoglycans (sGAGs) was measured by using [35S]sulfate incorporation; mRNA encoding the cartilage markers aggrecan and type II collagen, as well as IL-1β, were analyzed by RT–PCR. Furthermore, effects of the 3-tesla EMF were determined over the course of time directly after exposure (day 0) and at days 3 and 6. In addition, the influence of a 1.5-tesla EMF on cartilage sGAG synthesis was evaluated. Chondrocyte cell death was assessed by staining with Annexin V and TdT-mediated dUTP nick end labelling (TUNEL). Exposure to the EMF resulted in a significant decrease in cartilage macromolecule synthesis. Gene expression of both aggrecan and IL-1β, but not of collagen type II, was reduced in comparison with controls. Staining with Annexin V and TUNEL revealed no evidence of cell death. Interestingly, chondrocytes regained their biosynthetic activity within 3 days after exposure, as shown by proteoglycan synthesis rate and mRNA expression levels. Cartilage samples exposed to a 1.5-tesla EMF remained unaffected. Although MRI devices with a field strength of more than 1.5 T provide a better signal-to-noise ratio and thereby higher spatial resolution, their high field strength impairs the biosynthetic activity of articular chondrocytes in vitro. Although this decrease in biosynthetic activity seems to be transient, articular cartilage exposed to high-energy EMF may become vulnerable to damage. PMID:16831232

Impairment of chondrocyte biosynthetic activity by exposure to 3-tesla high-field magnetic resonance imaging is temporary.

PubMed

Sunk, Ilse-Gerlinde; Trattnig, Siegfried; Graninger, Winfried B; Amoyo, Love; Tuerk, Birgit; Steiner, Carl-Walter; Smolen, Josef S; Bobacz, Klaus

2006-01-01

The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissues is unknown. We investigated the effects of a 3-tesla electromagnetic field (EMF) on the biosynthetic activity of bovine articular cartilage. Bovine articular cartilage was obtained from juvenile and adult animals. Whole joints or cartilage explants were subjected to a pulsed 3-tesla EMF; controls were left unexposed. Synthesis of sulfated glycosaminoglycans (sGAGs) was measured by using [35S]sulfate incorporation; mRNA encoding the cartilage markers aggrecan and type II collagen, as well as IL-1beta, were analyzed by RT-PCR. Furthermore, effects of the 3-tesla EMF were determined over the course of time directly after exposure (day 0) and at days 3 and 6. In addition, the influence of a 1.5-tesla EMF on cartilage sGAG synthesis was evaluated. Chondrocyte cell death was assessed by staining with Annexin V and TdT-mediated dUTP nick end labelling (TUNEL). Exposure to the EMF resulted in a significant decrease in cartilage macromolecule synthesis. Gene expression of both aggrecan and IL-1beta, but not of collagen type II, was reduced in comparison with controls. Staining with Annexin V and TUNEL revealed no evidence of cell death. Interestingly, chondrocytes regained their biosynthetic activity within 3 days after exposure, as shown by proteoglycan synthesis rate and mRNA expression levels. Cartilage samples exposed to a 1.5-tesla EMF remained unaffected. Although MRI devices with a field strength of more than 1.5 T provide a better signal-to-noise ratio and thereby higher spatial resolution, their high field strength impairs the biosynthetic activity of articular chondrocytes in vitro. Although this decrease in biosynthetic activity seems to be transient, articular cartilage exposed to high-energy EMF may become vulnerable to damage.

Effects of overproduced ethylene on the contents of other phytohormones and expression of their key biosynthetic genes.

PubMed

Li, Weiqiang; Nishiyama, Rie; Watanabe, Yasuko; Van Ha, Chien; Kojima, Mikiko; An, Ping; Tian, Lei; Tian, Chunjie; Sakakibara, Hitoshi; Tran, Lam-Son Phan

2018-05-10

Ethylene is involved in regulation of various aspects of plant growth and development. Physiological and genetic analyses have indicated the existence of crosstalk between ethylene and other phytohormones, including auxin, cytokinin (CK), abscisic acid (ABA), gibberellin (GA), salicylic acid (SA), jasmonic acid (JA), brassinosteroid (BR) and strigolactone (SL) in regulation of different developmental processes. However, the effects of ethylene on the biosynthesis and contents of these hormones are not fully understood. Here, we investigated how overproduction of ethylene may affect the contents of other plant hormones using the ethylene-overproducing mutant ethylene-overproducer 1 (eto1-1). The contents of various hormones and transcript levels of the associated biosynthetic genes in the 10-day-old Arabidopsis eto1-1 mutant and wild-type (WT) plants were determined and compared. Higher levels of CK and ABA, while lower levels of auxin, SA and GA were observed in eto1-1 plants in comparison with WT, which was supported by the up- or down-regulation of their biosynthetic genes. Although we could not quantify the BR and SL contents in Arabidopsis, we observed that the transcript levels of the potential rate-limiting BR and SL biosynthetic genes were increased in the eto1-1 versus WT plants, suggesting that BR and SL levels might be enhanced by ethylene overproduction. JA level was not affected by overproduction of ethylene, which might be explained by unaltered expression level of the proposed rate-limiting JA biosynthetic gene allene oxide synthase. Taken together, our results suggest that ET affects the levels of auxin, CK, ABA, SA and GA, and potentially BR and SL, by influencing the expression of genes involved in the rate-limiting steps of their biosynthesis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Plant-derived isoprenoid sweeteners: recent progress in biosynthetic gene discovery and perspectives on microbial production.

PubMed

Seki, Hikaru; Tamura, Keita; Muranaka, Toshiya

2018-06-01

Increased public awareness of negative health effects associated with excess sugar consumption has triggered increasing interest in plant-derived natural sweeteners. Steviol glycosides are a group of highly sweet diterpene glycosides contained in the leaves of stevia (Stevia rebaudiana). Mogrosides, extracted from monk fruit (Siraitia grosvenorii), are a group of cucurbitane-type triterpenoid glycosides. Glycyrrhizin is an oleanane-type triterpenoid glycoside derived from the underground parts of Glycyrrhiza plants (licorice). This review focuses on the natural isoprenoid sweetening agents steviol glycosides, mogrosides, and glycyrrhizin, and describes recent progress in gene discovery and elucidation of the catalytic functions of their biosynthetic enzymes. Recently, remarkable progress has been made in engineering the production of various plant-specialized metabolites in microbial hosts such as Saccharomyces cerevisiae via the introduction of biosynthetic enzyme genes. Perspectives on the microbial production of plant-derived natural sweeteners are also discussed.

Biosynthetic approaches to creating bioactive fungal metabolites: Pathway engineering and activation of secondary metabolism.

PubMed

Motoyama, Takayuki; Osada, Hiroyuki

2016-12-15

The diversity of natural products is greater than that of combinatorial chemistry compounds and is similar to that of drugs. Compounds rich in sp 3 carbons, such as natural products, typically exhibit high structural complexity and high specificity to molecular targets. Microorganisms can synthesize such sp 3 carbon-rich compounds and can be used as excellent factories for making bioactive compounds. Here, we mainly focus on pathway engineering of two sp 3 carbon-rich bioactive indole alkaloids, fumitremorgin C and terpendole E. We also demonstrate the importance of activation of secondary metabolism by focusing on tenuazonic acid, a bioactive tetramic acid compound, as an example. Copyright © 2016 Elsevier Ltd. All rights reserved.

