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Sample records for carotenoid biosynthetic pathway

  1. Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

    PubMed Central

    Umeno, Daisuke; Tobias, Alexander V.; Arnold, Frances H.

    2005-01-01

    Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed. PMID:15755953

  2. THE CAROTENOID BIOSYNTHETIC PATHWAY: THINKING IN ALL DIMENSIONS

    PubMed Central

    Shumskaya, Maria; Wurtzel, Eleanore T.

    2013-01-01

    The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signalling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavour of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and predictability of metabolic engineering of carotenoids rests in the ability to target carotenoids for specific physiological purposes as well as to simultaneously modify carotenoids along with other desired traits. Here, we ask whether predictive metabolic engineering of the carotenoid pathway is indeed possible. Despite a long history of research on the pathway, at this point in time we can only describe the pathway as a parts list and have almost no knowledge of the location of the complete pathway, how it is assembled, and whether there exists any trafficking of the enzymes or the carotenoids themselves. We discuss the current state of knowledge regarding the “complete” pathway and make the argument that predictive metabolic engineering of the carotenoid pathway (and other pathways) will require investigation of the three dimensional state of the pathway as it may exist in plastids of different ultrastructures. Along with this message we point out the need to develop new types of visualization tools and resources that better reflect the dynamic nature of biosynthetic pathways. PMID:23683930

  3. 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.

  4. 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 ...

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

    PubMed Central

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

    2016-01-01

    Summary 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 S. 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 amongst 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. PMID:25329237

  6. 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.

  7. Carotenoid biosynthetic pathway: molecular phylogenies and evolutionary behavior of crt genes in eubacteria.

    PubMed

    Phadwal, Kanchan

    2005-01-17

    Phylogenetic analysis of carotenoid biosynthetic pathway genes and their evolutionary rate variations were studied among eubacterial taxa. The gene sequences for the enzymes involved in this pathway were obtained for major phylogenetic groups of eubacteria (green sulfur bacteria, green nonsulphur bacteria, Gram-positive bacteria, proteobacteria, flavobacteria, cyanobacteria) and archeabacteria. These gene datasets were distributed under five major steps of carotenoid biosynthesis in eubacteria; isoprenoid precursor biosynthesis, phytoene synthesis, dehydrogenation of phytoene, lycopene cyclization, formation of acyclic xanthophylls, formation of cyclic xanthophylls and carotenoid biosynthesis regulation. The NJ algorithm was used on protein coding DNA sequences to deduce the evolutionary relationship for the respective crt genes among different eubacterial lineages. The rate of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S)) were calculated for different clades of the respective phylogenetic tree for specific crt genes. The phylogenetic analysis suggests that evolutionary pattern of crt genes in eubacteria is characterized by lateral gene transfer and gene duplication events. The d(N) values indicate that carotenoid biosynthetic genes are more conserved in proteobacteria than in any other eubacterial phyla. Furthermore, of the genes involved in carotenoid biosynthesis pathway, structural genes evolve slowly than the regulatory genes in eubacteria.

  8. Complete Biosynthetic Pathway of the C50 Carotenoid Bacterioruberin from Lycopene in the Extremely Halophilic Archaeon Haloarcula japonica

    PubMed Central

    Yang, Ying; Ando, Ai; Miyoko, Nobuhiro; Fukui, Toshiaki; Takaichi, Shinichi; Nakamura, Satoshi

    2015-01-01

    ABSTRACT Haloarcula japonica, an extremely halophilic archaeon that requires high concentrations of NaCl for growth, accumulates the C50 carotenoid bacterioruberin (BR). By homology analysis, a gene cluster, including c0507, c0506, and c0505, was found and predicted to be involved in the synthesis of bacterioruberin. To elucidate the function of the encoded enzymes, we constructed Ha. japonica mutants of these genes and analyzed carotenoids produced by the mutants. Our research showed that c0507, c0506, and c0505 encoded a carotenoid 3,4-desaturase (CrtD), a bifunctional lycopene elongase and 1,2-hydratase (LyeJ), and a C50 carotenoid 2″,3″-hydratase (CruF), respectively. The above three carotenoid biosynthetic enzymes catalyze the reactions that convert lycopene to bacterioruberin in Ha. japonica. This is the first identification of functional CrtD and CruF in archaea and elucidation of the complete biosynthetic pathway of bacterioruberin from lycopene. IMPORTANCE Haloarcula japonica, an extremely halophilic archaeon, accumulates the C50 carotenoid bacterioruberin (BR). In this study, we have identified three BR biosynthetic enzymes and have elucidated their functions. Among them, two enzymes were found in an archaeon for the first time. Our results revealed the biosynthetic pathway responsible for production of BR in Ha. japonica and provide a basis for investigating carotenoid biosynthetic pathways in other extremely halophilic archaea. Elucidation of the carotenoid biosynthetic pathway in Ha. japonica may also prove useful for producing the C50 carotenoid BR efficiently by employing genetically modified haloarchaeal strains. PMID:25712483

  9. Gene Duplication in the Carotenoid Biosynthetic Pathway Preceded Evolution of the Grasses1

    PubMed Central

    Gallagher, Cynthia E.; Matthews, Paul D.; Li, Faqiang; Wurtzel, Eleanore T.

    2004-01-01

    Despite ongoing research on carotenoid biosynthesis in model organisms, there is a paucity of information on pathway regulation operating in the grasses (Poaceae), which include plants of world-wide agronomic importance. As a result, efforts to either breed for or metabolically engineer improvements in carotenoid content or composition in cereal crops have led to unexpected results. In comparison to maize (Zea mays), rice (Oryza sativa) accumulates no endosperm carotenoids, despite having a functional pathway in chloroplasts. To better understand why these two related grasses differ in endosperm carotenoid content, we began to characterize genes encoding phytoene synthase (PSY), since this nuclear-encoded enzyme appeared to catalyze a rate-controlling step in the plastid-localized biosynthetic pathway. The enzyme had been previously associated with the maize Y1 locus thought to be the only functional gene controlling PSY accumulation, though function of the Y1 gene product had never been demonstrated. We show that both maize and rice possess and express products from duplicate PSY genes, PSY1 (Y1) and PSY2; PSY1 transcript accumulation correlates with carotenoid-containing endosperm. Using a heterologous bacterial system, we demonstrate enzyme function of PSY1 and PSY2 that are largely conserved in sequence except for N- and C-terminal domains. By database mining and use of ortholog-specific universal PCR primers, we found that the PSY duplication is prevalent in at least eight subfamilies of the Poaceae, suggesting that this duplication event preceded evolution of the Poaceae. These findings will impact study of grass phylogeny and breeding of enhanced carotenoid content in an entire taxonomic group of plant crops critical for global food security. PMID:15247400

  10. Carotenoid profiling, in silico analysis and transcript profiling of miRNAs targeting carotenoid biosynthetic pathway genes in different developmental tissues of tomato.

    PubMed

    Koul, Archana; Yogindran, Sneha; Sharma, Deepak; Kaul, Sanjana; Rajam, Manchikatla Venkat; Dhar, Manoj K

    2016-11-01

    Carotenoid biosynthetic pathway is one of the highly significant and very well elucidated secondary metabolic pathways in plants. microRNAs are the potential regulators, widely known for playing a pivotal role in the regulation of various biological as well as metabolic processes. miRNAs may assist in the metabolic engineering of the secondary metabolites for the production of elite genotypes with increased biomass and content of various metabolites. miRNA mediated regulation of carotenoid biosynthetic genes has not been elucidated so far. To illustrate the potential regulatory role of miRNAs in carotenoid biosynthesis, transcript profiling of the known miRNAs and their possible target carotenoid genes was undertaken at eight different developmental stages of tomato, using stem-loop PCR approach combined with quantitative RT-PCR. The inter-relationship amongst carotenoid content, biosynthetic genes and miRNAs was studied in depth. Comparative expression profiles of miRNA and target genes showed variable expression in different tissues studied. The expression level of miRNAs and their target carotenoid genes displayed similar pattern in the vegetative tissues as compared to the reproductive ones, viz. fruit (different stages), indicating the possibility of regulation of carotenoid biosynthesis at various stages of fruit development. This was later confirmed by the HPLC analysis of the carotenoids. The present study has further enhanced the understanding of regulation of carotenoid biosynthetic pathway in plants. The identified miRNAs can be employed to manipulate the biosynthesis of different carotenoids, through metabolic engineering for the production of lycopene rich tomatoes.

  11. Carotenoids of Gemmatimonas aurantiaca (Gemmatimonadetes): identification of a novel carotenoid, deoxyoscillol 2-rhamnoside, and proposed biosynthetic pathway of oscillol 2,2'-dirhamnoside.

    PubMed

    Takaichi, Shinichi; Maoka, Takashi; Takasaki, Kazuto; Hanada, Satoshi

    2010-03-01

    Gemmatimonas aurantiaca strain T-27(T) is an orange-coloured, Gram-negative, facultatively aerobic, polyphosphate-accumulating bacterium belonging to a recently proposed phylum, Gemmatimonadetes. We purified its pigments and identified them as carotenoids and their glycoside derivatives using spectral data. The major carotenoid was (2S,2' S)-oscillol 2,2'-di-(alpha-l-rhamnoside), and the minor carotenoids were (2S)-deoxyoscillol 2-( alpha-l-rhamnoside) and didemethylspirilloxanthin. Deoxyoscillol 2-rhamnoside is a novel carotenoid. Oscillol 2,2'-diglycosides have hitherto only been reported in a limited number of cyanobacteria, and this is believed to be the first finding of such carotenoids in another bacterial phylum. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence, we propose a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. We propose the involvement of geranylgeranyl pyrophosphate synthase (CrtE), phytoene synthase (CrtB) and phytoene desaturase (CrtI) for lycopene synthesis; and of carotenoid 1,2-hydratase (CruF) and carotenoid 2-O-rhamnosyltransferase (CruG) for oscillol 2,2'-dirhamnoside synthesis. Further, isopentenyl pyrophosphate could be synthesized by a non-mevalonate pathway (DXP pathway).

  12. Differential expression of carotenoid biosynthetic pathway genes in two contrasting tomato genotypes for lycopene content.

    PubMed

    Pandurangaiah, Shilpa; Ravishankar, Kundapura V; Shivashankar, Kodthalu S; Sadashiva, Avverahally T; Pillakenchappa, Kavitha; Narayanan, Sunil Kumar

    2016-06-01

    Tomato (Solanum lycopersicum L.) is one of the model plant to study carotenoid biosynthesis. In the present study, the fruit carotenoid content were quantified at different developmental stages for two contrasting genotypes, viz. IIHR-249-1 and IIHR-2866 by UPLC. Lycopene content was high in IIHR-249-1 (19.45 mg/100 g fresh weight) compared to IIHR-2866 (1.88 mg/100 g fresh weight) at the ripe stage. qPCR was performed for genes that are involved in the carotenoid biosynthetic pathway to study the difference in lycopene content in fruits of both the genotypes. The expression of Phytoene synthase (PSY) increased by 36-fold and Phytoene desaturase (PDS) increased by 14-fold from immature green stage to ripe stage in IIHR-249-1. The expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta cyclase (CYC-B) decreased gradually from the initial stage to the ripe stage in IIHR-249-1. IIHR 249-1 showed 3- and 1.8-fold decrease in gene expression for Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) .The F2 hybrids derived from IIHR-249-1 and IIHR-2866 were analysed at the ripe stage for lycopene content. The gene expression of Chloroplast lycopene beta-cyclase (LCY-B) and Chromoplast lycopene beta-cyclase (CYC-B) in high and low lycopene lines from F2 progenies also showed the decrease in transcript levels of both the genes in high lycopene F2 lines. We wish to suggest that the differential expression of lycopene beta-cyclases can be used in marker-assisted breeding.

  13. 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

  14. Reconstruction of the Carotenoid Biosynthetic Pathway of Cronobacter sakazakii BAA894 in Escherichia coli

    PubMed Central

    Zhang, Wei; Hu, Xiaoqing; Wang, Liqin; Wang, Xiaoyuan

    2014-01-01

    Cronobacter sakazakii could form yellow-pigmented colonies. However, the chemical structure and the biosynthetic pathway of the yellow pigments have not been identified. In this study, the yellow pigments of C. sakazakii BAA894 were purified and analyzed. The major components of the yellow pigments were confirmed as zeaxanthin-monoglycoside and zeaxanthin-diglycoside. A gene cluster containing seven genes responsible for the yellow pigmentation in C. sakazakii BAA894 was identified. The seven genes of C. sakazakii BAA894 or parts of them were reconstructed in a heterologous host Escherichia coli DH5α. The pigments formed in these E. coli strains were isolated and analyzed by thin layer chromatography, UV-visible spectroscopy, high performance liquid chromatography, and electron spray ionization-mass spectrometry. These redesigned E. coli strains could produce different carotenoids. E. coli strain expressing all the seven genes could produce zeaxanthin-monoglycoside and zeaxanthin-diglycoside; E. coli strains expressing parts of the seven genes could produce lycopene, β-carotene, cryptoxanthin or zeaxanthin. This study identified the gene cluster responsible for the yellow pigmentation in C. sakazakii BAA894. PMID:24466219

  15. Flower color alteration in Lotus japonicus by modification of the carotenoid biosynthetic pathway.

    PubMed

    Suzuki, Sakae; Nishihara, Masahiro; Nakatsuka, Takashi; Misawa, Norihiko; Ogiwara, Isao; Yamamura, Saburo

    2007-07-01

    To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding beta-carotene ketolase (4,4'-beta-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.

  16. Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis, phylogenetic analysis, and expression profiling.

    PubMed

    Li, Peirong; Zhang, Shujiang; Zhang, Shifan; Li, Fei; Zhang, Hui; Cheng, Feng; Wu, Jian; Wang, Xiaowu; Sun, Rifei

    2015-07-03

    Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa. We identified 67 carotenoid biosynthetic genes in B. rapa, which were orthologs of the 47 carotenoid genes in A. thaliana. A high level of synteny was observed for carotenoid biosynthetic genes between A. thaliana and B. rapa. Out of 47 carotenoid biosynthetic genes in A. thaliana, 46 were successfully mapped to the 10 B. rapa chromosomes, and most of the genes retained more than one copy in B. rapa. The gene expansion was caused by the whole-genome triplication (WGT) event experienced by Brassica species. An expression analysis of the carotenoid biosynthetic genes suggested that their expression levels differed in root, stem, leaf, flower, callus, and silique tissues. Additionally, the paralogs of each carotenoid biosynthetic gene, which were generated from the WGT in B. rapa, showed significantly different expression levels among tissues, suggesting differentiated functions for these multi-copy genes in the carotenoid pathway. This first systematic study of carotenoid biosynthetic genes in B. rapa provides insights into the carotenoid metabolic mechanisms of Brassica crops. In addition, a better understanding of carotenoid biosynthetic genes in B. rapa will contribute to the development of conventional and transgenic B. rapa cultivars with enriched carotenoid levels in the future.

  17. 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.

  18. Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases▿ †

    PubMed Central

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

    2010-01-01

    We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C50 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 C40 lycopene, C45 nonaflavuxanthin, C50 flavuxanthin, and C50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C50 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 ɛ-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C50 carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ɛ-cyclic C50 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 ɛ- and γ-cyclic C50 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 C50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids. PMID:20802040

  19. A highly selective biosynthetic pathway to non-natural C50 carotenoids assembled from moderately selective enzymes.

    PubMed

    Furubayashi, Maiko; Ikezumi, Mayu; Takaichi, Shinichi; Maoka, Takashi; Hemmi, Hisashi; Ogawa, Takuya; Saito, Kyoichi; Tobias, Alexander V; Umeno, Daisuke

    2015-07-14

    Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C50 backbones produced. We then further extend the C50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C50 carotenoids, including ∼ 90% C50-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.

  20. A highly selective biosynthetic pathway to non-natural C50 carotenoids assembled from moderately selective enzymes

    PubMed Central

    Furubayashi, Maiko; Ikezumi, Mayu; Takaichi, Shinichi; Maoka, Takashi; Hemmi, Hisashi; Ogawa, Takuya; Saito, Kyoichi; Tobias, Alexander V; Umeno, Daisuke

    2015-01-01

    Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C50 backbones produced. We then further extend the C50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C50 carotenoids, including ∼90% C50-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes. PMID:26168783

  1. Construction and utilization of carotenoid reporter systems: identification of chromosomal integration sites that support suitable expression of biosynthetic genes and pathways.