Spatially organizing biochemistry: choosing a strategy to translate synthetic biology to the factory.

PubMed

Jakobson, Christopher M; Tullman-Ercek, Danielle; Mangan, Niall M

2018-05-29

Natural biochemical systems are ubiquitously organized both in space and time. Engineering the spatial organization of biochemistry has emerged as a key theme of synthetic biology, with numerous technologies promising improved biosynthetic pathway performance. One strategy, however, may produce disparate results for different biosynthetic pathways. We use a spatially resolved kinetic model to explore this fundamental design choice in systems and synthetic biology. We predict that two example biosynthetic pathways have distinct optimal organization strategies that vary based on pathway-dependent and cell-extrinsic factors. Moreover, we demonstrate that the optimal design varies as a function of kinetic and biophysical properties, as well as culture conditions. Our results suggest that organizing biosynthesis has the potential to substantially improve performance, but that choosing the appropriate strategy is key. The flexible design-space analysis we propose can be adapted to diverse biosynthetic pathways, and lays a foundation to rationally choose organization strategies for biosynthesis.

Discovery of a Phosphonoacetic Acid Derived Natural Product by Pathway Refactoring.

PubMed

Freestone, Todd S; Ju, Kou-San; Wang, Bin; Zhao, Huimin

2017-02-17

The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules. Although methods exist to elicit secondary metabolite gene clusters in native hosts, they require the strain to be amenable to genetic manipulation. One method to circumvent this is pathway refactoring, which we implemented in an effort to discover new phosphonic acids from a gene cluster from Streptomyces sp. strain NRRL F-525. By reengineering this cluster for expression in the production host Streptomyces lividans, utility of refactoring is demonstrated with the isolation of a novel phosphonic acid, O-phosphonoacetic acid serine, and the characterization of its biosynthesis. In addition, a new biosynthetic branch point is identified with a phosphonoacetaldehyde dehydrogenase, which was used to identify additional phosphonic acid gene clusters that share phosphonoacetic acid as an intermediate.

Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli

PubMed Central

Cheol Kim, Seong; Eun Min, Byung; Gyu Hwang, Hyun; Woo Seo, Sang; Yeol Jung, Gyoo

2015-01-01

L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5′-untranslated region (5′-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5′-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria. PMID:26346938

Fumonisin-nonproducing mutants exhibit differential expression of putative polyketide biosynthetic gene clusters in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

The maize pathogen Fusarium verticillioides produces a group of polyketide derived secondary metabolites called fumonisins. Fumonisins can cause diseases in animals, and have been correlated epidemiologically with esophageal cancer and birth defects in humans. The fumonisin biosynthetic gene clust...

Streptomyces scabies 87-22 contains a coronafacic acid-like biosynthetic cluster that contributes to plant-microbe interactions.

PubMed

Bignell, Dawn R D; Seipke, Ryan F; Huguet-Tapia, José C; Chambers, Alan H; Parry, Ronald J; Loria, Rosemary

2010-02-01

Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

IMG-ABC. A knowledge base to fuel discovery of biosynthetic gene clusters and novel secondary metabolites

DOE PAGES

Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; ...

2015-07-14

In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG’s comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve asmore » the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC’s focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in lphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG’s extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG

IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites.

PubMed

Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Szeto, Ernest; Huang, Jinghua; Reddy, T B K; Cimermančič, Peter; Fischbach, Michael A; Ivanova, Natalia N; Markowitz, Victor M; Kyrpides, Nikos C; Pati, Amrita

2015-07-14

In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of "big" genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to

Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis

PubMed Central

2013-01-01

Background The antifungal therapy caspofungin is a semi-synthetic derivative of pneumocandin B0, a lipohexapeptide produced by the fungus Glarea lozoyensis, and was the first member of the echinocandin class approved for human therapy. The nonribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) gene cluster responsible for pneumocandin biosynthesis from G. lozoyensis has not been elucidated to date. In this study, we report the elucidation of the pneumocandin biosynthetic gene cluster by whole genome sequencing of the G. lozoyensis wild-type strain ATCC 20868. Results The pneumocandin biosynthetic gene cluster contains a NRPS (GLNRPS4) and a PKS (GLPKS4) arranged in tandem, two cytochrome P450 monooxygenases, seven other modifying enzymes, and genes for L-homotyrosine biosynthesis, a component of the peptide core. Thus, the pneumocandin biosynthetic gene cluster is significantly more autonomous and organized than that of the recently characterized echinocandin B gene cluster. Disruption mutants of GLNRPS4 and GLPKS4 no longer produced the pneumocandins (A0 and B0), and the Δglnrps4 and Δglpks4 mutants lost antifungal activity against the human pathogenic fungus Candida albicans. In addition to pneumocandins, the G. lozoyensis genome encodes a rich repertoire of natural product-encoding genes including 24 PKSs, six NRPSs, five PKS-NRPS hybrids, two dimethylallyl tryptophan synthases, and 14 terpene synthases. Conclusions Characterization of the gene cluster provides a blueprint for engineering new pneumocandin derivatives with improved pharmacological properties. Whole genome estimation of the secondary metabolite-encoding genes from G. lozoyensis provides yet another example of the huge potential for drug discovery from natural products from the fungal kingdom. PMID:23688303

Unravelling the architecture and dynamics of tropane alkaloid biosynthesis pathways using metabolite correlation networks.

PubMed

Nguyen, Thi-Kieu-Oanh; Jamali, Arash; Lanoue, Arnaud; Gontier, Eric; Dauwe, Rebecca

2015-08-01

The tropane alkaloid spectrum in Solanaceae is highly variable within and between species. Little is known about the topology and the coordination of the biosynthetic pathways leading to the variety of tropine and pseudotropine derived esters in the alkaloid spectrum, or about the metabolic dynamics induced by tropane alkaloid biosynthesis stimulating conditions. A good understanding of the metabolism, including all ramifications, is however necessary for the development of strategies to increase the abundance of pharmacologically interesting compounds such as hyoscyamine and scopolamine. The present study explores the tropane alkaloid metabolic pathways in an untargeted approach involving a correlation-based network analysis. Using GC-MS metabolite profiling, the variation and co-variation among tropane alkaloids and primary metabolites was monitored in 60 Datura innoxia Mill. individuals, of which half were exposed to tropane alkaloid biosynthesis stimulating conditions by co-culture with Agrobacterium rhizogenes. Considerable variation was evident in the relative proportions of the tropane alkaloids. Remodeling of the tropane alkaloid spectrum under co-culture with A. rhizogenes involved a specific and strong increase of hyoscyamine production and revealed that the accumulation of hyoscyamine, 3-tigloyloxy-6,7-epoxytropane, and 3-methylbutyryloxytropane was controlled independently of the majority of tropane alkaloids. Based on correlations between metabolites, we propose a biosynthetic origin of hygrine, the order of esterification of certain di-oxygenated tropanes, and that the rate of acetoxylation contributes to control of hyoscyamine production. Overall, this study shows that the biosynthesis of tropane alkaloids may be far more complex and finely controlled than previously expected. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression Profiling of Regulatory and Biosynthetic Genes in Contrastingly Anthocyanin Rich Strawberry (Fragaria × ananassa) Cultivars Reveals Key Genetic Determinants of Fruit Color.