    PubMed

    Sharpe, Pamela L; DiCosimo, Deana J

    2012-01-01

    In order to metabolically engineer microorganisms to produce compounds of interest, it is often desirable to integrate foreign genes into the chromosome of the host. However, the consequences of these genetic alterations are not always predictable. The use of a reporter system can often assist in determining chromosomal locations for optimal expression of foreign biosynthetic genes. The method described here involves the construction and utilization of promoterless carotenoid transposons, which provides a colorimetric screen for identifying the best chromosomal integration sites for the expression of the genes of interest. The transposons (pUTmTn5::392W and pUTmTn5::392) contain the carotenoid genes required for the production of canthaxanthin and astaxanthin, respectively. Thus, when promoterless transposons insert into the host's genome, the color of the colonies will vary based on their chromosomal location. There is a correlation between the color intensity of the colonies and the expression of the carotenoid transposon. The transposon insertion site can be determined via direct chromosomal sequencing. This sequence information is used to guide the site-specific integration of biosynthetic genes and pathways of interest.

  2. 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

  3. Biosynthetic routes of hydroxylated carotenoids (xanthophylls) in Marchantia polymorpha, and production of novel and rare xanthophylls through pathway engineering in Escherichia coli.

    PubMed

    Takemura, Miho; Maoka, Takashi; Misawa, Norihiko

    2015-03-01

    MpBHY codes for a carotene β-ring 3(,3')-hydroxylase responsible for both zeaxanthin and lutein biosynthesis in liverwort. MpCYP97C functions as an ε-ring hydroxylase (zeinoxanthin 3'-hydroxylase) to produce lutein in liverwort. Xanthophylls are oxygenated or hydroxylated carotenes that are most abundant in the light-harvesting complexes of plants. The plant-type xanthophylls consist of α-xanthophyll (lutein) and β-xanthophylls (zeaxanthin, antheraxanthin, violaxanthin and neoxanthin). The α-xanthophyll and β-xanthophylls are derived from α-carotene and β-carotene by carotene hydroxylase activities, respectively. β-Ring 3,3'-hydroxylase that mediates the route of zeaxanthin from β-carotene via β-cryptoxanthin is present in higher plants and is encoded by the BHY (BCH) gene. On the other hand, CYP97A (or BHY) and CYP97C genes are responsible for β-ring 3-hydroxylation and ε-ring 3'-hydroxylation, respectively, in routes from α-carotene to lutein. To elucidate the evolution of the biosynthetic routes of such hydroxylated carotenoids from carotenes in land plants, we identified and functionally analyzed carotenoid hydroxylase genes of liverwort Marchantia polymorpha L. Three genes homologous to higher plants, BHY, CYP97A, and CYP97C, were isolated and named MpBHY, MpCYP97A, and MpCYP97C, respectively. MpBHY was found to code for β-ring hydroxylase, which is responsible for both routes starting from β-carotene and α-carotene. MpCYP97C functioned as an ε-ring hydroxylase not for α-carotene but for zeinoxanthin, while MpCYP97A showed no hydroxylation activity for β-carotene or α-carotene. These findings suggest the original functions of the hydroxylation enzymes of carotenes in land plants, which are thought to diversify in higher plants. In addition, we generated recombinant Escherichia coli cells, which produced rare and novel carotenoids such as α-echinenone and 4-ketozeinoxanthin, through pathway engineering using bacterial carotenogenic genes

  4. Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits.

    PubMed

    Smita, Shuchi; Rajwanshi, Ravi; Lenka, Sangram Keshari; Katiyar, Amit; Chinnusamy, Viswanathan; Bansal, Kailash Chander

    2013-12-01

    Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the beta-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype-Pusa Rohini. We found that expression of phytoene synthase and beta-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.

  5. 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...

  6. Timing and Biosynthetic Potential for Carotenoid Accumulation in Genetically Diverse Germplasm of Maize1[C][W][OA

    PubMed Central

    Vallabhaneni, Ratnakar; Wurtzel, Eleanore T.

    2009-01-01

    Enhancement of the carotenoid biosynthetic pathway in food crops benefits human health and adds commercial value of natural food colorants. However, predictable metabolic engineering or breeding is limited by the incomplete understanding of endogenous pathway regulation, including rate-controlling steps and timing of expression in carotenogenic tissues. The grass family (Poaceae) contains major crop staples, including maize (Zea mays), wheat (Triticum aestivum), rice (Oryza sativa), sorghum (Sorghum bicolor), and millet (Pennisetum glaucum). Maize carotenogenesis was investigated using a novel approach to discover genes encoding limiting biosynthetic steps in the nutritionally targeted seed endosperm. A combination of bioinformatics and cloning were first used to identify and map gene families encoding enzymes in maize and other grasses. These enzymes represented upstream pathways for isopentenyl diphosphate and geranylgeranyl diphosphate synthesis and the downstream carotenoid biosynthetic pathway, including conversion to abscisic acid. A maize germplasm collection was used for statistical testing of the correlation between carotenoid content and candidate gene transcript levels. Multiple pathway bottlenecks for isoprenoid biosynthesis and carotenoid biosynthesis were discovered in specific temporal windows of endosperm development. Transcript levels of paralogs encoding isoprenoid isopentenyl diphosphate and geranylgeranyl diphosphate-producing enzymes, DXS3, DXR, HDR, and GGPPS1, were found to positively correlate with endosperm carotenoid content. For carotenoid pathway enzymes, transcript levels for CrtISO inversely correlated with seed carotenoid content, as compared with positive correlation of PSY1 transcripts. Since zeaxanthin epoxidase (ZEP) depletes the carotenoid pool in subsequent conversion to abscisic acid, ZEP transcripts were examined. Carotenoid accumulation was found to be inversely associated with ZEP1 and ZEP2 transcript levels. Extension of

  7. Carotenoid profiling and the expression of carotenoid biosynthetic genes in developing coffee grain.

    PubMed

    Simkin, Andrew J; Kuntz, Marcel; Moreau, Helene; McCarthy, James

    2010-06-01

    Roasted coffee contains a complex array of volatile organic compounds (VOCs) which make an important contribution to the characteristic flavour and aroma of the final beverage. It is thought that a few of the potent coffee aroma components, such as "beta-damascenone", could be derived from carotenoid precursors. In order to further investigate the potential link between carotenoids and coffee aroma profiles, we have measured the carotenoid content in developing coffee grain. The data obtained confirms the presence of lutein in the grain, and additionally shows that the immature coffee grain also contains significant amounts of beta-carotene, alpha-carotene, violaxanthin, and neoxanthin. Complimentary quantitative gene expression analysis revealed that all the carotenoid biosynthetic genes examined are expressed in the grain, and that the transcript levels are gene and stage dependent. Furthermore, consistent with the reduction of the carotenoid levels at the last stage of grain development (mature-red), most of the transcript levels were also found to be lower at the final developmental stage. Quantitative expression analysis of the carotenoid genes was also carried out for the developing pericarp tissue of the coffee cherries. Again, all the genes examined were expressed, and in most cases, the highest transcript levels were detected around the large green-yellow stages, a period when carotenoid synthesis is probably greatest.

  8. Biosynthetic Pathways of Ergot Alkaloids

    PubMed Central

    Gerhards, Nina; Neubauer, Lisa; Tudzynski, Paul; Li, Shu-Ming

    2014-01-01

    Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes. PMID:25513893

  9. Accumulation of Carotenoids and Expression of Carotenoid Biosynthetic Genes during Maturation in Citrus Fruit1

    PubMed Central

    Kato, Masaya; Ikoma, Yoshinori; Matsumoto, Hikaru; Sugiura, Minoru; Hyodo, Hiroshi; Yano, Masamichi

    2004-01-01

    The relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation was investigated in three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). We cloned the cDNAs for phytoene synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO), lycopene β-cyclase (CitLCYb), β-ring hydroxylase (CitHYb), zeaxanthin (zea) epoxidase (CitZEP), and lycopene ε-cyclase (CitLCYe) from Satsuma mandarin, which shared high identities in nucleotide sequences with Valencia orange, Lisbon lemon, and other plant species. With the transition of peel color from green to orange, the change from β,ε-carotenoid (α-car and lutein) accumulation to β,β-carotenoid (β-car, β-cryptoxanthin, zea, and violaxanthin) accumulation was observed in the flavedos of Satsuma mandarin and Valencia orange, accompanying the disappearance of CitLCYe transcripts and the increase in CitLCYb transcripts. Even in green fruit, high levels of β,ε-carotenoids and CitLCYe transcripts were not observed in the juice sacs. As fruit maturation progressed in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive β,β-xanthophyll (β-cryptoxanthin, zea, and violaxanthin) accumulation in both the flavedo and juice sacs. The gene expression of CitCRTISO was kept low or decreased in the flavedo during massive β,β-xanthophyll accumulation. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phytoene was observed with a decrease in the transcript level for CitPDS. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination of the expression among carotenoid biosynthetic genes. In this paper, the mechanism leading to diversity in

  10. Heterologous carotenoid-biosynthetic enzymes: functional complementation and effects on carotenoid profiles in Escherichia coli.

    PubMed

    Song, Gyu Hyeon; Kim, Se Hyeuk; Choi, Bo Hyun; Han, Se Jong; Lee, Pyung Cheon

    2013-01-01

    A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product-3,4,3',4'-tetradehydrolycopene-along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.

  11. Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

    PubMed Central

    Song, Gyu Hyeon; Kim, Se Hyeuk; Choi, Bo Hyun; Han, Se Jong

    2013-01-01

    A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product—3,4,3′,4′-tetradehydrolycopene—along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does. PMID:23144136

  12. A biosynthetic pathway for anandamide

    PubMed Central

    Liu, Jie; Wang, Lei; Harvey-White, Judith; Osei-Hyiaman, Douglas; Razdan, Raj; Gong, Qian; Chan, Andrew C.; Zhou, Zhifeng; Huang, Bill X.; Kim, Hee-Yong; Kunos, George

    2006-01-01

    The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released “on demand” by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE–PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE–PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target. PMID:16938887

  13. The sub-cellular localisation of the potato (Solanum tuberosum L.) carotenoid biosynthetic enzymes, CrtRb2 and PSY2.

    PubMed

    Pasare, Stefania; Wright, Kathryn; Campbell, Raymond; Morris, Wayne; Ducreux, Laurence; Chapman, Sean; Bramley, Peter; Fraser, Paul; Roberts, Alison; Taylor, Mark

    2013-12-01

    Carotenoids are isoprenoids with important biological roles both for plants and animals. The yellow flesh colour of potato (Solanum tuberosum L.) tubers is a quality trait dependent on the types and levels of carotenoids that accumulate. The carotenoid biosynthetic pathway is well characterised, facilitating the successful engineering of carotenoid content in numerous crops including potato. However, a clear understanding concerning the factors regulating carotenoid accumulation and localisation in plant storage organs, such as tubers, is lacking. In the present study, the localisation of key carotenoid biosynthetic enzymes was investigated, as one of the unexplored factors that could influence the accumulation of carotenoids in potato tubers. Stable transgenic potato plants were generated by over-expressing β-CAROTENE HYDROXYLASE 2 (CrtRb2) and PHYTOENE SYNTHASE 2 (PSY2) genes, fused to red fluorescent protein (RFP). Gene expression and carotenoid levels were both significantly increased, confirming functionality of the fluorescently tagged proteins. Confocal microscopy studies revealed different sub-organellar localisations of CrtRb2-RFP and PSY2-RFP within amyloplasts. CrtRb2 was detected in small vesicular structures, inside amyloplasts, whereas PSY2 was localised in the stroma of amyloplasts. We conclude that it is important to consider the location of biosynthetic enzymes when engineering the carotenoid metabolic pathway in storage organs such as tubers.

  14. 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.

  15. Biosynthetic Pathways of Brassinolide in Arabidopsis1

    PubMed Central

    Noguchi, Takahiro; Fujioka, Shozo; Choe, Sunghwa; Takatsuto, Suguru; Tax, Frans E.; Yoshida, Shigeo; Feldmann, Kenneth A.

    2000-01-01

    Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone → 3-dehydro-6-deoxoteasterone → 6-deoxotyphasterol → 6-deoxocastasterone → 6α-hydroxycastasterone → castasterone → BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone → 3-dehydroteasterone → typhasterol → castasterone → BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants. PMID:10982435

  16. Biosynthetic pathways of brassinolide in Arabidopsis.

    PubMed

    Noguchi, T; Fujioka, S; Choe, S; Takatsuto, S; Tax, F E; Yoshida, S; Feldmann, K A

    2000-09-01

    Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone --> 3-dehydro-6-deoxoteasterone --> 6-deoxotyphasterol --> 6-deoxocastasterone --> 6alpha-hydroxycastasterone --> castasterone --> BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone --> 3-dehydroteasterone --> typhasterol --> castasterone --> BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.

  17. Effects of white, blue, and red light-emitting diodes on carotenoid biosynthetic gene expression levels and carotenoid accumulation in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.).

    PubMed

    Tuan, Pham Anh; Thwe, Aye Aye; Kim, Yeon Bok; Kim, Jae Kwang; Kim, Sun-Ju; Lee, Sanghyun; Chung, Sun-Ok; Park, Sang Un

    2013-12-18

    In this study, the optimum wavelengths of light required for carotenoid biosynthesis were determined by investigating the expression levels of carotenoid biosynthetic genes and carotenoid accumulation in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.) exposed to white, blue, and red light-emitting diodes (LEDs). Most carotenoid biosynthetic genes showed higher expression in sprouts irradiated with white light at 8 days after sowing than in those irradiated with blue and red lights. The dominant carotenoids in tartary buckwheat sprouts were lutein and β-carotene. The richest accumulation of total carotenoids was observed in sprouts grown under white light (1282.63 μg g(-1) dry weight), which was relatively higher than that in sprouts grown under blue and red lights (940.86 and 985.54 μg g(-1), respectively). This study might establish an effective strategy for maximizing the production of carotenoids and other important secondary metabolites in tartary buckwheat sprouts by using LED technology.

  18. Transformation of carotenoid biosynthetic genes using a micro-cross section method in kiwifruit (Actinidia deliciosa cv. Hayward).

    PubMed

    Kim, Misun; Kim, Seong-Cheol; Song, Kwan Jeong; Kim, Ho Bang; Kim, In-Jung; Song, Eun-Young; Chun, Seung-Jong

    2010-12-01

    Genetic transformation using a micro-cross section (MCS) technique was conducted to improve the carotenoid content in kiwifruit (Actinidia deliciosa cv. Hayward). The introduced carotenoid biosynthetic genes include geranylgeranyl diphosphate synthase (GGPS), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), β-carotene hydroxylase (CHX), and phytoene synthase (PSY). The transformed explants were selected on half-strength MS medium containing 0.001 mg l(-1) of 2,4-D and 0.1 mg l(-1) of zeatin, either 5 mg l(-1) hygromycin or 25 mg l(-1) kanamycin, and 500 mg l(-1) cefotaxime. The genomic PCR, genomic Southern blot analysis, and RT-PCR were performed to confirm the integration and expression of the transgenes. The transformation efficiencies of either kanamycin- or hygromycin-resistant shoots ranged from 2.9 to 22.1% depending on the target genes, and from 2.9 to 24.2% depending on the reporter genes. The selection efficiencies ranged from 66.7 to 100% for the target genes and from 95.8 to 100% for the reporter genes. Changes of carotenoid content in the several PCR-positive plants were determined by UPLC analysis. As a result, transgenic plants expressing either GGPS or PSY increased about 1.2- to 1.3-fold in lutein or β-carotene content compared to non-transgenic plants. Our results suggest that the Agrobacterium-mediated transformation efficiency of kiwifruit can be greatly increased by this MCS method and that the carotenoid biosynthetic pathway can be modified in kiwifruit by genetic transformation. Our results further suggest that GGPS and PSY genes could be major target genes to increase carotenoid contents in kiwifruit.

  19. Redesign, reconstruction, and directed extension of the Brevibacterium linens C40 carotenoid pathway in Escherichia coli.

    PubMed

    Kim, Se Hyeuk; Park, Yun Hee; Schmidt-Dannert, Claudia; Lee, Pyung Cheon

    2010-08-01

    In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY(2) and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed beta-carotene pathway synthesized an unexpected 7,8-dihydro-beta-carotene in addition to beta-carotene. Extension of the beta-carotene pathway with the B. linens beta-ring desaturase CrtU and Pantoea beta-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.

  20. Model of the haem biosynthetic pathway

    NASA Astrophysics Data System (ADS)

    Greaves-Brown, Jeanette; Williams, Tim J.; Parish, J. H.

    1995-03-01

    (delta) -Aminolaevulinic acid (ALA) is a photodynamic therapy (PDT) agent that utilizes the haem biosynthetic pathway to create therapeutic levels of photoactive agents within tissues. Photosensitizer dosimetry and drug concentrations in target tissues are areas of uncertainty within PDT research. A program is described that uses numerical methods to model mathematically the haem biosynthetic pathway from ALA to haem as a set of partial differential rate equations. The data generated allow analysis and correlation with functions describing the kinetic behavior governing the reactions. This analysis provides insight into the production of protoporphyrin IX and other photoactive agents from exogenous ALA and provides a method for optimizing parameters, and for highlighting metabolic steps to which the product formation is most sensitive.

  1. Biosynthetic route towards saxitoxin and shunt pathway

    PubMed Central

    Tsuchiya, Shigeki; Cho, Yuko; Konoki, Keiichi; Nagasawa, Kazuo; Oshima, Yasukatsu; Yotsu-Yamashita, Mari

    2016-01-01

    Saxitoxin, the most potent voltage-gated sodium channel blocker, is one of the paralytic shellfish toxins (PSTs) produced by cyanobacteria and dinoflagellates. Recently, putative biosynthetic genes of PSTs were reported in these microorganisms. We previously synthesized genetically predicted biosynthetic intermediates, Int-A’ and Int-C’2, and also Cyclic-C’ which was not predicted based on gene, and identified them all in the toxin-producing cyanobacterium Anabaena circinalis (TA04) and the dinoflagellate Alexandrium tamarense (Axat-2). This study examined the incorporation of 15N-labeled intermediates into PSTs (C1 and C2) in A. circinalis (TA04). Conversions from Int-A’ to Int-C’2, from Int-C’2 to Cyclic-C’, and from Int-A’ and Int-C’2 to C1 and C2 were indicated using high resolution-LC/MS. However, Cyclic-C’ was not converted to C1 and C2 and was detected primarily in the extracellular medium. These results suggest that Int-A’ and Int-C’2 are genuine precursors of PSTs, but Int-C’2 converts partially to Cyclic-C’ which is a shunt product excreted to outside the cells. This paper provides the first direct demonstration of the biosynthetic route towards saxitoxin and a shunt pathway. PMID:26842222

  2. Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    PubMed Central

    2013-01-01

    Background Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. Results Total carotenoids progressively increased during fruit ripening up to ~55 μg g-1 fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. Conclusions Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening. PMID:24219562

  3. Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit.

    PubMed

    Grassi, Stefania; Piro, Gabriella; Lee, Je Min; Zheng, Yi; Fei, Zhangjun; Dalessandro, Giuseppe; Giovannoni, James J; Lenucci, Marcello S

    2013-11-12

    Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. Total carotenoids progressively increased during fruit ripening up to ~55 μg g(-1) fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening.

  4. Bioretrosynthetic construction of a didanosine biosynthetic pathway

    PubMed Central

    Birmingham, William R.; Starbird, Chrystal A.; Panosian, Timothy D.; Nannemann, David P.; Iverson, T. M.; Bachmann, Brian O.

    2014-01-01

    Concatenation of engineered biocatalysts into multistep pathways dramatically increases their utility, but development of generalizable assembly methods remains a significant challenge. Herein we evaluate ‘bioretrosynthesis’, which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2,3-dideoxyinosine). Applying both directed evolution and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and 3-fold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase, but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase. PMID:24657930

  5. Structural Biology of the Purine Biosynthetic Pathway

    PubMed Central

    Zhang, Yang; Morar, Mariya; Ealick, Steven E.

    2008-01-01

    Purine biosynthesis requires ten enzymatic transformations to generate inosine monophosphate. PurF, PurD, PurL, PurM, PurC, and PurB are common to all pathways, while PurN or PurT, PurK/PurE-I or PurE-II, PurH or PurP, and PurJ or PurO catalyze the same steps in different organisms. X-ray crystal structures are available for all 15 purine biosynthetic enzymes, including seven ATP-dependent enzymes, two amidotransferases and two tetrahydrofolate-dependent enzymes. Here we summarize the structures of the purine biosynthetic enzymes, discuss similarities and differences, and present arguments for pathway evolution. Four of the ATP-dependent enzymes belong to the ATP-grasp superfamily and two to the PurM superfamily. The amidotransferases are unrelated with one utilizing an NTN-glutaminase and the other utilizing a triad glutaminase. Likewise the tetrahydrofolate-dependent enzymes are unrelated. Ancestral proteins may have included a broad specificity enzyme instead of PurD, PurT, PurK, PurC, and PurP, and a separate enzyme instead of PurM and PurL. PMID:18712276

  6. 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

  7. 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.

  8. Functional Expression and Extension of Staphylococcal Staphyloxanthin Biosynthetic Pathway in Escherichia coli*

    PubMed Central

    Kim, Se Hyeuk; Lee, Pyung Cheon

    2012-01-01

    The biosynthetic pathway for staphyloxanthin, a C30 carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4′-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4′-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4′-diaponeurosporen-4-al into 4,4′-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4′-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes. PMID:22535955

  9. 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.

  10. 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

  11. Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability.

    PubMed

    Zhang, Lei; Yang, Qiao; Luo, Xuesong; Fang, Chengxiang; Zhang, Qiuju; Tang, Yali

    2007-10-01

    Deinococcus radiodurans R1, a red-pigmented strain of the extremely radioresistant genus Deinococcus, contains a major carotenoid namely deinoxanthin. The high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV) has been widely reported. However, the possible antioxidant role of carotenoids in this strain has not been completely elucidated. In this study, we constructed two colorless mutants by knockout of crtB and crtI genes, respectively. Comparative analysis of the two colorless mutants and the wild type showed that the two colorless mutants were more sensitive to ionizing radiation, UV, and hydrogen peroxide, but not to mitomycin-C (MMC). With electron spin resonance (ESR) and spin trapping techniques, we observed that hydroxyl radical signals occurred in the suspensions of UV irradiated Deinococcus radiodurans cells and the intensity of signals was influenced by carotenoids levels. We further showed that the carotenoid extract from the wild type could obviously scavenge superoxide anions generated by the irradiated riboflavin/EDTA system. These results suggest that carotenoids in D. radiodurans R1 function as free radical scavengers to protect this organism against the deleterious effects of oxidative DNA-damaging agents.

  12. Metabolic modeling of Rosmarinic acid biosynthetic pathway

    PubMed Central

    Sundaram, Shanthy; Tripathi, Ashutosh; Gupta, Deepak K

    2010-01-01

    Rosmarinic acid (RA) is an ester of caffeic acid and 3, 4‐dihydroxyphenyllacticacid. It is commonly found in Coleus blumei, Salvia officinalis, Melissa officinalis and Rosmarinus officinalis. The biosynthesis of RA starts with precursor molecules L‐phenylalanine and L‐tyrosine. Simulation of RA biosynthetic pathway was done using Gepasi Software, includes the reaction kinetics of each step of the pathway and different integration methods such as Euler's method. Optimization of the significant parameters responsible for RA biosynthesis was carried out. As the goal of the work was to increase the productivity of i.e. to maximize the concentration of the RA, the final concentration of RA ([RA]t) was selected as an objective function and selected initial concentration of the Caffeoyl‐3’‐4’hydroxyphenyllactic acid (3’C4HPLA) as parameter constraint and varied its initial concentration as: 0≤ [3’C4HPLA]i ≤ 0.025. Several optimization methods such as Simulated annealing, Evolutionary algorithms and Genetic algorithms were used to optimize the objective function. After optimization the final concentration of RA was slightly higher (4.566132e‐002 mM) than before optimization (4.047119e‐ 002 mM). On the basis of results obtained, it is clear that 4‐hydroxyphenyllactic acid and 3’C4HPLA play major role in the high productivity of the RA. PMID:21364781

  13. Heterologous expression and manipulation of three tetracycline biosynthetic pathways.

    PubMed

    Wang, Peng; Kim, Woncheol; Pickens, Lauren B; Gao, Xue; Tang, Yi

    2012-10-29

    A very accommodating host: Three tetracycline biosynthetic pathways were overexpressed and manipulated in the heterologous host Streptomyces lividans K4-114. Through the inactivation of various genes and characterization of the resulting biosynthetic intermediates, new tetracycline-modifying enzymes were identified (see scheme). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Carotenoids

    USDA-ARS?s Scientific Manuscript database

    This short article indicated that greater understanding of the biological functions of carotenoids mediated via their oxidative metabolites through their effects on these important cellular pathways and molecular targets, as well as their significance to cancer prevention, is needed. In considering ...

  15. Phytochrome-mediated Carotenoids Biosynthesis in Ripening Tomatoes.

    PubMed

    Thomas, R L; Jen, J J

    1975-09-01

    Red light induced and far red light inhibited carotenoid biosynthesis in ripening tomatoes (Lycopersicon esculentum Mill.) when compared to controls kept in the dark. Red illumination following far red illumination reversed the inhibitory action of far red light on carotenoid biosynthesis, suggesting a phytochrome-mediated process. Quantitation of individual carotenoids favored the hypothesis of two separate carotenoid biosynthetic pathways in tomatoes.

  16. Modification of carotenoid levels by abscission agents and expression of carotenoid biosynthetic genes in 'valencia' sweet orange.

    PubMed

    Alferez, Fernando; Pozo, Luis V; Rouseff, Russell R; Burns, Jacqueline K

    2013-03-27

    The effect of 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) and ethephon on peel color, flavedo carotenoid gene expression, and carotenoid accumulation was investigated in mature 'Valencia' orange ( Citrus sinensis L. Osbeck) fruit flavedo at three maturation stages. Abscission agent application altered peel color. CMNP was more effective than ethephon in promoting green-to-red (a) and blue-to-yellow (b) color at the middle and late maturation stages and total carotenoid changes at all maturation stages. Altered flow of carotenoid precursors during maturation due to abscission agents was suggested by changes in phytoene desaturase (Pds) and ζ-carotene desaturase (Zds) gene expression. However, each abscission agent affected downstream expression differentially. Ethephon application increased β-carotene hydroxilase (β-Chx) transcript accumulation 12-fold as maturation advanced from the early to middle and late stages. CMNP markedly increased β- and ε-lycopene cyclase (Lcy) transcript accumulation 45- and 15-fold, respectively, at midmaturation. Patterns of carotenoid accumulation in flavedo were supported in part by gene expression changes. CMNP caused greater accumulation of total flavedo carotenoids at all maturation stages when compared with ethephon or controls. In general, CMNP treatment increased total red carotenoids more than ethephon or the control but decreased total yellow carotenoids at each maturation stage. In control fruit flavedo, total red carotenoids increased and yellow carotenoids decreased as maturation progressed. Trends in total red carotenoids during maturation were consistent with measured a values. Changes in carotenoid accumulation and expression patterns in flavedo suggest that regulation of carotenoid accumulation is under transcriptional, translational, and post-translational control.

  17. 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.

  18. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms.

    PubMed

    Wang, Yan; Wang, Liuqing; Liu, Fei; Wang, Qi; Selvaraj, Jonathan Nimal; Xing, Fuguo; Zhao, Yueju; Liu, Yang

    2016-03-21

    Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms.

  19. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms

    PubMed Central

    Wang, Yan; Wang, Liuqing; Liu, Fei; Wang, Qi; Selvaraj, Jonathan Nimal; Xing, Fuguo; Zhao, Yueju; Liu, Yang

    2016-01-01

    Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms. PMID:27007394

  20. De novo genetic engineering of the camalexin biosynthetic pathway.

    PubMed

    Møldrup, Morten E; Salomonsen, Bo; Geu-Flores, Fernando; Olsen, Carl E; Halkier, Barbara A

    2013-09-10

    Camalexin is a tryptophan-derived phytoalexin that is induced in the model plant Arabidopsis thaliana upon pathogen attack. Only few genes in the biosynthetic pathway of camalexin remain unidentified, however, investigation of candidate genes for these steps has proven particularly difficult partly because of redundancy in the genome of Arabidopsis. Here we describe metabolic engineering of the camalexin biosynthetic pathway in the transient Nicotiana benthamiana expression system. Camalexin accumulated in levels corresponding to what is seen in induced Arabidopsis thaliana. We have used this system to evaluate candidate genes suggested to be involved in the camalexin pathway. This has provided biochemical evidence for CYP71A12 conducting same reaction as CYP71A13 in the pathway. We discuss the prospects of using metabolic engineering of camalexin, both with respect to engineering plant defense and as a tool for screening yet unidentified candidate genes in the camalexin pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Bioengineering natural product biosynthetic pathways for therapeutic applications.

    PubMed

    Wu, Ming-Cheng; Law, Brian; Wilkinson, Barrie; Micklefield, Jason

    2012-12-01

    With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications.

  2. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids.

    PubMed

    Kishimoto, Shinji; Sato, Michio; Tsunematsu, Yuta; Watanabe, Kenji

    2016-08-18

    Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

  3. Mining and engineering natural-product biosynthetic pathways.

    PubMed

    Wilkinson, Barrie; Micklefield, Jason

    2007-07-01

    Natural products continue to fulfill an important role in the development of therapeutic agents. In addition, with the advent of chemical genetics and high-throughput screening platforms, these molecules have become increasingly valuable as tools for interrogating fundamental aspects of biological systems. To access the vast portion of natural-product structural diversity that remains unexploited for these and other applications, genome mining and microbial metagenomic approaches are proving particularly powerful. When these are coupled with recombineering and related genetic tools, large biosynthetic gene clusters that remain intractable or cryptic in the native host can be more efficiently cloned and expressed in a suitable heterologous system. For lead optimization and the further structural diversification of natural-product libraries, combinatorial biosynthetic engineering has also become indispensable. However, our ability to rationally redesign biosynthetic pathways is often limited by our lack of understanding of the structure, dynamics and interplay between the many enzymes involved in complex biosynthetic pathways. Despite this, recent structures of fatty acid synthases should allow a more accurate prediction of the likely architecture of related polyketide synthase and nonribosomal peptide synthetase multienzymes.

  4. A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus.

    PubMed

    Galpaz, Navot; Ronen, Gil; Khalfa, Zehava; Zamir, Dani; Hirschberg, Joseph

    2006-08-01

    Carotenoids and their oxygenated derivatives xanthophylls play essential roles in the pigmentation of flowers and fruits. Wild-type tomato (Solanum lycopersicum) flowers are intensely yellow due to accumulation of the xanthophylls neoxanthin and violaxanthin. To study the regulation of xanthophyll biosynthesis, we analyzed the mutant white-flower (wf). It was found that the recessive wf phenotype is caused by mutations in a flower-specific beta-ring carotene hyroxylase gene (CrtR-b2). Two deletions and one exon-skipping mutation in different CrtR-b2 wf alleles abolish carotenoid biosynthesis in flowers but not leaves, where the homologous CrtR-b1 is constitutively expressed. A second beta-carotene hydroxylase enzyme as well as flower- and fruit-specific geranylgeranyl diphosphate synthase, phytoene synthase, and lycopene beta-cyclase together define a carotenoid biosynthesis pathway active in chromoplasts only, underscoring the crucial role of gene duplication in specialized plant metabolic pathways. We hypothesize that this pathway in tomato was initially selected during evolution to enhance flower coloration and only later recruited to enhance fruit pigmentation. The elimination of beta-carotene hydroxylation in wf petals results in an 80% reduction in total carotenoid concentration, possibly caused by the inability of petals to store high concentrations of carotenoids other than xanthophylls and by degradation of beta-carotene, which accumulates as a result of the wf mutation but is not due to altered expression of genes in the biosynthetic pathway.

  5. The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments.

    PubMed

    Colasuonno, Pasqualina; Lozito, Maria Luisa; Marcotuli, Ilaria; Nigro, Domenica; Giancaspro, Angelica; Mangini, Giacomo; De Vita, Pasquale; Mastrangelo, Anna Maria; Pecchioni, Nicola; Houston, Kelly; Simeone, Rosanna; Gadaleta, Agata; Blanco, Antonio

    2017-01-31

    In plants carotenoids play an important role in the photosynthetic process and photo-oxidative protection, and are the substrate for the synthesis of abscisic acid and strigolactones. In addition to their protective role as antioxidants and precursors of vitamin A, in wheat carotenoids are important as they influence the colour (whiteness vs. yellowness) of the grain. Understanding the genetic basis of grain yellow pigments, and identifying associated markers provide the basis for improving wheat quality by molecular breeding. Twenty-four candidate genes involved in the biosynthesis and catabolism of carotenoid compounds have been identified in wheat by comparative genomics. Single nucleotide polymorphisms (SNPs) found in the coding sequences of 19 candidate genes allowed their chromosomal location and accurate map position on two reference consensus maps to be determined. The genome-wide association study based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs validated quantitative trait loci (QTLs) previously detected in biparental populations and discovered new QTLs for grain colour-related traits. Ten carotenoid genes mapped in chromosome regions underlying pigment content QTLs indicating possible functional relationships between candidate genes and the trait. The availability of linked, candidate gene-based markers can facilitate breeding wheat cultivars with desirable levels of carotenoids. Identifying QTLs linked to carotenoid pigmentation can contribute to understanding genes underlying carotenoid accumulation in the wheat kernels. Together these outputs can be combined to exploit the genetic variability of colour-related traits for the nutritional and commercial improvement of wheat products.