PubMed

Hossain, Mohammad Rashed; Kim, Hoy-Taek; Shanmugam, Ashokraj; Nath, Ujjal Kumar; Goswami, Gayatri; Song, Jae-Young; Park, Jong-In; Nou, Ill-Sup

2018-02-26

Anthocyanins are the resultant end-point metabolites of phenylapropanoid/flavonoid (F/P) pathway which is regulated at transcriptional level via a series of structural genes. Identifying the key genes and their potential interactions can provide us with the clue for novel points of intervention for improvement of the trait in strawberry. We profiled the expressions of putative regulatory and biosynthetic genes of cultivated strawberry in three developmental and characteristically colored stages of fruits of contrastingly anthocyanin rich cultivars: Tokun, Maehyang and Soelhyang. Besides FaMYB10, a well-characterized positive regulator, FaMYB5 , FabHLH3 and FabHLH3-delta might also act as potential positive regulators, while FaMYB11 , FaMYB9 , FabHLH33 and FaWD44-1 as potential negative regulators of anthocyanin biosynthesis in these high-anthocyanin cultivars. Among the early BGs, Fa4CL7 , FaF3H , FaCHI1 , FaCHI3 , and FaCHS, and among the late BGs, FaDFR4-3 , FaLDOX , and FaUFGT2 showed significantly higher expression in ripe fruits of high anthocyanin cultivars Maehyang and Soelhyang. Multivariate analysis revealed the association of these genes with total anthocyanins. Increasingly higher expressions of the key genes along the pathway indicates the progressive intensification of pathway flux leading to final higher accumulation of anthocyanins. Identification of these key genetic determinants of anthocyanin regulation and biosynthesis in Korean cultivars will be helpful in designing crop improvement programs.

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

PubMed Central

Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; Niedzwiedzki, Dariusz M.; Zhang, Hao; Qian, Pu; Vasilev, Cvetelin; Grayson, Katie J.; Jackson, Philip J.; Martin, Elizabeth C.; Li, Ying; Holten, Dewey; Neil Hunter, C.

2015-01-01

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon–carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N = 10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2′-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC–LH1–PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2′-diketo-spirilloxanthin (15 conjugated C 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 C bonds; N = 15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N = 9; 94%), spheroidene (N = 10; 96%) and spheroidenone (N = 11; 95%), whereas intermediate values were measured for lycopene (N = 11; 64%), rhodopin (N = 11; 62%) and spirilloxanthin (N = 13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoeamore » agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated CC bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). In conclusion, the variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.« less

Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway

DOE PAGES

Chi, Shuang C.; Mothersole, David J.; Dilbeck, Preston; ...

2014-10-27

Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoeamore » agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated CC bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). In conclusion, the variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.« less

Genome wide transcriptome profiling reveals differential gene expression in secondary metabolite pathway of Cymbopogon winterianus.

PubMed

Devi, Kamalakshi; Mishra, Surajit K; Sahu, Jagajjit; Panda, Debashis; Modi, Mahendra K; Sen, Priyabrata

2016-02-15

Advances in transcriptome sequencing provide fast, cost-effective and reliable approach to generate large expression datasets especially suitable for non-model species to identify putative genes, key pathway and regulatory mechanism. Citronella (Cymbopogon winterianus) is an aromatic medicinal grass used for anti-tumoral, antibacterial, anti-fungal, antiviral, detoxifying and natural insect repellent properties. Despite of having number of utilities, the genes involved in terpenes biosynthetic pathway is not yet clearly elucidated. The present study is a pioneering attempt to generate an exhaustive molecular information of secondary metabolite pathway and to increase genomic resources in Citronella. Using high-throughput RNA-Seq technology, root and leaf transcriptome was analysed at an unprecedented depth (11.7 Gb). Targeted searches identified majority of the genes associated with metabolic pathway and other natural product pathway viz. antibiotics synthesis along with many novel genes. Terpenoid biosynthesis genes comparative expression results were validated for 15 unigenes by RT-PCR and qRT-PCR. Thus the coverage of these transcriptome is comprehensive enough to discover all known genes of major metabolic pathways. This transcriptome dataset can serve as important public information for gene expression, genomics and function genomics studies in Citronella and shall act as a benchmark for future improvement of the crop.

Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae

PubMed Central

2011-01-01

Microalgae have presented themselves as a strong candidate to replace diminishing oil reserves as a source of lipids for biofuels. Here we describe successful modifications of terrestrial plant lipid content which increase overall lipid production or shift the balance of lipid production towards lipid varieties more useful for biofuel production. Our discussion ranges from the biosynthetic pathways and rate limiting steps of triacylglycerol formation to enzymes required for the formation of triacylglycerol containing exotic lipids. Secondarily, we discuss techniques for genetic engineering and modification of various microalgae which can be combined with insights gained from research in higher plants to aid in the creation of production strains of microalgae. PMID:22047615

Characterization and developmental expression of genes encoding the early carotenoid biosynthetic enzymes in Citrus paradisi Macf.

PubMed

Costa, Marcio G C; Moreira, Cristina D; Melton, John R; Otoni, Wagner C; Moore, Gloria A

2012-02-01

In the present study, the full-length cDNA sequences of PSY, PDS, and ZDS, encoding the early carotenoid biosynthetic enzymes in the carotenoid pathway of grapefruit (Citrus paradisi), were isolated and characterized for the first time. CpPSY contained a 1311-bp open reading frame (ORF) encoding a polypeptide of 436 amino acids, CpPDS contained a 1659-bp ORF encoding a polypeptide of 552 amino acids, and CpZDS contained a 1713-bp ORF encoding a polypeptide of 570 amino acids. Phylogenetic analysis indicated that CpPSY shares homology with PSYs from Citrus, tomato, pepper, Arabidopsis, and the monocot PSY1 group, while CpPDS and CpZDS are most closely related to orthologs from Citrus and tomato. Expression analysis revealed fluctuations in CpPSY, CpPDS, and CpZDS transcript abundance and a non-coordinated regulation between the former and the two latter genes during fruit development in albedo and juice vesicles of white ('Duncan') and red ('Flame') grapefruits. A 3× higher upregulation of CpPSY expression in juice vesicles of red-fleshed 'Flame' as compared to white-fruited 'Duncan' was observed in the middle stages of fruit development, which correlates with the well documented accumulation pattern of lycopene in red grapefruit. Together with previous data, our results suggest that the primary mechanism controlling lycopene accumulation in red grapefruit involves the transcriptional upregulation of CpPSY, which controls the flux into the carotenoid pathway, and the downregulated expression of CpLCYB2, which controls the step of cyclization of lycopene in chromoplasts during fruit ripening. A correlation between CpPSY expression and fruit color evolution in red grapefruit is demonstrated.