  6. Manipulating Natural Product Biosynthetic Pathways via DNA Assembler

    PubMed Central

    Shao, Zengyi; Zhao, Huimin

    2014-01-01

    DNA assembler is an efficient synthetic biology method for constructing and manipulating biochemical pathways. The rapidly increasing number of sequenced genomes provides a rich source for discovery of gene clusters involved in synthesizing new natural products. However, both discovery and economical production are hampered by our limited knowledge in manipulating most organisms and the corresponding pathways. By taking advantage of yeast in vivo homologous recombination, DNA assembler synthesizes an entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication. Here we use the spectinabilin clusters originated from two hosts as examples to illustrate the guidelines of using DNA assembler for cluster characterization and silent cluster activation. Such strategies offer unprecedented versatility in cluster manipulation, bypass the traditional laborious strategies to elicit pathway expression, and provide a new platform for de novo cluster assembly and genome mining for discovering new natural products. PMID:24903884

  7. Manipulating natural product biosynthetic pathways via DNA assembler.

    PubMed

    Shao, Zengyi; Zhao, Huimin

    2014-06-03

    DNA assembler is an efficient synthetic biology method for constructing and manipulating biochemical pathways. The rapidly increasing number of sequenced genomes provides a rich source for discovery of gene clusters involved in synthesizing new natural products. However, both discovery and economical production are hampered by our limited knowledge in manipulating most organisms and the corresponding pathways. By taking advantage of yeast in vivo homologous recombination, DNA assembler synthesizes an entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication. Here we use the spectinabilin clusters originated from two hosts as examples to illustrate the guidelines of using DNA assembler for cluster characterization and silent cluster activation. Such strategies offer unprecedented versatility in cluster manipulation, bypass the traditional laborious strategies to elicit pathway expression, and provide a new platform for de novo cluster assembly and genome mining for discovering new natural products.

  8. Heterologous activation of the actinorhodin biosynthetic pathway in Streptomyces lividans.

    PubMed Central

    Romero, N M; Parro, V; Malpartida, F; Mellado, R P

    1992-01-01

    A DNA fragment of Streptomyces fradiae is able to activate the antibiotic actinorhodin biosynthetic pathway when cloned in Streptomyces lividans. The activator DNA region has been sequenced and its transcription initiation and termination sites accurately mapped in vivo. This DNA encodes a 132 nucleotides long transcript which is apparently responsible for the actinorhodin production phenotype, possibly acting as an antisense RNA. The sequence of the activator gene revealed no homology with any other known Streptomyces coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Images PMID:1614864

  9. Substrate specificity of the sialic acid biosynthetic pathway

    SciTech Connect

    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.

    2001-07-18

    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

  10. Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway.

    PubMed

    Liu, Qing; Manzano, David; Tanić, Nikola; Pesic, Milica; Bankovic, Jasna; Pateraki, Irini; Ricard, Lea; Ferrer, Albert; de Vos, Ric; van de Krol, Sander; Bouwmeester, Harro

    2014-05-01

    Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.4μgg(-1) parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding a costunolide and parthenolide 3β-hydroxylase was identified opening up further options to improve the water solubility of parthenolide and therefore its potential as a drug. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  11. Hyperglycemia exacerbates colon cancer malignancy through hexosamine biosynthetic pathway.

    PubMed

    Vasconcelos-Dos-Santos, A; Loponte, H F B R; Mantuano, N R; Oliveira, I A; de Paula, I F; Teixeira, L K; de-Freitas-Junior, J C M; Gondim, K C; Heise, N; Mohana-Borges, R; Morgado-Díaz, J A; Dias, W B; Todeschini, A R

    2017-03-20

    Hyperglycemia is a common feature of diabetes mellitus, considered as a risk factor for cancer. However, its direct effects in cancer cell behavior are relatively unexplored. Herein we show that high glucose concentration induces aberrant glycosylation, increased cell proliferation, invasion and tumor progression of colon cancer. By modulating the activity of the rate-limiting enzyme, glutamine-fructose-6-phosphate amidotransferase (GFAT), we demonstrate that hexosamine biosynthetic pathway (HBP) is involved in those processes. Biopsies from patients with colon carcinoma show increased levels of GFAT and consequently aberrant glycans' expression suggesting an increase of HBP flow in human colon cancer. All together, our results open the possibility that HBP links hyperglycemia, aberrant glycosylation and tumor malignancy, and suggest this pathway as a potential therapeutic target for colorectal cancer.

  12. Convergent biosynthetic pathways to β-lactam antibiotics

    PubMed Central

    Townsend, Craig A.

    2016-01-01

    Five naturally-occurring β-lactams have inspired a class of drugs that constitute >60% of the antimicrobials used in human medicine. Their biosynthetic pathways reveal highly individualized synthetic strategies that yet converge on a common azetidinone ring assembled in structural contexts that confer selective binding and inhibition of D,D-transpeptidases that play essential roles in bacterial cell wall (peptidoglycan) biosynthesis. These enzymes belong to a single “clan” of evolutionarily distinct serine hydrolases whose active site geometry and mechanism of action is specifically matched by these antibiotics for inactivation that is kinetically competitive with their native function. Unusual enzyme-mediated reactions and catalytic multitasking in these pathways are discussed with particular attention to the diverse ways the β-lactam itself is generated, and more broadly how the intrinsic reactivity of this core structural element is modulated in natural systems through the introduction of ring strain and electronic effects. PMID:27693891

  13. Evolution of the isoprene biosynthetic pathway in kudzu.

    PubMed

    Sharkey, Thomas D; Yeh, Sansun; Wiberley, Amy E; Falbel, Tanya G; Gong, Deming; Fernandez, Donna E

    2005-02-01

    Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.

  14. 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.

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

    DTIC Science & Technology

    2011-10-17

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

  16. Detection of additional genes of the patulin biosynthetic pathway in Penicillium griseofulvum

    USDA-ARS?s Scientific Manuscript database

    Genes in the patulin biosynthetic pathway are likely to be arranged in a cluster as has been found for biosynthetic pathways of other mycotoxins. The mycotoxin patulin, common in apples and apple juice, is most often associated with Penicillium expansum. However, of 15 fungal species capable of sy...

  17. 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.

  18. BIOSYNTHESIS OF YEAST CAROTENOIDS

    PubMed Central

    Simpson, Kenneth L.; Nakayama, T. O. M.; Chichester, C. O.

    1964-01-01

    Simpson, Kenneth L. (University of California, Davis), T. O. M. Nakayama, and C. O. Chichester. Biosynthesis of yeast carotenoids. J. Bacteriol. 88:1688–1694. 1964.—The biosynthesis of carotenoids was followed in Rhodotorula glutinis and in a new strain, 62-506. The treatment of the growing cultures by methylheptenone, or ionone, vapors permitted observations of the intermediates in the biosynthetic pathway. On the basis of concentration changes and accumulation in blocked pathways, the sequence of carotenoid formation is postulated as phytoene, phytofluene, ζ-carotene, neurosporene, β-zeacarotene, γ-carotene, torulin, a C40 aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  19. Chemically inducible expression of the PHB biosynthetic pathway in Arabidopsis.

    PubMed

    Kourtz, Lauralynn; Dillon, Kevin; Daughtry, Sean; Peoples, Oliver P; Snell, Kristi D

    2007-12-01

    Arabidopsis plants were transformed with a multi-gene construct for expression of the polyhydroxybutyrate (PHB) biosynthetic pathway containing a gene switch that can be activated by commercially available non-steroidal ecdysone analogs approved for use on some crops as pesticides. T(1) progeny of transgenic Arabidopsis plants were isolated and screened for PHB production in the presence of ecdysone analogs. T(2) progeny derived from selected T(1) lines were subjected to further analysis by comparing PHB production levels prior to treatment with inducing agent and 21 days after initiation of induction. Significant PHB production was delayed in many of the engineered plants until after induction. PHB levels of up to 14.3% PHB per unit dry weight were observed in young leaves harvested from engineered T(2) plants after applications of the commercial ecdysone analog Mimic. PHB in older leaves reached levels of up to 7% PHB per unit dry weight. This study represents a first step towards engineering a chemically inducible gene switch for PHB production in plants using inducing agents that are approved for field use.

  20. 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

  1. An alternative carotenoid-to-bacteriochlorophyll energy transfer pathway in photosynthetic light harvesting.

    PubMed

    Papagiannakis, Emmanouil; Kennis, John T M; van Stokkum, Ivo H M; Cogdell, Richard J; van Grondelle, Rienk

    2002-04-30

    Blue and green sunlight become available for photosynthetic energy conversion through the light-harvesting (LH) function of carotenoids, which involves transfer of carotenoid singlet excited states to nearby (bacterio)chlorophylls (BChls). The excited-state manifold of carotenoids usually is described in terms of two singlet states, S(1) and S(2), of which only the latter can be populated from the ground state by the absorption of one photon. Both states are capable of energy transfer to (B)Chl. We recently showed that in the LH1 complex of the purple bacterium Rhodospirillum rubrum, which is rather inefficient in carotenoid-to-BChl energy transfer, a third additional carotenoid excited singlet state is formed. This state, which we termed S*, was found to be a precursor on an ultrafast fission reaction pathway to carotenoid triplet state formation. Here we present evidence that S* is formed with significant yield in the LH2 complex of Rhodobacter sphaeroides, which has a highly efficient carotenoid LH function. We demonstrate that S* is actively involved in the energy transfer process to BChl and thus have uncovered an alternative pathway of carotenoid-to-BChl energy transfer. In competition with energy transfer to BChl, fission occurs from S*, leading to ultrafast formation of carotenoid triplets. Analysis in terms of a kinetic model indicates that energy transfer through S* accounts for 10-15% of the total energy transfer to BChl, and that inclusion of this pathway is necessary to obtain a highly efficient LH function of carotenoids.

  2. Physiological factors affecting transcription of genes involved in the flavonoid biosynthetic pathway in different rice varieties.

    PubMed

    Chen, Xiaoqiong; Itani, Tomio; Wu, Xianjun; Chikawa, Yuuki; Irifune, Kohei

    2013-01-01

    Flavonoids play an important role in the grain color and flavor of rice. Since their characterization in maize, the flavonoid biosynthetic genes have been extensively studied in grape, Arabidopsis, and Petunia. However, we are still a long way from understanding the molecular features and mechanisms underlying the flavonoid biosynthetic pathway. The present study was undertaken to understand the physiological factors affecting the transcription and regulation of these genes. We report that the expression of CHI, CHS, DFR, LAR, and ANS, the 5 flavonoid biosynthetic genes in different rice varieties, differ dramatically with respect to the stage of development, white light, and sugar concentrations. We further demonstrate that white light could induce the transcription of the entire flavonoid biosynthetic gene pathway; however, differences were observed in the degrees of sensitivity and the required illumination time. Our study provides valuable insights into understanding the regulation of the flavonoid biosynthetic pathway.

  3. Specific estradiol biosynthetic pathway in choriocarcinoma (JEG-3) cell line.

    PubMed

    Samson, Mélanie; Labrie, Fernand; Luu-The, Van

    2009-09-01

    Estradiol (E2) plays a crucial role in all reproduction processes. In the placenta, it is well recognized that E2 is synthesized from fetal dehydroepiandrosterone sulfate (DHEAS). However, there is some controversy about the biosynthetic pathway involved, some authors suggest that E2 is produced by aromatization of testosterone (T), while others suggest that E2 is produced by the conversion of estrone (E1) into E2 by type 1 17beta-HSD, subsequent to the aromatization of 4-androstenedione (4-dione) into E1. In the present report, using the precursor [(14)C]DHEA, inhibitors of steroidogenic enzymes (chemical inhibitors and siRNA) and a choriocarcinoma (JEG-3) cell line that expresses all the enzymes necessary to transform DHEA into E2, we could determine the sequential steps and the specific steroidogenic enzymes involved in the transformation of DHEA into E2. Quantification of mRNA expression levels using real-time PCR, strongly suggests that type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD1), aromatase and type 1 17beta-HSD (17beta-HSD1) that are highly expressed in JEG-3 cells are the enzymes responsible for the transformation of DHEA into E2. Analysis of the intermediates produced in the absence and presence of 3beta-HSD, aromatase and 17beta-HSD1 inhibitors permits to determine the following sequential steps: DHEA is transformed into 4-dione by 3beta-HSD1, then 4-dione is aromatized into E1 by aromatase and E1 is finally transformed into E2 by 17beta-HSD1. Our data are clearly in favor of the pathway in which the step of aromatization precedes the step of reduction by 17beta-HSD.

  4. The Magnesium Branch of the Tetrapyrrole Biosynthetic Pathway

    SciTech Connect

    Beale, S. I.

    2004-05-11

    It should be noted that the focus of the research changed somewhat during the course of the current award. The initial focus is indicated by the title of the current grant, ''The Magnesium Branch of the Chlorophyll Biosynthetic Pathway''. During the current grant period, Dr. Robert Willows, a postdoctoral associate, joined the faculty of McQuarie University in Australia. When he left my lab, we decided that he should independently pursue research on structure/function relationships in Mg chelatase and that our laboratories would collaborate on regulatory studies of this enzyme. Also, during the current award period, I began collaborating with Dr. Ariane Atteia and Mr. Robert van Lis, who were at the time located at the Autonomous University of Mexico. Dr. Atteia has since joined my laboratory and Mr. van Lis will also do so when he obtains his Ph.D. in the near future. These individuals bring to the laboratory their interests and expertise in the respiratory components of Chlamydomonas and their desire to become experts in tetrapyrrole metabolism. Recently, in a collaboration with Dr. David Bollivar, a former postdoctoral associate who is now at Illinois Wesleyan University, and Dr. Caroline Walker, who was at Clemson University but has since left this research area, we recently made a major breakthrough on the oxygen-independent cyclase reaction, which has now become an important component of the current proposal. Finally, our research on phycobilin biosynthesis in Synechucystis has revealed that this organism can grow at very low oxygen concentrations and its genome contains several genes that may encode for enzymes that catalyze alternative oxygen-independent reactions for tetrapyrrole biosynthesis, so characterizing the genes, their enzymes, and regulation of expression have also become parts of the current proposal.

  5. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis

    USDA-ARS?s Scientific Manuscript database

    Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control caro...

  6. 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

  7. Pathway engineering strategies for production of beneficial carotenoids in microbial hosts.

    PubMed

    Ye, Victor M; Bhatia, Sujata K

    2012-08-01

    Carotenoids, such as lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.

  8. Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between C. reticulata and C. limon: the case of pulp carotenoid biosynthesis pathway.

    PubMed

    Bassene, Jean Baptiste; Froelicher, Yann; Dhuique-Mayer, Claudie; Mouhaya, Waffa; Ferrer, Rosa Mar; Ancillo, Gema; Morillon, Raphael; Navarro, Luis; Ollitrault, Patrick

    2009-11-01

    Allopolyploidy is known to induce novel patterns of gene expression and often gives rise to new phenotypes. Here we report on the first attempt to relate phenotypic inheritance in an allotetraploid somatic hybrid with gene expression. Carotenoid compounds in the fruit pulp of the two parental species and the hybrid were evaluated quantitatively by HPLC. Only very low levels of beta-carotene and beta-cryptoxanthin were observed in Citrus limon, while beta-cryptoxanthin was a major component of C. reticulata, which also displayed high levels of phytoene, phytofluene, beta-carotene, lutein, zeaxantin and violaxanthin. Total carotenoid content in mandarin juice sacs was 60 times greater than that in lemon. The allotetraploid hybrid produced all the same compounds as mandarin but at very low levels. Transgressive concentration of abscisic acid (ABA) was observed in the somatic hybrid. Real-time RT-PCR of total RNA from juice sacs was used to study expression of seven genes (CitDxs, CitPsy, CitPds, CitZds, CitLcy-b, CitChx-b, and CitZep) of the carotenoid biosynthetic pathway and two genes (CitNced1 and CitNced2) involved in abscisic acid synthesis from carotenoid. Gene expression was significantly higher for mandarin than lemon for seven of the nine genes analyzed. Lemon under expression was partially dominant in the somatic hybrid for three upstream steps of the biosynthetic pathway, particularly for CitDxs. Transgressive over expression was observed for the two CitNced genes. A limitation of the upstream steps of the pathway and a downstream higher consumption of carotenoids may explain the phenotype of the somatic hybrid.