Ultraviolet Radiation-Elicited Enhancement of Isoflavonoid Accumulation, Biosynthetic Gene Expression, and Antioxidant Activity in Astragalus membranaceus Hairy Root Cultures.

PubMed

Jiao, Jiao; Gai, Qing-Yan; Wang, Wei; Luo, Meng; Gu, Cheng-Bo; Fu, Yu-Jie; Ma, Wei

2015-09-23

In this work, Astragalus membranaceus hairy root cultures (AMHRCs) were exposed to ultraviolet radiation (UV-A, UV-B, and UV-C) for promoting isoflavonoid accumulation. The optimum enhancement for isoflavonoid production was achieved in 34-day-old AMHRCs elicited by 86.4 kJ/m(2) of UV-B. The resulting isoflavonoid yield was 533.54 ± 13.61 μg/g dry weight (DW), which was 2.29-fold higher relative to control (232.93 ± 3.08 μg/g DW). UV-B up-regulated the transcriptional expressions of all investigated genes involved in isoflavonoid biosynthetic pathway. PAL and C4H were found to be two potential key genes that controlled isoflavonoid biosynthesis. Moreover, a significant increase was noted in antioxidant activity of extracts from UV-B-elicited AMHRCs (IC50 values = 0.85 and 1.08 mg/mL) in comparison with control (1.38 and 1.71 mg/mL). Overall, this study offered a feasible elicitation strategy to enhance isoflavonoid accumulation in AMHRCs and also provided a basis for metabolic engineering of isoflavonoid biosynthesis in the future.

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin.

PubMed

Bernstein, Jeffrey R; Bulter, Thomas; Liao, James C

2008-01-01

This work demonstrates the transfer of the five-gene cyclohexane carboxylate (CHC) degradation pathway from the high-GC alphaproteobacterium Rhodopseudomonas palustris to Escherichia coli, a gammaproteobacterium. The degradation product of this pathway is pimeloyl-CoA, a key metabolite in E. coli's biotin biosynthetic pathway. This pathway is useful for biotin overproduction in E. coli; however, the expression of GC-rich genes is troublesome in this host. When the native R. palustris CHC degradation pathway is transferred to a DeltabioH pimeloyl-CoA auxotroph of E. coli, it is unable to complement growth in the presence of CHC. To overcome this expression problem we redesigned the operon with decreased GC content and removed stretches of high-GC intergenic DNA which comprise the 5' untranslated region of each gene, replacing these features with shorter low-GC sequences. We show this synthetic construct enables growth of the DeltabioH strain in the presence of CHC. When the synthetic degradation pathway is overexpressed in conjunction with the downstream genes for biotin biosynthesis, we measured significant accumulation of biotin in the growth medium, showing that the pathway transfer is successfully integrated with the host metabolism.

Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data.

PubMed

Kawasaki, Regiane; Baraúna, Rafael A; Silva, Artur; Carepo, Marta S P; Oliveira, Rui; Marques, Rodolfo; Ramos, Rommel T J; Schneider, Maria P C

2016-01-01

Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.

Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data

PubMed Central

Kawasaki, Regiane; Carepo, Marta S. P.; Oliveira, Rui; Marques, Rodolfo; Ramos, Rommel T. J.; Schneider, Maria P. C.

2016-01-01

Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments. PMID:27595107

Mono- and Dialkyl Glycerol Ether Lipids in Anaerobic Bacteria: Biosynthetic Insights from the Mesophilic Sulfate Reducer Desulfatibacillum alkenivorans PF2803T

PubMed Central

Mollex, Damien; Vinçon-Laugier, Arnauld; Hakil, Florence; Pacton, Muriel; Cravo-Laureau, Cristiana

2015-01-01

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1′-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1′-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria. PMID:25724965

High GC Content Cas9-Mediated Genome-Editing and Biosynthetic Gene Cluster Activation in Saccharopolyspora erythraea.

PubMed

Liu, Yong; Wei, Wen-Ping; Ye, Bang-Ce

2018-05-18

The overexpression of bacterial secondary metabolite biosynthetic enzymes is the basis for industrial overproducing strains. Genome editing tools can be used to further improve gene expression and yield. Saccharopolyspora erythraea produces erythromycin, which has extensive clinical applications. In this study, the CRISPR-Cas9 system was used to edit genes in the S. erythraea genome. A temperature-sensitive plasmid containing the PermE promoter, to drive Cas9 expression, and the Pj23119 and PkasO promoters, to drive sgRNAs, was designed. Erythromycin esterase, encoded by S. erythraea SACE_1765, inactivates erythromycin by hydrolyzing the macrolactone ring. Sequencing and qRT-PCR confirmed that reporter genes were successfully inserted into the SACE_1765 gene. Deletion of SACE_1765 in a high-producing strain resulted in a 12.7% increase in erythromycin levels. Subsequent PermE- egfp knock-in at the SACE_0712 locus resulted in an 80.3% increase in erythromycin production compared with that of wild type. Further investigation showed that PermE promoter knock-in activated the erythromycin biosynthetic gene clusters at the SACE_0712 locus. Additionally, deletion of indA (SACE_1229) using dual sgRNA targeting without markers increased the editing efficiency to 65%. In summary, we have successfully applied Cas9-based genome editing to a bacterial strain, S. erythraea, with a high GC content. This system has potential application for both genome-editing and biosynthetic gene cluster activation in Actinobacteria.

In Vitro Reconstitution of Metabolic Pathways: Insights into Nature’s Chemical Logic

PubMed Central

Lowry, Brian; Walsh, Christopher T.

2015-01-01

In vitro analysis of metabolic pathways is becoming a powerful method to gain a deeper understanding of Nature’s core biochemical transformations. With astounding advancements in biotechnology, purification of a metabolic pathway’s constitutive enzymatic components is becoming a tractable problem, and such in vitro studies allow scientists to capture the finer details of enzymatic reaction mechanisms, kinetics, and the identity of organic product molecules. In this review, we present eleven metabolic pathways that have been the subject of in vitro reconstitution studies in the literature in recent years. In addition, we have selected and analyzed subset of four case studies within these eleven examples that exemplify remarkable organic chemistry occurring within biology. These examples serves as tangible reminders that Nature’s biochemical routes obey the fundamental principles of organic chemistry, and the chemical mechanisms are reminiscent of those featured in traditional synthetic organic routes. The illustrations of biosynthetic chemistry depicted in this review may inspire the development of biomimetic chemistries via abiotic chemical techniques. PMID:26207083

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

PubMed Central

Yoshida, Kiyohito; Hashimoto, Mikako; Hori, Ryuji; Adachi, Takumi; Okuyama, Hidetoshi; Orikasa, Yoshitake; Nagamine, Tadashi; Shimizu, Satoru; Ueno, Akio; Morita, Naoki

2016-01-01

The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed. PMID:27187420

Characterization of the fumonisin B2 biosynthetic gene cluster in Aspergillus niger and A. awamori.