  9. The strigolactone germination stimulants of the plant-parasitic Striga and Orobanche spp. are derived from the carotenoid pathway.

    PubMed

    Matusova, Radoslava; Rani, Kumkum; Verstappen, Francel W A; Franssen, Maurice C R; Beale, Michael H; Bouwmeester, Harro J

    2005-10-01

    The seeds of parasitic plants of the genera Striga and Orobanche will only germinate after induction by a chemical signal exuded from the roots of their host. Up to now, several of these germination stimulants have been isolated and identified in the root exudates of a series of host plants of both Orobanche and Striga spp. In most cases, the compounds were shown to be isoprenoid and belong to one chemical class, collectively called the strigolactones, and suggested by many authors to be sesquiterpene lactones. However, this classification was never proven; hence, the biosynthetic pathways of the germination stimulants are unknown. We have used carotenoid mutants of maize (Zea mays) and inhibitors of isoprenoid pathways on maize, cowpea (Vigna unguiculata), and sorghum (Sorghum bicolor) and assessed the effects on the root exudate-induced germination of Striga hermonthica and Orobanche crenata. Here, we show that for these three host and two parasitic plant species, the strigolactone germination stimulants are derived from the carotenoid pathway. Furthermore, we hypothesize how the germination stimulants are formed. We also discuss this finding as an explanation for some phenomena that have been observed for the host-parasitic plant interaction, such as the effect of mycorrhiza on S. hermonthica infestation.

  10. The Strigolactone Germination Stimulants of the Plant-Parasitic Striga and Orobanche spp. Are Derived from the Carotenoid Pathway1

    PubMed Central

    Matusova, Radoslava; Rani, Kumkum; Verstappen, Francel W.A.; Franssen, Maurice C.R.; Beale, Michael H.; Bouwmeester, Harro J.

    2005-01-01

    The seeds of parasitic plants of the genera Striga and Orobanche will only germinate after induction by a chemical signal exuded from the roots of their host. Up to now, several of these germination stimulants have been isolated and identified in the root exudates of a series of host plants of both Orobanche and Striga spp. In most cases, the compounds were shown to be isoprenoid and belong to one chemical class, collectively called the strigolactones, and suggested by many authors to be sesquiterpene lactones. However, this classification was never proven; hence, the biosynthetic pathways of the germination stimulants are unknown. We have used carotenoid mutants of maize (Zea mays) and inhibitors of isoprenoid pathways on maize, cowpea (Vigna unguiculata), and sorghum (Sorghum bicolor) and assessed the effects on the root exudate-induced germination of Striga hermonthica and Orobanche crenata. Here, we show that for these three host and two parasitic plant species, the strigolactone germination stimulants are derived from the carotenoid pathway. Furthermore, we hypothesize how the germination stimulants are formed. We also discuss this finding as an explanation for some phenomena that have been observed for the host-parasitic plant interaction, such as the effect of mycorrhiza on S. hermonthica infestation. PMID:16183851

  11. Carotenoid biosynthesis in extremophilic Deinococcus-Thermus bacteria.

    PubMed

    Tian, Bing; Hua, Yuejin

    2010-11-01

    Bacteria from the phylum Deinococcus-Thermus are known for their resistance to extreme stresses including radiation, oxidation, desiccation and high temperature. Cultured Deinococcus-Thermus bacteria are usually red or yellow pigmented because of their ability to synthesize carotenoids. Unique carotenoids found in these bacteria include deinoxanthin from Deinococcus radiodurans and thermozeaxanthins from Thermus thermophilus. Investigations of carotenogenesis will help to understand cellular stress resistance of Deinococcus-Thermus bacteria. Here, we discuss the recent progress toward identifying carotenoids, carotenoid biosynthetic enzymes and pathways in some species of Deinococcus-Thermus extremophiles. In addition, we also discuss the roles of carotenoids in these extreme bacteria.

  12. New biosynthetic pathway for pink pigments from uncultured oceanic viruses.

    PubMed

    Ledermann, Benjamin; Béjà, Oded; Frankenberg-Dinkel, Nicole

    2016-12-01

    The pink open-chain tetrapyrrole pigment phycoerythrobilin (PEB) is employed by marine cyanobacteria, red algae and cryptophytes as a light-harvesting chromophore in phycobiliproteins. Genes encoding biosynthesis proteins for PEB have also been discovered in cyanophages, viruses that infect cyanobacteria, and mimic host pigment biosynthesis with the exception of PebS which combines the enzymatic activities of two host enzymes. In this study, we have identified novel members of the PEB biosynthetic enzyme families, heme oxygenases and ferredoxin-dependent bilin reductases. Encoding genes were found in metagenomic datasets and could be traced back to bacteriophage but not cyanophage origin. While the heme oxygenase exhibited standard activity, a new bilin reductase with highest homology to the teal pigment producing enzyme PcyA revealed PEB biosynthetic activity. Although PcyX possesses PebS-like activity both enzymes share only 9% sequence identity and likely catalyze the reaction via two independent mechanisms. Our data point towards the presence of phycobilin biosynthetic genes in phages that probably infect alphaproteobacteria and, therefore, further support a role of phycobilins outside oxygenic phototrophs.

  13. Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway.

    PubMed

    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

    2015-02-01

    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 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%). 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.

  14. 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

  15. Enzymic Pathways for Formation of Carotenoid Cleavage Products

    NASA Astrophysics Data System (ADS)

    Fleischmann, Peter; Zorn, Holger

    Degraded carotenoids (apocarotenoids, norisoprenoids) have been a subject of intensive research for several decades. From the perspective of human physiology and nutrition, the retinoids, acting as vitamins, signalling molecules, and visual pigments, attracted the greatest attention (Chapters 15 and 16). Plant scientists, however, detected a wealth of different apocarotenoids, presumably derived by the excentric cleavage of carotenoids in various species, the plant hormone abscisic acid (1, Scheme 6) being the best-investigated example. With the onset of fruit ripening, flower opening or senescence of green tissues, carotenoids are degraded oxidatively to smaller, volatile compounds. The natural biological functions of the reaction products are outlined in Chapter 15. As many of these apocarotenoids act as potent flavour compounds, food chemists and flavourists worldwide have investigated meticulously their structural and sensory properties. Many aspects of carotenoid metabolites and breakdown products as aroma compounds are presented in a comprehensive book [1].

  16. Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae.

    PubMed

    Newman, Adam G; Townsend, Craig A

    2016-03-30

    Perylenequinones are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens that notably produce reactive oxygen species with visible light. The best-studied perylenequinone is cercosporin-a product of the Cercospora species. While the cercosporin biosynthetic gene cluster has been described in the tobacco pathogen Cercospora nicotianae, little is known of the metabolite's biosynthesis. Furthermore, in vitro investigations of the polyketide synthase central to cercosporin biosynthesis identified the naphthopyrone nor-toralactone as its direct product-an observation in conflict with published biosynthetic proposals. Here, we present an alternative biosynthetic pathway to cercosporin based on metabolites characterized from a series of biosynthetic gene knockouts. We show that nor-toralactone is the key polyketide intermediate and the substrate for the unusual didomain protein CTB3. We demonstrate the unique oxidative cleavage activity of the CTB3 monooxygenase domain in vitro. These data advance our understanding of perylenequinone biosynthesis and expand the biochemical repertoire of flavin-dependent monooxygenases.

  17. The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L.

    PubMed Central

    2012-01-01

    Background Carotenoids are a heterogeneous group of plant isoprenoids primarily involved in photosynthesis. In plants the cleavage of carotenoids leads to the formation of the phytohormones abscisic acid and strigolactone, and C13-norisoprenoids involved in the characteristic flavour and aroma compounds in flowers and fruits and are of specific importance in the varietal character of grapes and wine. This work extends the previous reports of carotenoid gene expression and photosynthetic pigment analysis by providing an up-to-date pathway analysis and an important framework for the analysis of carotenoid metabolic pathways in grapevine. Results Comparative genomics was used to identify 42 genes putatively involved in carotenoid biosynthesis/catabolism in grapevine. The genes are distributed on 16 of the 19 chromosomes and have been localised to the physical map of the heterozygous ENTAV115 grapevine sequence. Nine of the genes occur as single copies whereas the rest of the carotenoid metabolic genes have more than one paralogue. The cDNA copies of eleven corresponding genes from Vitis vinifera L. cv. Pinotage were characterised, and four where shown to be functional. Microarrays provided expression profiles of 39 accessions in the metabolic pathway during three berry developmental stages in Sauvignon blanc, whereas an optimised HPLC analysis provided the concentrations of individual carotenoids. This provides evidence of the functioning of the lutein epoxide cycle and the respective genes in grapevine. Similarly, orthologues of genes leading to the formation of strigolactone involved in shoot branching inhibition were identified: CCD7, CCD8 and MAX1. Moreover, the isoforms typically have different expression patterns, confirming the complex regulation of the pathway. Of particular interest is the expression pattern of the three VvNCEDs: Our results support previous findings that VvNCED3 is likely the isoform linked to ABA content in berries. Conclusions The

  18. Development of fruit color in Solanaceae: a story of two biosynthetic pathways.

    PubMed

    Dhar, Manoj K; Sharma, Rupali; Koul, Archana; Kaul, Sanjana

    2015-05-01

    This review highlights the major differences between the regulation of two important pathways namely anthocyanin and carotenoid pathways, responsible for fruit color generation in Solanaceae mediated by transcription factors (TFs). The anthocyanin pathway is regulated by a common set of TFs (MYB, MYC and WD40) belonging to specific families of DNA-binding proteins. Their regulation is aimed at controlling the type and amount of pigments produced and the physiological conditions (like pH) at which they are finally stored. In the carotenoid pathway, the color diversity depends on the quantity of pigment produced and the point where the pathway is arrested. TFs in the latter case are accordingly found to influence the sequestration and degradation of these pigments, which determines their final concentration in the tissue. TFs (phytochrome interacting factors, MADS-BOX, HB-ZIP and B-ZIP) also regulate important rate-determining steps, which decide the direction in which the pathway proceeds and the point at which it is terminated. In the absence of a clear pattern of TF-mediated regulation, it is suggested that the carotenoid pathway is more significantly influenced by other regulatory methods which need to be explored. It is expected that common factors affecting these pathways are the ones acting much before the initiation of the biosynthesis of respective pigments.

  19. Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

    PubMed Central

    Ohyama, Kiyoshi; Suzuki, Masashi; Kikuchi, Jun; Saito, Kazuki; Muranaka, Toshiya

    2009-01-01

    The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-13C2H3]mevalonate with Arabidopsis seedlings. Applying 13C-{1H}{2H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol “the lanosterol pathway.” LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as β-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites. PMID:19139393

  20. Carotenoid profiling and biosynthetic gene expression in flesh and peel of wild-type and hp-1 tomato fruit under UV-B depletion.

    PubMed

    Lazzeri, Valerio; Calvenzani, Valentina; Petroni, Katia; Tonelli, Chiara; Castagna, Antonella; Ranieri, Annamaria

    2012-05-16

    Although light is recognized as one of the main factors influencing fruit carotenogenesis, the specific role of UV-B radiation has been poorly investigated. The present work is addressed to assess the molecular events underlying carotenoid accumulation in presence or absence of ultraviolet-B (UV-B) light in tomato fruits of wild-type and high pigment-1 (hp-1), a mutant characterized by exaggerated photoresponsiveness and increased fruit pigmentation. Gene expression analyses indicated that in wild-type fruits UV-B radiation mainly negatively affects the carotenoid biosynthetic genes encoding enzymes downstream of lycopene both in flesh and peel, suggesting that the down-regulation of genes CrtL-b and CrtL-e and the subsequent accumulation of lycopene during tomato ripening are determined at least in part by UV-B light. In contrast to wild-type, UV-B depletion did not greatly affect carotenoid accumulation in hp-1 and generally determined minor differences in gene expression between control and UV-B-depleted conditions.

  1. Phylogenetic and Evolutionary Patterns in Microbial Carotenoid Biosynthesis Are Revealed by Comparative Genomics

    PubMed Central

    Klassen, Jonathan L.

    2010-01-01

    Background Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data. Methodology/Principal Findings Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection. Conclusions/Significance Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This

  2. Subchromoplast sequestration of carotenoids affects regulatory mechanisms in tomato lines expressing different carotenoid gene combinations.

    PubMed

    Nogueira, Marilise; Mora, Leticia; Enfissi, Eugenia M A; Bramley, Peter M; Fraser, Paul D

    2013-11-01

    Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.

  3. Exploring Drug Targets in Isoprenoid Biosynthetic Pathway for Plasmodium falciparum.

    PubMed

    Qidwai, Tabish; Jamal, Farrukh; Khan, Mohd Y; Sharma, Bechan

    2014-01-01

    Emergence of rapid drug resistance to existing antimalarial drugs in Plasmodium falciparum has created the need for prediction of novel targets as well as leads derived from original molecules with improved activity against a validated drug target. The malaria parasite has a plant plastid-like apicoplast. To overcome the problem of falciparum malaria, the metabolic pathways in parasite apicoplast have been used as antimalarial drug targets. Among several pathways in apicoplast, isoprenoid biosynthesis is one of the important pathways for parasite as its multiplication in human erythrocytes requires isoprenoids. Therefore targeting this pathway and exploring leads with improved activity is a highly attractive approach. This report has explored progress towards the study of proteins and inhibitors of isoprenoid biosynthesis pathway. For more comprehensive analysis, antimalarial drug-protein interaction has been covered.

  4. 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

  5. PERTURBATIONS OF THE LIGNIN BIOSYNTHETIC PATHWAY AND THEIR POTENTIAL TO IMPACT PLANT CELL WALL UTILIZATION

    USDA-ARS?s Scientific Manuscript database

    The effects on lignification of perturbing most of the genes for enzymes on the monolignol biosynthetic pathway have now been reasonably well studied, particularly in angiosperms. Early studies sought to reduce lignin content with the idea of targeting the key barrier to efficient utilization of pla...

  6. Linking Biosynthetic Gene Clusters to their Metabolites via Pathway-Targeted Molecular Networking

    PubMed Central

    Trautman, Eric P.; Crawford, Jason M.

    2016-01-01

    The connection of microbial biosynthetic gene clusters to the small molecule metabolites they encode is central to the discovery and characterization of new metabolic pathways with ecological and pharmacological potential. With increasing microbial genome sequence information being deposited into publicly available databases, it is clear that microbes have the coding capacity for many more biologically active small molecules than previously realized. Of increasing interest are the small molecules encoded by the human microbiome, as these metabolites likely mediate a variety of currently uncharacterized human-microbe interactions that influence health and disease. In this mini-review, we describe the ongoing biosynthetic, structural, and functional characterizations of the genotoxic colibactin pathway in gut bacteria as a thematic example of linking biosynthetic gene clusters to their metabolites. We also highlight other natural products that are produced through analogous biosynthetic logic and comment on some current disconnects between bioinformatics predictions and experimental structural characterizations. Lastly, we describe the use of pathway-targeted molecular networking as a tool to characterize secondary metabolic pathways within complex metabolomes and to aid in downstream metabolite structural elucidation efforts. PMID:26456470

  7. 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

  8. Computational genomic identification and functional reconstitution of plant natural product biosynthetic pathways

    PubMed Central

    2016-01-01

    Covering: 2003 to 2016 The last decade has seen the first major discoveries regarding the genomic basis of plant natural product biosynthetic pathways. Four key computationally driven strategies have been developed to identify such pathways, which make use of physical clustering, co-expression, evolutionary co-occurrence and epigenomic co-regulation of the genes involved in producing a plant natural product. Here, we discuss how these approaches can be used for the discovery of plant biosynthetic pathways encoded by both chromosomally clustered and non-clustered genes. Additionally, we will discuss opportunities to prioritize plant gene clusters for experimental characterization, and end with a forward-looking perspective on how synthetic biology technologies will allow effective functional reconstitution of candidate pathways using a variety of genetic systems. PMID:27321668

  9. Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes.

    PubMed

    Wheeler, Glen; Ishikawa, Takahiro; Pornsaksit, Varissa; Smirnoff, Nicholas

    2015-03-13

    Ascorbic acid (vitamin C) is an enzyme co-factor in eukaryotes that also plays a critical role in protecting photosynthetic eukaryotes against damaging reactive oxygen species derived from the chloroplast. Many animal lineages, including primates, have become ascorbate auxotrophs due to the loss of the terminal enzyme in their biosynthetic pathway, L-gulonolactone oxidase (GULO). The alternative pathways found in land plants and Euglena use a different terminal enzyme, L-galactonolactone dehydrogenase (GLDH). The evolutionary processes leading to these differing pathways and their contribution to the cellular roles of ascorbate remain unclear. Here we present molecular and biochemical evidence demonstrating that GULO was functionally replaced with GLDH in photosynthetic eukaryote lineages following plastid acquisition. GULO has therefore been lost repeatedly throughout eukaryote evolution. The formation of the alternative biosynthetic pathways in photosynthetic eukaryotes uncoupled ascorbate synthesis from hydrogen peroxide production and likely contributed to the rise of ascorbate as a major photoprotective antioxidant.