USDA-ARS?s Scientific Manuscript database

Aspergillus niger and A. awamori strains isolated from grapes cultivated in Mediterranean basin were examined for fumonisin B2 (FB2) production and presence/absence of sequences within the fumonisin biosynthetic gene (fum) cluster. Presence of 13 regions in the fum cluster was evaluated by PCR assay...

Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux.

PubMed

Ost, Mario; Keipert, Susanne; van Schothorst, Evert M; Donner, Verena; van der Stelt, Inge; Kipp, Anna P; Petzke, Klaus-Jürgen; Jove, Mariona; Pamplona, Reinald; Portero-Otin, Manuel; Keijer, Jaap; Klaus, Susanne

2015-04-01

Recent studies on mouse and human skeletal muscle (SM) demonstrated the important link between mitochondrial function and the cellular metabolic adaptation. To identify key compensatory molecular mechanisms in response to chronic mitochondrial distress, we analyzed mice with ectopic SM respiratory uncoupling in uncoupling protein 1 transgenic (UCP1-TG) mice as model of muscle-specific compromised mitochondrial function. Here we describe a detailed metabolic reprogramming profile associated with mitochondrial perturbations in SM, triggering an increased protein turnover and amino acid metabolism with induced biosynthetic serine/1-carbon/glycine pathway and the longevity-promoting polyamine spermidine as well as the trans-sulfuration pathway. This is related to an induction of NADPH-generating pathways and glutathione metabolism as an adaptive mitohormetic response and defense against increased oxidative stress. Strikingly, consistent muscle retrograde signaling profiles were observed in acute stress states such as muscle cell starvation and lipid overload, muscle regeneration, and heart muscle inflammation, but not in response to exercise. We provide conclusive evidence for a key compensatory stress-signaling network that preserves cellular function, oxidative stress tolerance, and survival during conditions of increased SM mitochondrial distress, a metabolic reprogramming profile so far only demonstrated for cancer cells and heart muscle. © FASEB.

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

PubMed Central

Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H.; Møller, Birger Lindberg; Rook, Fred

2016-01-01

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters. PMID:27841372

Expanding the Bioactive Chemical Space of Anthrabenzoxocinones through Engineering the Highly Promiscuous Biosynthetic Modification Steps.

PubMed

Mei, Xianyi; Yan, Xiaoli; Zhang, Hui; Yu, Mingjia; Shen, Guangqing; Zhou, Linjun; Deng, Zixin; Lei, Chun; Qu, Xudong

2018-01-19

Anthrabenzoxocinones (ABXs) including (-)-ABXs and (+)-ABXs are a group of bacterial FabF-specific inhibitors with potent antimicrobial activity of resistant strains. Optimization of their chemical structures is a promising method to develop potent antibiotics. Through biosynthetic investigation, we herein identified and characterized two highly promiscuous enzymes involved in the (-)-ABX structural modification. The promiscuous halogenase and methyltransferase can respectively introduce halogen-modifications into various positions of the ABX scaffolds and methylation to highly diverse substrates. Manipulation of their activity in both of the (-)-ABXs and (+)-ABXs biosyntheses led to the generation of 14 novel ABX analogues of both enantiomers. Bioactivity assessment revealed that a few of the analogues showed significantly improved antimicrobial activity, with the C3-hydroxyl and chlorine substitutions critical for their activity. This study enormously expands the bioactive chemical space of the ABX family and FabF-specific inhibitors. The disclosed broad-selective biosynthetic machineries and structure-activity relationship provide a solid basis for further generation of potent antimicrobial agents.

Controlling cell-free metabolism through physiochemical perturbations.

PubMed

Karim, Ashty S; Heggestad, Jacob T; Crowe, Samantha A; Jewett, Michael C

2018-01-01

Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore

RiboFACSeq: A new method for investigating metabolic and transport pathways in bacterial cells by combining a riboswitch-based sensor, fluorescence-activated cell sorting and next-generation sequencing

PubMed Central

Li, Yingfu

2017-01-01

The elucidation of the cellular processes involved in vitamin and cofactor biosynthesis is a challenging task. The conventional approaches to these investigations rely on the discovery and purification of the products (i.e proteins and metabolites) of a particular transport or biosynthetic pathway, prior to their subsequent analysis. However, the purification of low-abundance proteins or metabolites is a formidable undertaking that presents considerable technical challenges. As a solution, we present an alternative approach to such studies that circumvents the purification step. The proposed approach takes advantage of: (1) the molecular detection capabilities of a riboswitch-based sensor to detect the cellular levels of its cognate molecule, as a means to probe the integrity of the transport and biosynthetic pathways of the target molecule in cells, (2) the high-throughput screening ability of fluorescence-activated cell sorters to isolate cells in which only these specific pathways are disrupted, and (3) the ability of next-generation sequencing to quickly identify the genes of the FACS-sorted populations. This approach was named “RiboFACSeq”. Following their identification by RiboFACSeq, the role of these genes in the presumed pathway needs to be verified through appropriate functional assays. To demonstrate the utility of our approach, an adenosylcobalamin (AdoCbl)-responsive riboswitch-based sensor was used in this study to demonstrate that RiboFACSeq can be used to track and sort cells carrying genetic mutations in known AdoCbl transport and biosynthesis genes with desirable sensitivity and specificity. This method could potentially be used to elucidate any pathway of interest, as long as a suitable riboswitch-based sensor can be created. We believe that RiboFACSeq would be especially useful for the elucidation of biological pathways in which the proteins and/or their metabolites are present at very low physiological concentrations in cells, as is the

Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges

USDA-ARS?s Scientific Manuscript database

Carotenoid accumulation and biosynthetic gene expression levels during fruit maturation were compared between ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV). The two cultivars exhibited different carotenoid profiles and regulatory mechanisms in flavedo and...

A new member of the 4-methylideneimidazole-5-one–containing aminomutase family from the enediyne kedarcidin biosynthetic pathway

PubMed Central

Huang, Sheng-Xiong; Lohman, Jeremy R.; Huang, Tingting; Shen, Ben

2013-01-01

4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of l-α-amino acids to β-amino acids with either an (R) or an (S) configuration. l-Phenylalanine and l-tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an (R)-2-aza-3-chloro-β-tyrosine moiety reminiscent of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for (R)-2-aza-3-chloro-β-tyrosine biosynthesis starting from 2-aza-l-tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza-l-tyrosine to (R)-2-aza-β-tyrosine, exhibiting no detectable activity toward 2-aza-l-phenylalanine or l-tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of l-tyrosine to (S)-β-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza-l-tyrosine as an alternative substrate but generating (S)-2-aza-β-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity. PMID:23633564

A new member of the 4-methylideneimidazole-5-one-containing aminomutase family from the enediyne kedarcidin biosynthetic pathway.