  10. A simple biosynthetic pathway for large product generation from small substrate amounts.

    PubMed

    Djordjevic, Marko; Djordjevic, Magdalena

    2012-10-01

    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways.

  11. A simple biosynthetic pathway for large product generation from small substrate amounts

    NASA Astrophysics Data System (ADS)

    Djordjevic, Marko; Djordjevic, Magdalena

    2012-10-01

    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways.

  12. Accumulation and bioavailability of dietary carotenoids in vegetable crops.

    PubMed

    Kopsell, Dean A; Kopsell, David E

    2006-10-01

    Carotenoids are lipid-soluble pigments found in many vegetable crops that are reported to have the health benefits of cancer and eye disease reduction when consumed in the diet. Research shows that environmental and genetic factors can significantly influence carotenoid concentrations in vegetable crops, and that changing cultural management strategies could be advantageous, resulting in increased vegetable carotenoid concentrations. Improvements in vegetable carotenoid levels have been achieved using traditional breeding methods and molecular transformations to stimulate biosynthetic pathways. Postharvest and processing activities can alter carotenoid chemistry, and ultimately affect bioavailability. Bioavailability data emphasize the importance of carotenoid enhancement in vegetable crops and the need to characterize potential changes in carotenoid composition during cultivation, storage and processing before consumer purchase.

  13. 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

  14. Reassembled biosynthetic pathway for large-scale carbohydrate synthesis: alpha-Gal epitope producing "superbug".

    PubMed

    Chen, Xi; Liu, Ziye; Zhang, Jianbo; Zhang, Wei; Kowal, Przemyslaw; Wang, Peng George

    2002-01-04

    A metabolic pathway engineered Escherichia coli strain (superbug) containing one plasmid harboring an artificial gene cluster encoding all the five enzymes in the biosynthetic pathway of Galalpha l,3Lac through galactose metabolism has been developed. The plasmid contains a lambda promoter, a c1857 repressor gene, an ampicillin resistance gene, and a T7 terminator. Each gene was preceded by a Shine - Dalgarno sequence for ribosome binding. In a reaction catalyzed by the recombinant E. coli strain, Galalpha 1,3Lac trisaccharide accumulated at concentrations of 14.2 mM (7.2 gL(-1)) in a reaction mixture containing galactose, glucose, lactose, and a catalytic amount of uridine 5'-diphosphoglucose. This work demonstrates that large-scale synthesis of complex oligosaccharides can be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism.

  15. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli.

    PubMed

    Stahlhut, Steen G; Siedler, Solvej; Malla, Sailesh; Harrison, Scott J; Maury, Jérôme; Neves, Ana Rute; Forster, Jochen

    2015-09-01

    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer's disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  16. A biosynthetic regulated secretory pathway in constitutive secretory cells

    PubMed Central

    1996-01-01

    It has frequently been proposed that while the constitutive secretory pathway is present in all cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine, or exocrine types. In this study we provide evidence that suggests that this distinction is not as restrictive as proposed. We have identified a population of post-Golgi storage vesicles in several constitutive secretory cells using [35S]SO4-labeled glycosaminoglycan (GAG) chains as a marker. A fraction of this pool of vesicles can undergo exocytosis in response to stimuli such as cytoplasmic Ca2+ and phorbol esters. The effect of Ca2+ was demonstrated both in intact cells in the presence of the ionophore A23187 and in streptolysin-O-permeabilized semi-intact cells. N-ethylmaleiimide, under conditions known to block regulated and constitutive secretion, inhibited the stimulated secretion from these cells, suggesting that the observed release of labeled GAG chains was not due to a leakage artefact. Subcellular fractionation revealed that the stored GAG chains were in low-density membrane granules (d approximately 1.12 g/ml), whose size was greater than that of synaptic- like vesicles found in PC12 cells. In addition, in CHO cells that express epitope-tagged rab 3D, the labeled GAG chains were found to cofractionate with the exogenous rab protein. When expressed in the regulated cell line AtT-20, this tagged rab protein was found to colocalize with ACTH-containing dense-core granules by indirect immunofluorescence. Taken together, these results provide evidence for the presence of a cryptic regulated secretory pathway in "constitutive" cells and suggest that the regulated secretory pathway is more widespread amongst different cell types than previously believed. PMID:8682857

  17. Engineering biosynthetic pathways for deoxysugars: branched-chain sugar pathways and derivatives from the antitumor tetracenomycin.

    PubMed

    Lombó, Felipe; Gibson, Miranda; Greenwell, Lisa; Braña, Alfredo F; Rohr, Jürgen; Salas, José A; Méndez, Carmen

    2004-12-01

    Sugar biosynthesis cassette genes have been used to construct plasmids directing the biosynthesis of branched-chain deoxysugars: pFL942 (NDP-L-mycarose), pFL947 (NDP-4-deacetyl-L-chromose B), and pFL946/pFL954 (NDP-2,3,4-tridemethyl-L-nogalose). Expression of pFL942 and pFL947 in S. lividans 16F4, which harbors genes for elloramycinone biosynthesis and the flexible ElmGT glycosyltransferase of the elloramycin biosynthetic pathway, led to the formation of two compounds: 8-alpha-L-mycarosyl-elloramycinone and 8-demethyl-8-(4-deacetyl)-alpha-L-chromosyl-tetracenomycin C, respectively. Expression of pFL946 or pFL954 failed to produce detectable amounts of a novel glycosylated tetracenomycin derivative. Formation of these two compounds represents examples of the sugar cosubstrate flexibility of the ElmGT glycosyltransferase. The use of these cassette plasmids also provided insights into the substrate flexibility of deoxysugar biosynthesis enzymes as the C-methyltransferases EryBIII and MtmC, the epimerases OleL and EryBVII, and the 4-ketoreductases EryBIV and OleU.

  18. In silico tools for the analysis of antibiotic biosynthetic pathways.

    PubMed

    Weber, Tilmann

    2014-05-01

    Natural products of bacteria and fungi are the most important source for antimicrobial drug leads. For decades, such compounds were exclusively found by chemical/bioactivity-guided screening approaches. The rapid progress in sequencing technologies only recently allowed the development of novel screening methods based on the genome sequences of potential producing organisms. The basic principle of such genome mining approaches is to identify genes, which are involved in the biosynthesis of such molecules, and to predict the products of the identified pathways. Thus, bioinformatics methods and tools are crucial for genome mining. In this review, a comprehensive overview is given on programs and databases for the identification and analysis of antibiotic biosynthesis gene clusters in genomic data. Copyright © 2014 Elsevier GmbH. All rights reserved.

  19. 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

  20. A biosynthetic pathway for a prominent class of microbiota-derived bile acids

    PubMed Central

    Devlin, A. Sloan; Fischbach, Michael A.

    2015-01-01

    The gut bile acid pool is millimolar in concentration, varies widely in composition among individuals, and is linked to metabolic disease and cancer. Although these molecules derive almost exclusively from the microbiota, remarkably little is known about which bacterial species and genes are responsible for their biosynthesis. Here, we report a biosynthetic pathway for the second most abundant class in the gut, iso (3β-hydroxy) bile acids, whose levels exceed 300 µM in some humans and are absent in others. We show, for the first time, that iso bile acids are produced by Ruminococcus gnavus, a far more abundant commensal than previously known producers; and that the iso bile acid pathway detoxifies deoxycholic acid, favoring the growth of the keystone genus Bacteroides. By revealing the biosynthetic genes for an abundant class of bile acids, our work sets the stage for predicting and rationally altering the composition of the bile acid pool. PMID:26192599

  1. The Sphingolipid Biosynthetic Pathway Is a Potential Target for Chemotherapy against Chagas Disease

    PubMed Central

    Koeller, Carolina Macedo; Heise, Norton

    2011-01-01

    The protozoan parasite Trypanosoma cruzi is the causative agent of human Chagas disease, for which there currently is no cure. The life cycle of T. cruzi is complex, including an extracellular phase in the triatomine insect vector and an obligatory intracellular stage inside the vertebrate host. These phases depend on a variety of surface glycosylphosphatidylinositol-(GPI-) anchored glycoconjugates that are synthesized by the parasite. Therefore, the surface expression of GPI-anchored components and the biosynthetic pathways of GPI anchors are attractive targets for new therapies for Chagas disease. We identified new drug targets for chemotherapy by taking the available genome sequence information and searching for differences in the sphingolipid biosynthetic pathways (SBPs) of mammals and T. cruzi. In this paper, we discuss the major steps of the SBP in mammals, yeast and T. cruzi, focusing on the IPC synthase and ceramide remodeling of T. cruzi as potential therapeutic targets for Chagas disease. PMID:21603271

  2. A biosynthetic pathway for a prominent class of microbiota-derived bile acids.

    PubMed

    Devlin, A Sloan; Fischbach, Michael A

    2015-09-01

    The gut bile acid pool is millimolar in concentration, varies widely in composition among individuals and is linked to metabolic disease and cancer. Although these molecules are derived almost exclusively from the microbiota, remarkably little is known about which bacterial species and genes are responsible for their biosynthesis. Here we report a biosynthetic pathway for the second most abundant class in the gut, 3β-hydroxy(iso)-bile acids, whose levels exceed 300 μM in some humans and are absent in others. We show, for the first time, that iso-bile acids are produced by Ruminococcus gnavus, a far more abundant commensal than previously known producers, and that the iso-bile acid pathway detoxifies deoxycholic acid and thus favors the growth of the keystone genus Bacteroides. By revealing the biosynthetic genes for an abundant class of bile acids, our work sets the stage for predicting and rationally altering the composition of the bile acid pool.

  3. Streptomyces turgidiscabies possesses a functional cytokinin biosynthetic pathway and produces leafy galls.

    PubMed

    Joshi, Madhumita V; Loria, Rosemary

    2007-07-01

    Streptomyces turgidiscabies, a cause of potato scab, possesses a mobilizable pathogenicity island containing multiple virulence genes and a cytokinin biosynthetic pathway. These biosynthetic genes are homologous and collinear with the fas operon in Rhodococcus fascians. Reverse-transcriptase polymerase chain reaction of S. turgidiscabies demonstrated that all six genes were transcribed in oat bran broth with and without glucose, though transcription was partially repressed by glucose. The supernatant of S. turgidiscabies cultures had cytokinin activity in callus initiation and differentiation assays. Arabidopsis and tobacco plants inoculated with a thaxtomin-deficient mutant (deltanos) produced leafy galls, indistinguishable from those produced by R. fascians. Deletion of the ipt gene in the pathway eliminated gall phenotype. Other symptoms on tobacco included production of hairy roots and de novo meristems.

  4. Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative

    PubMed Central

    Bleeker, Petra M.; Mirabella, Rossana; Diergaarde, Paul J.; VanDoorn, Arjen; Tissier, Alain; Kant, Merijn R.; Prins, Marcel; de Vos, Martin; Haring, Michel A.; Schuurink, Robert C.

    2012-01-01

    Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance. PMID:23169639

  5. Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative.

    PubMed

    Bleeker, Petra M; Mirabella, Rossana; Diergaarde, Paul J; VanDoorn, Arjen; Tissier, Alain; Kant, Merijn R; Prins, Marcel; de Vos, Martin; Haring, Michel A; Schuurink, Robert C

    2012-12-04

    Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.

  6. Biosynthetic Pathways, Gene Replacement and the Antiquity of Life

    NASA Astrophysics Data System (ADS)

    Blankenship, R.; Raymond, J.

    2004-12-01

    Our understanding of the evolution and diversity of life during the Archean has been profoundly influenced by geological signatures including microfossils, stromatolites, and fractionated carbon and sulfur isotopes. These biosignatures have been extensively debated on the grounds of competing abiotic processes. Hopane and squalene biomarkers have been taken as evidence that by 2700 Ma, all three domains of life had emerged, that cyanobacteria, the `inventors' of oxygenic photosynthesis, had diverged from their bacterial ancestors, heralding the impending oxidation of Earth's atmosphere, and that O2 was already available, as it is required explicitly in modern organisms for the synthesis of squalene. We argue that, based on corollaries present throughout enzymology, the oxidization of squalene during the Archean could just as likely have been carried out by an anaerobic enzyme that either remains to be characterized or that may have been totally lost as eukaryotes assumed primarily aerobic lifestyles. The transition from an anoxic to an oxic world had unfathomable consequences for early life; molecular oxygen and its derivatives went from being poisonous in an obligately anaerobic world, to assuming a central role in the subsequent development of complex, macroscopic life. Though the aftermath was global in scale, the biological transition to aerobiosis was effected at the molecular level, which included the evolution of a wealth of new enzymes, the extensive modification of many existing enzymes, and the `rewiring' of central biochemical pathways.

  7. Contribution of trehalose biosynthetic pathway to drought stress tolerance of Capparis ovata Desf.

    PubMed

    Ilhan, S; Ozdemir, F; Bor, M

    2015-03-01

    Trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Among recent findings for trehalose and its metabolism, the role of signalling in the regulation of growth and development and its potential for use as a storage energy source can be listed. The xerophytic plant Capparis ovata (caper) is well adapted to drought and high temperature stress in arid and semi-arid regions of the Mediterranean. The contribution of trehalose and the trehalose biosynthetic pathway to drought stress responses and tolerance in C. ovata are not known. We investigated the effects of PEG-mediated drought stress in caper plants and analysed physiological parameters and trehalose biosynthetic pathway components, trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), trehalase activity, trehalose and proline content in drought stress-treated and untreated plants. Our results indicated that trehalose and the trehalose biosynthetic pathway contributed to drought stress tolerance of C. ovata. Overall growth and leaf water status were not dramatically affected by drought, as both high relative growth rate and relative water content were recorded even after 14 days of drought stress. Trehalose accumulation increased in parallel to induced TPS and TPP activities and decreased trehalase activity in caper plants on day 14. Constitutive trehalose levels were 28.75 to 74.75 μg·g·FW(-1) , and drought stress significantly induced trehalose accumulation (385.25 μg·g·FW(-1) on day 14) in leaves of caper. On day 14 of drought, proline levels were lower than on day 7. Under drought stress the discrepancy between trehalose and proline accumulation trends might result from the mode of action of these osmoprotectant molecules in C. ovata. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  8. Conserved biosynthetic pathways for phosalacine, bialaphos and newly discovered phosphonic acid natural products.

    PubMed

    Blodgett, Joshua A V; Zhang, Jun Kai; Yu, Xiaomin; Metcalf, William W

    2016-01-01

    Natural products containing phosphonic or phosphinic acid functionalities often display potent biological activities with applications in medicine and agriculture. The herbicide phosphinothricin-tripeptide (PTT) was the first phosphinate natural product discovered, yet despite numerous studies, questions remain surrounding key transformations required for its biosynthesis. In particular, the enzymology required to convert phosphonoformate to carboxyphosphonoenolpyruvate and the mechanisms underlying phosphorus methylation remain poorly understood. In addition, the model for non-ribosomal peptide synthetase assembly of the intact tripeptide product has undergone numerous revisions that have yet to be experimentally tested. To further investigate the biosynthesis of this unusual natural product, we completely sequenced the PTT biosynthetic locus from Streptomyces hygroscopicus and compared it with the orthologous cluster from Streptomyces viridochromogenes. We also sequenced and analyzed the closely related phosalacine (PAL) biosynthetic locus from Kitasatospora phosalacinea. Using data drawn from the comparative analysis of the PTT and PAL pathways, we also evaluate three related recently discovered phosphonate biosynthetic loci from Streptomyces sviceus, Streptomyces sp. WM6386 and Frankia alni. Our observations address long-standing biosynthetic questions related to PTT and PAL production and suggest that additional members of this pharmacologically important class await discovery.

  9. Conserved biosynthetic pathways for phosalacine, bialaphos and newly discovered phosphonic acid natural products

    PubMed Central

    Blodgett, Joshua A. V; Zhang, Jun Kai; Yu, Xiaomin; Metcalf, William W.

    2015-01-01

    Natural products containing phosphonic or phosphinic acid functionalities often display potent biological activities with applications in medicine and agriculture. The herbicide phosphinothricin-tripeptide (PTT) was the first phosphinate natural product discovered, yet despite numerous studies, questions remain surrounding key transformations required for its biosynthesis. In particular, the enzymology required to convert phosphonoformate to carboxyphosphonoenolpyruvate and the mechanisms underlying phosphorus-methylation remain poorly understood. In addition, the model for NRPS assembly of the intact tripeptide product has undergone numerous revisions that have yet to be experimentally tested. To further investigate the biosynthesis of this unusual natural product, we completely sequenced the PTT biosynthetic locus from Streptomyces hygroscopicus and compared it to the orthologous cluster from Streptomyces viridochromogenes. We also sequenced and analysed the closely related phosalacine (PAL) biosynthetic locus from Kitasatospora phosalacinea. Using data drawn from the comparative analysis of the PTT and PAL pathways, we also evaluate three related recently discovered phosphonate biosynthetic loci from Streptomyces sviceus, Streptomyces sp. WM6386 and Frankia alni. Our observations address long-standing biosynthetic questions related to PTT and PAL production and suggest that additional members of this pharmacologically important class await discovery. PMID:26328935

  10. Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae

    PubMed Central

    Newman, Adam G.; Townsend, Craig A.

    2016-01-01

    Perylenequinones are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens that notably produce reactive oxygen species with visible light. The best-studied perylenequinone is cercosporin—a product of the Cercospora species. While the cercosporin biosynthetic gene cluster has been described in the tobacco pathogen Cercospora nicotianae, little is known of the metabolite’s biosynthesis. Furthermore, in vitro investigations of the polyketide synthase central to cercosporin biosynthesis identified the naphthopyrone nor-toralactone as its direct product—an observation in conflict with published biosynthetic proposals. Here, we present an alternative biosynthetic pathway to cercosporin based on metabolites characterized from a series of biosynthetic gene knockouts. We show that nor-toralactone is the key polyketide intermediate and the substrate for the unusual didomain protein CTB3. We demonstrate the unique oxidative cleavage activity of the CTB3 monooxygenase domain in vitro. These data advance our understanding of perylenequinone biosynthesis and expand the biochemical repertoire of flavin-dependent monooxygenases. PMID:26938470

  11. Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

    PubMed Central

    Kim, Seung Young; Ju, Kou-San; Metcalf, William W.; Evans, Bradley S.; Kuzuyama, Tomohisa

    2012-01-01

    Fosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent. PMID:22615277

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

    PubMed

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

    2015-07-07

    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 (13)C NMR spectrometry make possible the measurement of the nonstatistical position-specific natural abundance distribution of (13)C (δ(13)Ci) within the molecule with better than 1‰ precision. Very substantial variation in the (13)C positional distribution is found: between δ(13)Ci = -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 (18)O/(16)O 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 (13)C 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.

  13. 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

  14. Genomics-Enabled Discovery of Phosphonate Natural Products and their Biosynthetic Pathways

    PubMed Central

    Ju, Kou-San; Doroghazi, James R.; Metcalf, William W.

    2014-01-01

    Phosphonate natural products have proven to be a rich source of useful pharmaceutical, agricultural and biotechnology products, whereas study of their biosynthetic pathways has revealed numerous intriguing enzymes that catalyze unprecedented biochemistry. Here we review the history of phosphonate natural product discovery, highlighting technological advances that have played a key role in the recent advances in their discovery. Central to these developments has been the application of genomics, which allowed discovery and development of a global phosphonate metabolic framework to guide research efforts. This framework suggests that the future of phosphonate natural products remains bright, with many new compounds and pathways yet to be discovered. PMID:24271089

  15. Subchromoplast Sequestration of Carotenoids Affects Regulatory Mechanisms in Tomato Lines Expressing Different Carotenoid Gene Combinations[C][W

    PubMed Central

    Nogueira, Marilise; Mora, Leticia; Enfissi, Eugenia M.A.; Bramley, Peter M.; Fraser, Paul D.

    2013-01-01

    Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed. PMID:24249831

  16. The thioesterase domain from the pimaricin and erythromycin biosynthetic pathways can catalyze hydrolysis of simple thioester substrates.

    PubMed

    Sharma, Krishna K; Boddy, Christopher N

    2007-06-01

    The recombinant polyketide synthase thioesterase domains from the pimaricin and 6-deoxyerythronolide B biosynthetic pathways catalyze hydrolysis of a number of simple N-acetylcysteamine thioester derivatives. This study demonstrates that thioesterases are not highly substrate selective in formation of the acyl-enzyme intermediate, in contrast to non-ribosomal peptide synthase thioesterase domains that show very high specificity for substrate loading. This observation has important implications for the engineering of biosynthetic pathways to produce polyketide products.

  17. 2-Deoxystreptamine-containing aminoglycoside antibiotics: recent advances in the characterization and manipulation of their biosynthetic pathways.

    PubMed

    Park, Sung Ryeol; Park, Je Won; Ban, Yeon Hee; Sohng, Jae Kyung; Yoon, Yeo Joon

    2013-01-01

    The 2-deoxystreptamine-containing aminoglycosides, such as neomycin, kanamycin and gentamicin, are an important class of antibiotics. A detailed understanding of the complete biosynthetic pathway of aminoglycosides and their biosynthetic enzymes will allow us to not only generate more robust antibiotic agents or drugs with other altered biological activities, but also to produce clinically important semi-synthetic antibiotics by direct fermentation. This Highlight focuses on recent advances in the characterization of their biosynthetic enzymes and pathway as well as some chemo-enzymatic and metabolic engineering approaches for the biological production of natural, semi-synthetic, and novel aminoglycosides.

  18. Maize phytoene desaturase and zeta-carotene desaturase catalyse a poly-Z desaturation pathway: implications for genetic engineering of carotenoid content among cereal crops.

    PubMed

    Matthews, Paul D; Luo, RuiBai; Wurtzel, Eleanore T

    2003-10-01

    Carotene desaturation, an essential step in the biosynthesis of coloured carotenoids, has received much attention (1) as a target of bleaching herbicide action, (2) as a determinant of geometric isomer states of carotenoids and their metabolites, and (3) as a key modulator of accumulation and structural variability of carotenoids. Having previously isolated and functionally characterized the cDNA encoding the first enzyme in maize carotene desaturation, phytoene desaturase (PDS), the isolation and functional characterization of the second desaturase, a maize endosperm cDNA (2265 bp) encoding zetacarotene (zeta-carotene) desaturase (ZDS) is reported here. Functional analysis of the concerted actions of maize PDS and ZDS ex situ showed these enzymes to mediate a poly-Z desaturation pathway to the predominate geometric isomer 7,9,7',9'-tetra-Z-lycopene (poly-Z-lycopene or prolycopene), and not the all-trans substrate required of the downstream lycopene cyclase enzymes. This finding suggests a rate-controlling isomerase associated with the carotene desaturases as a corollary of a default poly-Z carotenoid biosynthetic pathway active in planta for maize. Comparative gene analysis between maize and rice revealed that genes encoding PDS and ZDS are single copy; the Zds cDNA characterized here was mapped to maize chromosome 7S and vp9 is suggested as a candidate locus for the structural gene while y9 is ruled out. Classical genetic resources were used to dissect the desaturation steps further and hydroxyphenylpyruvate dioxygenase was linked to the vp2 locus, narrowing candidate loci for an obligate isomerase in maize to only a few. Since the first functional analysis of the paired carotene desaturases for a cereal crop is reported here, the implications for the genetic modification of the pro-vitamin A content in cereal crops such as rice and maize, are discussed.

  19. 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.

  20. Distribution of. delta. -aminolevulinic acid biosynthetic pathways among phototrophic and related bacteria

    SciTech Connect

    Avissar, Y.J.; Beale, S.I. ); Ormerod, J.G. )

    1989-04-01

    Two biosynthetic pathways are known for the universal tetrapyrrole precursor, {delta}-aminolevulinic acid (ALA): condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO{sub 2}, and conversion of the intact carbon skeleton of glutamate to ALA in a process requiring tRNA{sup Glu}, ATP, Mg{sup 2+}, NADPH, and pyridoxal phosphate. The distribution of the two ALA biosynthetic pathways among various bacterial genera was determined, using cell-free extracts obtained from representative organisms. Evidence for the operation of the glutamate pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA using 3,4-({sup 3}H)glutamate and 1-({sup 14}C)glutamate as substrate. The glycine pathway was indicated by RNase-insensitive incorporation of level from 2-({sup 14}C)glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously phylogenetic relationships and clearly indicates that the glutamate pathway is the more ancient process, whereas the glycine pathway probably evolved much later. The glutamate pathway is the more widely utilized one among bacteria, while the glycine pathway is apparently limited to the {alpha} subgroup of purple bacteria (including Rhodobacter, Rhodospirillum, and Rhizobium). E. coli was found ALA via the glutamate pathway. The ALA-requiring hemA mutant of E. coli was determined to lack the dehydrogenase activity that utilizes glutamyl-tRNA as a substrate.

  1. Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway.

    PubMed

    Yao, Yuan-Feng; Wang, Chang-Song; Qiao, Jianjun; Zhao, Guang-Rong

    2013-09-01

    Salvianic acid A, a valuable derivative from L-tyrosine biosynthetic pathway of the herbal plant Salvia miltiorrhiza, is well known for its antioxidant activities and efficacious therapeutic potential on cardiovascular diseases. Salvianic acid A was traditionally isolated from plant root or synthesized by chemical methods, both of which had low efficiency. Herein, we developed an unprecedented artificial biosynthetic pathway of salvianic acid A in E. coli, enabling its production from glucose directly. In this pathway, 4-hydroxyphenylpyruvate was converted to salvianic acid A via D-lactate dehydrogenase (encoding by d-ldh from Lactobacillus pentosus) and hydroxylase complex (encoding by hpaBC from E. coli). Furthermore, we optimized the pathway by a modular engineering approach and deleting genes involved in the regulatory and competing pathways. The metabolically engineered E. coli strain achieved high productivity of salvianic acid A (7.1g/L) with a yield of 0.47mol/mol glucose. © 2013 Elsevier Inc. All rights reserved.

  2. Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera

    PubMed Central

    Pandey, Akansha; Swarnkar, Vishakha; Pandey, Tushar; Srivastava, Piush; Kanojiya, Sanjeev; Mishra, Dipak Kumar; Tripathi, Vineeta

    2016-01-01

    Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG’s were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes. PMID:27703261

  3. A branched biosynthetic pathway is involved in production of roquefortine and related compounds in Penicillium chrysogenum.

    PubMed

    Ali, Hazrat; Ries, Marco I; Nijland, Jeroen G; Lankhorst, Peter P; Hankemeier, Thomas; Bovenberg, Roel A L; Vreeken, Rob J; Driessen, Arnold J M

    2013-01-01

    Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.

  4. Combinatorial genetic transformation of cereals and the creation of metabolic libraries for the carotenoid pathway.

    PubMed

    Farre, Gemma; Naqvi, Shaista; Sanahuja, Georgina; Bai, Chao; Zorrilla-López, Uxue; Rivera, Sol M; Canela, Ramon; Sandman, Gerhard; Twyman, Richard M; Capell, Teresa; Zhu, Changfu; Christou, Paul

    2012-01-01

    Combinatorial nuclear transformation is used to generate populations of transgenic plants containing random selections from a collection of input transgenes. This is a useful approach because it provides the means to test different combinations of genes without the need for separate transformation experiments, allowing the comprehensive analysis of metabolic pathways and other genetic systems requiring the coordinated expression of multiple genes. The principle of combinatorial nuclear transformation is demonstrated in this chapter through protocols developed in our laboratory that allow combinations of genes encoding enzymes in the carotenoid biosynthesis pathway to be introduced into rice and a white-endosperm variety of corn. These allow the accumulation of carotenoids to be screened initially by the colour of the endosperm, which ranges from white through various shades of yellow and orange depending on the types and quantities of carotenoids present. The protocols cover the preparation of DNA-coated metal particles, the transformation of corn and rice plants by particle bombardment, the regeneration of transgenic plants, the extraction of carotenoids from plant tissues, and their analysis by high-performance liquid chromatography.

  5. Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

    PubMed Central

    Stevens, David Cole; Conway, Kyle R.; Pearce, Nelson; Villegas-Peñaranda, Luis Roberto; Garza, Anthony G.; Boddy, Christopher N.

    2013-01-01

    Background Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. Methodology We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. Conclusions We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products. PMID:23724102

  6. Analysis of Polygala tenuifolia Transcriptome and Description of Secondary Metabolite Biosynthetic Pathways by Illumina Sequencing

    PubMed Central

    Tian, Hongling; Xu, Xiaoshuang; Zhang, Fusheng; Wang, Yaoqin; Guo, Shuhong; Qin, Xuemei; Du, Guanhua

    2015-01-01

    Radix polygalae, the dried roots of Polygala tenuifolia and P. sibirica, is one of the most well-known traditional Chinese medicinal plants. Radix polygalae contains various saponins, xanthones, and oligosaccharide esters and these compounds are responsible for several pharmacological properties. To provide basic breeding information, enhance molecular biological analysis, and determine secondary metabolite biosynthetic pathways of P. tenuifolia, we applied Illumina sequencing technology and de novo assembly. We also applied this technique to gain an overview of P. tenuifolia transcriptome from samples with different years. Using Illumina sequencing, approximately 67.2% of unique sequences were annotated by basic local alignment search tool similarity searches against public sequence databases. We classified the annotated unigenes by using Nr, Nt, GO, COG, and KEGG databases compared with NCBI. We also obtained many candidates CYP450s and UGTs by the analysis of genes in the secondary metabolite biosynthetic pathways, including putative terpenoid backbone and phenylpropanoid biosynthesis pathway. With this transcriptome sequencing, future genetic and genomics studies related to the molecular mechanisms associated with the chemical composition of P. tenuifolia may be improved. Genes involved in the enrichment of secondary metabolite biosynthesis-related pathways could enhance the potential applications of P. tenuifolia in pharmaceutical industries. PMID:26543847

  7. Coordinate regulation of the tryptophan biosynthetic pathway and indolic phytoalexin accumulation in Arabidopsis.

    PubMed

    Zhao, J; Last, R L

    1996-12-01

    Little is known about the mechanisms that couple regulation of secondary metabolic pathways to the synthesis of primary metabolic precursors. Camalexin, an indolic secondary metabolite, appears to be the major phytoalexin in Arabidopsis. It was previously shown that camalexin accumulation is caused by infection with plant pathogens, by abiotic elicitors, and in spontaneous lesions in the accelerated cell death mutant acd2. We demonstrate that the accumulation of this phytoalexin is accompanied by the induction of the mRNAs and proteins for all of the tryptophan biosynthetic enzymes tested. A strong correlation was observed between the magnitude of camalexin accumulation and the induction of tryptophan biosynthetic proteins, indicating coordinate regulation of these processes. Production of disease symptoms is not sufficient for the response because systemic infection with cauliflower mosaic virus or cucumber mosaic virus did not induce the tryptophan pathway enzymes or camalexin accumulation. Salicylic acid appears to be required, but unlike other documented pathogenesis-related proteins, it is not sufficient for the coordinate induction. Results with trp mutants suggest that the tryptophan pathway is not rate limiting for camalexin accumulation. Taken together, these results are consistent with the hypothesis that the regulation of the tryptophan pathway in plants responds to needs for biosynthesis of secondary metabolites.

  8. Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes

    PubMed Central

    Wheeler, Glen; Ishikawa, Takahiro; Pornsaksit, Varissa; Smirnoff, Nicholas

    2015-01-01

    Ascorbic acid (vitamin C) is an enzyme co-factor in eukaryotes that also plays a critical role in protecting photosynthetic eukaryotes against damaging reactive oxygen species derived from the chloroplast. Many animal lineages, including primates, have become ascorbate auxotrophs due to the loss of the terminal enzyme in their biosynthetic pathway, l-gulonolactone oxidase (GULO). The alternative pathways found in land plants and Euglena use a different terminal enzyme, l-galactonolactone dehydrogenase (GLDH). The evolutionary processes leading to these differing pathways and their contribution to the cellular roles of ascorbate remain unclear. Here we present molecular and biochemical evidence demonstrating that GULO was functionally replaced with GLDH in photosynthetic eukaryote lineages following plastid acquisition. GULO has therefore been lost repeatedly throughout eukaryote evolution. The formation of the alternative biosynthetic pathways in photosynthetic eukaryotes uncoupled ascorbate synthesis from hydrogen peroxide production and likely contributed to the rise of ascorbate as a major photoprotective antioxidant. DOI: http://dx.doi.org/10.7554/eLife.06369.001 PMID:25768426

  9. ProCarDB: a database of bacterial carotenoids.

    PubMed

    Nupur, L N U; Vats, Asheema; Dhanda, Sandeep Kumar; Raghava, Gajendra P S; Pinnaka, Anil Kumar; Kumar, Ashwani

    2016-05-26

    Carotenoids have important functions in bacteria, ranging from harvesting light energy to neutralizing oxidants and acting as virulence factors. However, information pertaining to the carotenoids is scattered throughout the literature. Furthermore, information about the genes/proteins involved in the biosynthesis of carotenoids has tremendously increased in the post-genomic era. A web server providing the information about microbial carotenoids in a structured manner is required and will be a valuable resource for the scientific community working with microbial carotenoids. Here, we have created a manually curated, open access, comprehensive compilation of bacterial carotenoids named as ProCarDB- Prokaryotic Carotenoid Database. ProCarDB includes 304 unique carotenoids arising from 50 biosynthetic pathways distributed among 611 prokaryotes. ProCarDB provides important information on carotenoids, such as 2D and 3D structures, molecular weight, molecular formula, SMILES, InChI, InChIKey, IUPAC name, KEGG Id, PubChem Id, and ChEBI Id. The database also provides NMR data, UV-vis absorption data, IR data, MS data and HPLC data that play key roles in the identification of carotenoids. An important feature of this database is the extension of biosynthetic pathways from the literature and through the presence of the genes/enzymes in different organisms. The information contained in the database was mined from published literature and databases such as KEGG, PubChem, ChEBI, LipidBank, LPSN, and Uniprot. The database integrates user-friendly browsing and searching with carotenoid analysis tools to help the user. We believe that this database will serve as a major information centre for researchers working on bacterial carotenoids.