PubMed

Huang, Sheng-Xiong; Lohman, Jeremy R; Huang, Tingting; Shen, Ben

2013-05-14

4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of L-α-amino acids to β-amino acids with either an (R) or an (S) configuration. L-phenylalanine and L-tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an (R)-2-aza-3-chloro-β-tyrosine moiety reminiscent of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for (R)-2-aza-3-chloro-β-tyrosine biosynthesis starting from 2-aza-L-tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza-L-tyrosine to (R)-2-aza-β-tyrosine, exhibiting no detectable activity toward 2-aza-L-phenylalanine or L-tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of L-tyrosine to (S)-β-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza-L-tyrosine as an alternative substrate but generating (S)-2-aza-β-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity.

Biosynthetic Potential of Phylogenetically Unique Endophytic Actinomycetes from Tropical Plants▿ †

PubMed Central

Janso, Jeffrey E.; Carter, Guy T.

2010-01-01

The culturable diversity of endophytic actinomycetes associated with tropical, native plants is essentially unexplored. In this study, 123 endophytic actinomycetes were isolated from tropical plants collected from several locations in Papua New Guinea and Mborokua Island, Solomon Islands. Isolates were found to be prevalent in roots but uncommon in leaves. Initially, isolates were dereplicated to the strain level by ribotyping. Subsequent characterization of 105 unique strains by 16S rRNA gene sequence analysis revealed that 17 different genera were represented, and rare genera, such as Sphaerisporangium and Planotetraspora, which have never been previously reported to be endophytic, were quite prevalent. Phylogenetic analyses grouped many of the strains into clades distinct from known genera within Thermomonosporaceae and Micromonosporaceae, indicating that they may be unique genera. Bioactivity testing and liquid chromatography-mass spectrometry (LC-MS) profiling of crude fermentation extracts were performed on 91 strains. About 60% of the extracts exhibited bioactivity or displayed LC-MS profiles with spectra indicative of secondary metabolites. The biosynthetic potential of 29 nonproductive strains was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. Despite their lack of detectable secondary metabolite production in fermentation, most were positive for type I (66%) and type II (79%) PKS genes, and all were positive for NRPS genes. These results suggest that tropical plants from New Guinea and the adjacent archipelago are hosts to unique endophytic actinomycetes that possess significant biosynthetic potential. PMID:20472734

Antimicrobial Activity and Cell Selectivity of Synthetic and Biosynthetic Cationic Polymers

PubMed Central

Venkatesh, Mayandi; Barathi, Veluchamy Amutha; Goh, Eunice Tze Leng; Anggara, Raditya; Fazil, Mobashar Hussain Urf Turabe; Ng, Alice Jie Ying; Harini, Sriram; Aung, Thet Tun; Fox, Stephen John; Liu, Shouping; Barkham, Timothy Mark Sebastian; Loh, Xian Jun

2017-01-01

ABSTRACT The mammalian and microbial cell selectivity of synthetic and biosynthetic cationic polymers has been investigated. Among the polymers with peptide backbones, polymers containing amino side chains display greater antimicrobial activity than those with guanidine side chains, whereas ethylenimines display superior activity over allylamines. The biosynthetic polymer ε-polylysine (εPL) is noncytotoxic to primary human dermal fibroblasts at concentrations of up to 2,000 μg/ml, suggesting that the presence of an isopeptide backbone has greater cell selectivity than the presence of α-peptide backbones. Both εPL and linear polyethylenimine (LPEI) exhibit bactericidal properties by depolarizing the cytoplasmic membrane and disrupt preformed biofilms. εPL displays broad-spectrum antimicrobial properties against antibiotic-resistant Gram-negative and Gram-positive strains and fungi. εPL elicits rapid bactericidal activity against both Gram-negative and Gram-positive bacteria, and its biocompatibility index is superior to those of cationic antiseptic agents and LPEI. εPL does not interfere with the wound closure of injured rabbit corneas. In a rabbit model of bacterial keratitis, the topical application of εPL (0.3%, wt/vol) decreases the bacterial burden and severity of infections caused by Pseudomonas aeruginosa and Staphylococcus aureus strains. In vivo imaging studies confirm that εPL-treated corneas appeared transparent and nonedematous compared to untreated infected corneas. Taken together, our results highlight the potential of εPL in resolving topical microbial infections. PMID:28784676

The Auxin Biosynthetic TRYPTOPHAN AMINOTRANSFERASE RELATED TaTAR2.1-3A Increases Grain Yield of Wheat.

PubMed

Shao, An; Ma, Wenying; Zhao, Xueqiang; Hu, Mengyun; He, Xue; Teng, Wan; Li, Hui; Tong, Yiping

2017-08-01

Controlling the major auxin biosynthetic pathway to manipulate auxin content could be a target for genetic engineering of crops with desired traits, but little progress had been made because low or high auxin contents often cause developmental inhibition. Here, we performed a genome-wide analysis of bread wheat ( Triticum aestivum ) to identify the Tryptophan Aminotransferase of Arabidopsis1/Tryptophan Aminotransferase-Related (TAA1/TAR) genes that function in the tryptophan-dependent pathway of auxin biosynthesis. Sequence mining together with gene cloning identified 15 TaTAR genes, among which 12 and three genes were phylogenetically close to Arabidopsis ( Arabidopsis thaliana ) AtTAR2 and AtTAR3, respectively. TaTAR2.1 had the most abundant transcripts in the TaTAR2 genes and was expressed mainly in roots and up-regulated by low nitrogen (N) availability. Knockdown of TaTAR2.1 caused vegetative and reproductive deficiencies and impaired lateral root (LR) growth under both high- and low-N conditions. Overexpressing TaTAR2.1-3A in wheat enhanced LR branching, plant height, spike number, grain yield, and aerial N accumulation under different N supply levels. In addition, overexpressing TaTAR2.1-3A in Arabidopsis elevated auxin accumulation in the primary root tip, LR tip, LR primordia, and cotyledon and hypocotyl and increased primary root length, visible LR number, and shoot fresh weight under high- and low-N conditions. Our results indicate that TaTAR2.1 is critical for wheat growth and also shows potential for genetic engineering to reach the aim of improving the grain yield of wheat. © 2017 American Society of Plant Biologists. All Rights Reserved.

Complete genome sequence of the bacteriochlorophyll a-containing Roseibacterium elongatum type strain (DSM 19469T), a representative of the Roseobacter group isolated from Australian coast sand

PubMed Central

Riedel, Thomas; Fiebig, Anne; Göker, Markus; Klenk, Hans-Peter

2014-01-01

Roseibacterium elongatum Suzuki et al. 2006 is a pink-pigmented and bacteriochlorophyll a-producing representative of the Roseobacter group within the alphaproteobacterial family Rhodobacteraceae. Representatives of the marine ‘Roseobacter group’ were found to be abundant in the ocean and play an important role in global and biogeochemical processes. In the present study we describe the features of R. elongatum strain OCh 323T together with its genome sequence and annotation. The 3,555,102 bp long genome consists of one circular chromosome with no extrachromosomal elements and is one of the smallest known Roseobacter genomes. It contains 3,540 protein-coding genes and 59 RNA genes. Genome analysis revealed the presence of a photosynthetic gene cluster, which putatively enables a photoheterotrophic lifestyle. Gene sequences associated with quorum sensing, motility, surface attachment, and thiosulfate and carbon monoxide oxidation could be detected. The genome was sequenced as part of the activities of the Transregional Collaborative Research Centre 51 (TRR51) funded by the German Research Foundation (DFG). PMID:25197467

Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis.

PubMed

Anderson, Donald D; Woeller, Collynn F; Chiang, En-Pei; Shane, Barry; Stover, Patrick J

2012-03-02

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.

Genome Engineering of the 2,3-Butanediol Biosynthetic Pathway for Tight Regulation in Cyanobacteria.

PubMed

Nozzi, Nicole E; Atsumi, Shota

2015-11-20

Cyanobacteria have gained popularity among the metabolic engineering community as a tractable photosynthetic host for renewable chemical production. However, though a number of successfully engineered production systems have been reported, long-term genetic stability remains an issue for cyanobacterial systems. The genetic engineering toolbox for cyanobacteria is largely lacking inducible systems for expression control. The characterization of tight regulation systems for use in cyanobacteria may help to alleviate this problem. In this work we explore the function of the IPTG inducible promoter P(L)lacO1 in the model cyanobacterium Synechococcus elongatus PCC 7942 as well as the effect of gene order within an operon on pathway expression. According to our experiments, P(L)lacO1 functions well as an inducible promoter in S. elongatus. Additionally, we found that gene order within an operon can strongly influence control of expression of each gene.

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

PubMed Central

2012-01-01

Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants. PMID:22883984

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia.

PubMed

Zuiter, Afnan Saeid; Sawwan, Jammal; Al Abdallat, Ayed

2012-08-10

Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining.

PubMed

Tang, Xiaoyu; Li, Jie; Millán-Aguiñaga, Natalie; Zhang, Jia Jia; O'Neill, Ellis C; Ugalde, Juan A; Jensen, Paul R; Mantovani, Simone M; Moore, Bradley S

2015-12-18

Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs and (ii) how to rapidly connect genes to biosynthesized small molecules. Here, we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes colocalized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and autoresistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis.

Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

PubMed

Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

2016-09-01

Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Studies Towards the Leucetta-derived Alkaloids Spirocalcaridine A and B - Possible Biosynthetic Implications.

PubMed

Koswatta, Panduka B; Das, Jayanta; Yousufuddin, Muhammed; Lovely, Carl J

2015-04-01

An exploration of an abiotic approach to spirocalcaridines A and B is described centered on electrophile-induced dearomatizing spirocyclization of aryl enyne derivatives. Elaboration of the α-iodoenone via an Ullmann-like, copper-catalyzed amidation provided a formamide which upon treatment with methylamine undergoes a dienol-arene rearrangement, providing the corresponding kealiinine-like framework. This observation suggests a possible biosynthetic links between the spirocalcaridines and the naphthimidazole group of Leucetta alkaloids.

Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

USDA-ARS?s Scientific Manuscript database

Biosynthesis of ecdysteroids involves sequential enzymatic hydroxylations by microsomal enzymes and mitochondrial cytochrome P450’s. Enzymes of the pathway are collectively known as Halloween genes. Complete sequences for three Halloween genes, spook (Vdspo), disembodied (Vddib) and shade (Vdshd), w...

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants

DOE PAGES

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan; ...

2017-04-01

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we will need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can bemore » used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.« less

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schläpfer, Pascal; Zhang, Peifen; Wang, Chuan

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we will need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can bemore » used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.« less

Two pathways for pyrrole formation in coumermycin A(1) biosynthesis: the central pyrrole moiety is formed from L-threonine.

PubMed

Siebenberg, Stefanie; Burkard, Nadja; Knuplesch, Anna; Gust, Bertolt; Grond, Stephanie; Heide, Lutz

2011-11-25

Coumermycin A(1) is an aminocoumarin antibiotic produced by Streptomyces rishiriensis. It contains three pyrrole rings, that is, two terminal 5-methyl-pyrrole-2-carboxyl moieties and a central 3-methylpyrrole-2,4-dicarboxylic acid moiety. The biosynthesis of the terminal pyrrole moieties has been elucidated previously. However, the biosynthetic precursors of the central pyrrole moiety have remained unknown, and none of the genes or enzymes involved in its formation has been identified. We now show that five genes, contained in a contiguous 4.7 kb region within the coumermycin biosynthetic gene cluster, are required for the biosynthesis of this central pyrrole moiety. Each of these genes was deleted individually, resulting in a strong reduction or an abolishment of coumermycin production. External feeding of the central pyrrole moiety restored coumermycin production. One of these genes shows similarity to L-threonine kinase genes. Feeding of [U-(13)C,(15) N]L-threonine and (13)C NMR analysis of the resulting compound unequivocally proved that threonine was incorporated intact into the central pyrrole (19 % enrichment) to provide the heterocyclic nitrogen as well as four of the seven carbons of this moiety. Therefore, this pyrrole is formed via a new, hitherto unknown biosynthetic pathway. A hypothesis for the reaction sequence leading to the central pyrrole moiety of coumermycin A(1) is presented. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes

DOE PAGES

Hadjithomas, Michalis; Chen, I-Min A.; Chu, Ken; ...

2016-11-29

Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic genemore » clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery.« less

IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadjithomas, Michalis; Chen, I-Min A.; Chu, Ken

Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic genemore » clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery.« less

Unique pathway of expression of an opal suppressor phosphoserine tRNA.

PubMed Central

Lee, B J; de la Peña, P; Tobian, J A; Zasloff, M; Hatfield, D

1987-01-01

An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. We have shown that the product of this gene functions as a suppressor in an in vitro assay, and we have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, we show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of any known eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells. Images PMID:3114749

Structure and Functional Analysis of ClbQ, an Unusual Intermediate-Releasing Thioesterase from the Colibactin Biosynthetic Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guntaka, Naga Sandhya; Healy, Alan R.; Crawford, Jason M.

Colibactin is a genotoxic hybrid nonribosomal peptide/polyketide secondary metabolite produced by various pathogenic and probiotic bacteria residing in the human gut. The presence of colibactin metabolites has been correlated to colorectal cancer formation in several studies. The specific function of many gene products in the colibactin gene cluster can be predicted. However, the role of ClbQ, a type II editing thioesterase, has not been established. The importance of ClbQ has been demonstrated by genetic deletions that abolish colibactin cytotoxic activity, and recent studies suggest an atypical role in releasing pathway intermediates from the assembly line. Here we report the 2.0more » Å crystal structure and biochemical characterization of ClbQ. Our data reveal that ClbQ exhibits greater catalytic efficiency toward acyl-thioester substrates as compared to precolibactin intermediates and does not discriminate among carrier proteins. Cyclized pyridone-containing colibactins, which are off-pathway derivatives, are not viable substrates for ClbQ, while linear precursors are, supporting a role of ClbQ in facilitating the promiscuous off-loading of premature precolibactin metabolites and novel insights into colibactin biosynthesis.« less

Fine Tuning of Antibiotic Activity by a Tailoring Hydroxylase in a Trans-AT Polyketide Synthase Pathway.

PubMed

Mohammad, Hadi H; Connolly, Jack A; Song, Zhongshu; Hothersall, Joanne; Race, Paul R; Willis, Christine L; Simpson, Thomas J; Winn, Peter J; Thomas, Christopher M

2018-04-16

The addition or removal of hydroxy groups modulates the activity of many pharmacologically active biomolecules. It can be integral to the basic biosynthetic factory or result from associated tailoring steps. For the anti-MRSA antibiotic mupirocin, removal of a C8-hydroxy group late in the biosynthetic pathway gives the active pseudomonic acid A. An extra hydroxylation, at C4, occurs in the related but more potent antibiotic thiomarinol A. We report here in vivo and in vitro studies that show that the putative non-haem-iron(II)/α-ketoglutaratedependent dioxygenase TmuB, from the thiomarinol cluster, 4-hydroxylates various pseudomonic acids whereas C8-OH, and other substituents around the tetrahydropyran ring, block enzyme action but not substrate binding. Molecular modelling suggested a basis for selectivity, but mutation studies had a limited ability to rationally modify TmuB substrate specificity. 4-Hydroxylation had opposite effects on the potency of mupirocin and thiomarinol. Thus, TmuB can be added to the toolbox of polyketide tailoring technologies for the in vivo generation of new antibiotics in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Variation in the Apparent Biosynthetic Fractionation for N-alkane δD Among Terrestrial Plants: Patterns, Mechanisms, and Implications

NASA Astrophysics Data System (ADS)

Johnson, J. E.; Tipple, B. J.; Betancourt, J. L.; Ehleringer, J. R.; Leavitt, S. W.; Monson, R. K.

2016-12-01

Long-chain normal alkanes (n-alkanes) are a component of the leaf cuticle of all terrestrial plants. Since the hydrogen in the n-alkanes is derived from the hydrogen in plants' water sources and is non-exchangeable, the stable hydrogen isotopic composition (δD) of the n-alkanes provides information about the δD of environmental waters. While this relationship creates opportunities for using n-alkane δD for process-based reconstructions of δD of environmental waters, progress in this direction is currently constrained by the observation that terrestrial plants exhibit a startlingly wide range of apparent biosynthetic fractionations. To understand the mechanisms responsible for variation in the apparent biosynthetic fractionations, we compared measurements and models of δD for n-C29 in a water-limited ecosystem where the timing of primary and secondary cuticle deposition is closely coupled to water availability (Tumamoc Hill, Tucson, Arizona, USA). During the 2014-2015 hydrologic year, the most widespread and abundant plant species at this site exhibited δD for n-C29 varying over a total range of 102‰. Discrete samples of leaf water collected at the same time as the n-C29 samples exhibited δD varying over a total range of only 53‰, but a continuous model of leaf water through the annual cycle predicted δD varying over a total range of 190‰. These results indicate that the observed variation in the apparent biosynthetic fractionation for n-C29 δD could be primarily attributable to leaf water dynamics that are temporally uncoupled from primary and secondary cuticle deposition. If a single biosynthetic fractionation does describe the relationship between the δD of n-alkanes and leaf water during intervals of cuticle deposition, it will facilitate process-based interpretations of n-alkane δD values in ecological, hydrological, and climatological studies of modern and ancient terrestrial environments.

Metabolic engineering of E.coli for the production of a precursor to artemisinin, an anti-malarial drug [Chapter 25 in Manual of Industrial Microbiology and Biotechnology, 3rd edition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petzold, Christopher; Keasling, Jay

This document is Chapter 25 in the Manual of Industrial Microbiology and Biotechnology, 3rd edition. Topics covered include: Incorporation of Amorpha-4,11-Diene Biosynthetic Pathway into E. coli; Amorpha-4,11-Diene Pathway Optimization; "-Omics" Analyses for Increased Amorpha-4,11-Diene Production; Biosynthetic Oxidation of Amorpha-4,11-Diene.

P5CDH affects the pathways contributing to Pro synthesis after ProDH activation by biotic and abiotic stress conditions

PubMed Central

Rizzi, Yanina S.; Monteoliva, Mariela I.; Fabro, Georgina; Grosso, Carola L.; Laróvere, Laura E.; Alvarez, María E.

2015-01-01

Plants facing adverse conditions usually alter proline (Pro) metabolism, generating changes that help restore the cellular homeostasis. These organisms synthesize Pro from glutamate (Glu) or ornithine (Orn) by two-step reactions that share Δ1 pyrroline-5-carboxylate (P5C) as intermediate. In the catabolic process, Pro is converted back to Glu using a different pathway that involves Pro dehydrogenase (ProDH), P5C dehydrogenase (P5CDH), and P5C as intermediate. Little is known about the coordination of the catabolic and biosynthetic routes under stress. To address this issue, we analyzed how P5CDH affects the activation of Pro synthesis, in Arabidopsis tissues that increase ProDH activity by transient exposure to exogenous Pro, or infection with Pseudomonas syringae pv. tomato. Wild-type (Col-0) and p5cdh mutant plants subjected to these treatments were used to monitor the Pro, Glu, and Orn levels, as well as the expression of genes from Pro metabolism. Col-0 and p5cdh tissues consecutively activated ProDH and Pro biosynthetic genes under both conditions. However, they manifested a different coordination between these routes. When external Pro supply was interrupted, wild-type leaves degraded Pro to basal levels at which point Pro synthesis, mainly via Glu, became activated. Under the same condition, p5cdh leaves sustained ProDH induction without reducing the Pro content but rather increasing it, apparently by stimulating the Orn pathway. In response to pathogen infection, both genotypes showed similar trends. While Col-0 plants seemed to induce both Pro biosynthetic routes, p5cdh mutant plants may primarily activate the Orn route. Our study contributes to the functional characterization of P5CDH in biotic and abiotic stress conditions, by revealing its capacity to modulate the fate of P5C, and prevalence of Orn or Glu as Pro precursors in tissues that initially consumed Pro. PMID:26284090

Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity.

PubMed

Fan, Pengxiang; Miller, Abigail M; Liu, Xiaoxiao; Jones, A Daniel; Last, Robert L

2017-12-12

Plants produce hundreds of thousands of structurally diverse specialized metabolites via multistep biosynthetic networks, including compounds of ecological and therapeutic importance. These pathways are restricted to specific plant groups, and are excellent systems for understanding metabolic evolution. Tomato and other plants in the nightshade family synthesize protective acylated sugars in the tip cells of glandular trichomes on stems and leaves. We describe a metabolic innovation in wild tomato species that contributes to acylsucrose structural diversity. A small number of amino acid changes in two acylsucrose acyltransferases alter their acyl acceptor preferences, resulting in reversal of their order of reaction and increased product diversity. This study demonstrates how small numbers of amino acid changes in multiple pathway enzymes can lead to diversification of specialized metabolites in plants. It also highlights the power of a combined genetic, genomic and in vitro biochemical approach to identify the evolutionary mechanisms leading to metabolic novelty.

Polyamines and their biosynthetic enzymes during somatic embryo development in red spruce (Picea rubens Sarg.)

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