  10. Chemistry and biotechnology of carotenoids.

    PubMed

    Namitha, K K; Negi, P S

    2010-09-01

    Carotenoids are one of the most widespread groups of pigments in nature and more than 600 of these have been identified. Beside provitamin A activity, carotenoids are important as antioxidants and protective agents against various diseases. They are isoprenoids with a long polyene chain containing 3 to 15 conjugated double bonds, which determines their absorption spectrum. Cyclization at one or both ends occurs in hydrocarbon carotene, while xanthophylls are formed by the introduction of oxygen. In addition, modifications involving chain elongation, isomerization, or degradation are also found. The composition of carotenoids in food may vary depending upon production practices, post-harvest handling, processing, and storage. In higher plants they are synthesized in the plastid. Both mevalonate dependent and independent pathway for the formation of isopentenyl diphosphate are known. Isopentenyl diphosphate undergoes a series of addition and condensation reactions to form phytoene, which gets converted to lycopene. Cyclization of lycopene either leads to the formation of β-carotene and its derivative xanthophylls, β-cryptoxanthin, zeaxanthin, antheraxanthin, and violaxanthin or α-carotene and lutein. Even though most of the carotenoid biosynthetic genes have been cloned and identified, some aspects of carotenoid formation and manipulation in higher plants especially remain poorly understood. In order to enhance the carotenoid content of crop plants to a level that will be required for the prevention of diseases, there is a need for research in both the basic and the applied aspects.

  11. Biosynthesis of 2-Hydroxyethylphosphonate, an Unexpected Intermediate Common to Multiple Phosphonate Biosynthetic Pathways*S⃞

    PubMed Central

    Shao, Zengyi; Blodgett, Joshua A. V.; Circello, Benjamin T.; Eliot, Andrew C.; Woodyer, Ryan; Li, Gongyong; van der Donk, Wilfred A.; Metcalf, William W.; Zhao, Huimin

    2008-01-01

    Phosphonic acids encompass a common yet chemically diverse class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences among many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate, which is synthesized from phosphonoacetaldehyde by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34–37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide, and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities. These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure. Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters. Thus, the reduction of phosphonoacetaldehyde to hydroxyethyl-phosphonate may represent a common step in the biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new C–P containing secondary metabolites. PMID:18544530

  12. A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study.

    PubMed

    Cunningham, Francis X; Gantt, Elisabeth

    2007-05-01

    Carotenoids are indispensable pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are produced, as well, by many bacteria and fungi. Elucidation of biochemical pathways leading to the carotenoids that function in the photosynthetic membranes of land plants has been greatly aided by the use of carotenoid-accumulating strains of Escherichia coli as heterologous hosts for functional assays, in vivo, of the otherwise difficult to study membrane-associated pathway enzymes. This same experimental approach is uniquely well-suited to the discovery and characterization of yet-to-be identified enzymes that lead to carotenoids of the photosynthetic membranes in algal cells, to the multitude of carotenoids found in nongreen plant tissues, and to the myriad flavor and aroma compounds that are derived from carotenoids in plant tissues. A portfolio of plasmids suitable for the production in E. coli of a variety of carotenoids is presented herein. The use of these carotenoid-producing E. coli for the identification of cDNAs encoding enzymes of carotenoid and isoprenoid biosynthesis, for characterization of the enzymes these cDNAs encode, and for the production of specific carotenoids for use as enzyme substrates and reference standards, is described using the flowering plant Adonis aestivalis to provide examples. cDNAs encoding nine different A. aestivalis enzymes of carotenoid and isoprenoid synthesis were identified and the enzymatic activity of their products verified. Those cDNAs newly described include ones that encode phytoene synthase, beta-carotene hydroxylase, deoxyxylulose-5-phosphate synthase, isopentenyl diphosphate isomerase, and geranylgeranyl diphosphate synthase.

  13. Differential Activities of Thalidomide and Isoprenoid Biosynthetic Pathway Inhibitors in Multiple Myeloma Cells

    PubMed Central

    Holstein, Sarah A.; Tong, Huaxiang; Hohl, Raymond J.

    2013-01-01

    Thalidomide has emerged as an effective agent for treating multiple myeloma, however the precise mechanism of action remains unknown. Agents known to target the isoprenoid biosynthetic pathway (IBP) can have cytotoxic effects in myeloma cells. The interactions between thalidomide and IBP inhibitors in human multiple myeloma cells were evaluated. Enhanced cytotoxicity and induction of apoptosis was observed in RPMI-8226 cells. Examination of intracellular levels of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) revealed a wide variance in basal levels and response to IBP inhibitors. These findings provide a mechanism for the differential sensitivity of myeloma cells to pharmacologic manipulation of the IBP. PMID:19646757

  14. Neopikromycin and novapikromycin from the pikromycin biosynthetic pathway of Streptomyces venezuelae.

    PubMed

    Lee, Sang Kil; Park, Je Won; Kim, Ji Won; Jung, Won Seok; Park, Sung Ryeol; Choi, Cha Yong; Kim, Eung Soo; Kim, Beom Seok; Ahn, Jong Seog; Sherman, David H; Yoon, Yeo Joon

    2006-05-01

    Two new macrolides from the pikromycin biosynthetic pathway of Streptomyces venezuelae, neopikromycin (9) and novapikromycin (10), were identified and structurally characterized through mass spectrometry and NMR spectroscopy. The established structures showed that 9 and 10 have hydroxyl groups at C-14 (9) and at both C-12 and C-14 (10), on the basis of a comparison with narbomycin (7). The purified PikC cytochrome P450 monooxygenase catalyzes the in vitro hydroxylation of 7 and pikromycin (8) to yield 9 and 10, respectively, thus expanding the substrate- and regio-flexibility of this enzyme.

  15. Divergent evolutionary pattern of starch biosynthetic pathway genes in grasses and dicots.

    PubMed

    Li, Chun; Li, Qi-Gang; Dunwell, Jim M; Zhang, Yuan-Ming

    2012-10-01

    Starch is the most widespread and abundant storage carbohydrate in crops and its production is critical to both crop yield and quality. In regard to the starch content in the seeds of crop plants, there is a distinct difference between grasses (Poaceae) and dicots. However, few studies have described the evolutionary pattern of genes in the starch biosynthetic pathway in these two groups of plants. In this study, therefore, an attempt was made to compare evolutionary rate, gene duplication, and selective pattern of the key genes involved in this pathway between the two groups, using five grasses and five dicots as materials. The results showed 1) distinct differences in patterns of gene duplication and loss between grasses and dicots; duplication in grasses mainly occurred before the divergence of grasses, whereas duplication mostly occurred in individual species within the dicots; there is less gene loss in grasses than in dicots, 2) a considerably higher evolutionary rate in grasses than in dicots in most gene families analyzed, and 3) evidence of a different selective pattern between grasses and dicots; positive selection may have occurred asymmetrically in grasses in some gene families, for example, ADP-glucose pyrophosphorylase small subunit. Therefore, we deduced that gene duplication contributes to, and a higher evolutionary rate is associated with, the higher starch content in grasses. In addition, two novel aspects of the evolution of the starch biosynthetic pathway were observed.

  16. 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.

  17. 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.

  18. Analysis of the biosynthetic pathway for sulfoquinovosyldiacylglycerol in the purple bacterium R. sphaeroides

    SciTech Connect

    Benning, C.; Somerville, C.R. )

    1990-05-01

    The membrane lipid sulfoquinovosyldiacylglycerol (SQD) can be found in all photosynthetically active membranes studied. In a green leaf, about 50% of the organic sulfur is bound in SQD. Therefore, this sulfolipid constitutes a major component of the global sulfur cycle. However, since the discovery of SQD, very little progress has been made towards the elucidation of the biosynthetic pathway. For a genetical analysis of the pathway of SQD, we selected the photosynthetic purple-nonsulfur bacterium R. sphaeroides, as our model system. We have been able to isolate several classes of mutants, which show reduced levels of SQD. Some of the mutants accumulate {sup 35}S-labeled, water soluble compounds. We expect that these compounds are related to precursors or are precursors, which accumulate due to a specific block in the biosynthetic pathway of SQD. To study the possible precursor function of these compounds, we developed an in vitro SQD biosynthesis system using cell free extracts from R. sphaeroides. In addition, we have been able to isolate cosmids prepared from WT DNA, which complement three classes of SQD mutants in R. sphaeroides. The analysis and expression of the genes encoded by these cosmids should facilitate the characterization of the proteins involved in SQD biosynthesis.

  19. Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

    PubMed

    Yamamoto, Tsuyoshi; Matsui, Hidenori; Yamaji, Kenzaburo; Takahashi, Tetsufumi; Øverby, Anders; Nakamura, Masahiko; Matsumoto, Atsuko; Nonaka, Kenichi; Sunazuka, Toshiaki; Ōmura, Satoshi; Nakano, Hirofumi

    2016-09-01

    We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 μM), DHA (100 μM), or siamycin I (2.5 μM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis. Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  20. Evolution of the Tetrapyrrole Biosynthetic Pathway in Secondary Algae: Conservation, Redundancy and Replacement

    PubMed Central

    Horák, Aleš; Oborník, Miroslav

    2016-01-01

    Tetrapyrroles such as chlorophyll and heme are indispensable for life because they are involved in energy fixation and consumption, i.e. photosynthesis and oxidative phosphorylation. In eukaryotes, the tetrapyrrole biosynthetic pathway is shaped by past endosymbioses. We investigated the origins and predicted locations of the enzymes of the heme pathway in the chlorarachniophyte Bigelowiella natans, the cryptophyte Guillardia theta, the “green” dinoflagellate Lepidodinium chlorophorum, and three dinoflagellates with diatom endosymbionts (“dinotoms”): Durinskia baltica, Glenodinium foliaceum and Kryptoperidinium foliaceum. Bigelowiella natans appears to contain two separate heme pathways analogous to those found in Euglena gracilis; one is predicted to be mitochondrial-cytosolic, while the second is predicted to be plastid-located. In the remaining algae, only plastid-type tetrapyrrole synthesis is present, with a single remnant of the mitochondrial-cytosolic pathway, a ferrochelatase of G. theta putatively located in the mitochondrion. The green dinoflagellate contains a single pathway composed of mostly rhodophyte-origin enzymes, and the dinotoms hold two heme pathways of apparently plastidal origin. We suggest that heme pathway enzymes in B. natans and L. chlorophorum share a predominantly rhodophytic origin. This implies the ancient presence of a rhodophyte-derived plastid in the chlorarachniophyte alga, analogous to the green dinoflagellate, or an exceptionally massive horizontal gene transfer. PMID:27861576

  1. 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

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

    PubMed

    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; Cañas, Luis A

    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.

  3. exo-Brevicomin biosynthetic pathway enzymes from the Mountain Pine Beetle, Dendroctonus ponderosae.

    PubMed

    Song, Minmin; Delaplain, Patrick; Nguyen, Trang T; Liu, Xibei; Wickenberg, Leah; Jeffrey, Christopher; Blomquist, Gary J; Tittiger, Claus

    2014-10-01

    exoBrevicomin (exo-7-ethyl-5-methyl-6,8-dioxabicyclo[3.2.1]octane) is an important semiochemical for a number of beetle species, including the highly destructive Mountain Pine Beetle (Dendroctonus ponderosae). It is also found in other insects and the African elephant. Despite its significance, very little is known about its biosynthesis. A recent microarray analysis implicated a small cluster of three D. ponderosae genes in exo-brevicomin biosynthesis, two of which had identifiable open reading frames (Aw et al., 2010; BMC Genomics 11:215). Here we report further expression profiling of two genes in that cluster and functional analysis of their recombinantly-produced enzymes. One encodes a short-chain dehydrogenase that used NAD(P)(+) as a co-factor to catalyze the oxidation of (Z)-6-nonen-2-ol to (Z)-6-nonen-2-one. We therefore named the enzyme (Z)-6-nonen-2-ol dehydrogenase (ZnoDH). The other encodes the cytochrome P450, CYP6CR1, which epoxidized (Z)-6-nonen-2-one to 6,7-epoxynonan-2-one with very high specificity and substrate selectivity. Both the substrates and products of the two enzymes are intermediates in the exo-brevicomin biosynthetic pathway. Thus, ZnoDH and CYP6CR1 are enzymes that apparently catalyze the antepenultimate and penultimate steps in the exo-brevicomin biosynthetic pathway, respectively.

  4. Expression of parsley flavone synthase I establishes the flavone biosynthetic pathway in Arabidopsis thaliana.

    PubMed

    Yun, Choong-Soo; Yamamoto, Tomio; Nozawa, Akira; Tozawa, Yuzuru

    2008-04-01

    Arabidopsis thaliana lacks the flavone biosynthetic pathway, probably because of a lack or low activity of a flavone synthase. To establish this biosynthetic pathway in Arabidopsis, we subjected this model plant to transformation with the parsley gene for flavone synthase type I (FNS-I). Transgenic seedlings expressing FNS-I were cultured in liquid medium with or without naringenin, and plant extracts were then analyzed by high-performance liquid chromatography. In contrast to wild-type seedlings, the transgenic seedlings accumulated substantial amounts of apigenin, which is produced from naringenin by FNS-I, and the apigenin level correlated with the abundance of FNS-I mRNA in three different transgenic lines. These results indicate that the FNS-I transgene produces a functional enzyme that catalyzes the conversion of naringenin to apigenin in Arabidopsis. These FNS-I transgenic lines should prove useful in investigating the in vivo functions of enzymes that mediate the synthesis of the wide variety of flavones found in other plants.

  5. Expression of genes of the aflatoxin biosynthetic pathway in Aspergillus flavus isolates from keratitis.

    PubMed

    Leema, George; Chou, Duen-Suey; Jesudasan, Christadoss A Nelson; Geraldine, Pitchairaj; Thomas, Philip A

    2011-01-01

    To document transcriptional activation (expression) of key aflatoxin biosynthetic pathway genes in corneal isolates of Aspergillus flavus. The expression of certain regulatory (aflatoxin regulatory [aflR] and aflatoxin J [aflJ]) and structural (polyketide synthase acetate [pksA] and norsolonic acid-1 [nor-1]) genes in four corneal A. flavus isolates was evaluated by reverse transcription PCR. The aflatoxin-producing potential of each strain was determined by thin-layer chromatography and quantified by spectrophotometry. Four environmental isolates were used for comparison. The mean expression levels of these genes were compared in the corneal and environmental A. flavus isolates. In addition, the mean expression levels were also correlated with the aflatoxin production levels. All isolates expressed aflJ, nor-1, and pksA, while all but one expressed aflR. Overall, significantly higher mean expression levels occurred in aflatoxigenic than in non-aflatoxigenic corneal isolates. A significant positive correlation was noted between the mean expression level of aflR and the quantum of aflatoxin production by the corneal isolates. Essentially similar patterns of expression of these genes were noted in four environmental A. flavus isolates used for comparison. For the first time, isolates of A. flavus from human keratitis patients have been shown to express regulatory and structural aflatoxin biosynthetic pathway genes. Further studies are needed to clarify the precise influence of the corneal microenvironment on expression of these genes and aflatoxin production by A. flavus infecting the cornea.

  6. Structure of DnmZ, a nitrososynthase in the Streptomyces peucetius anthracycline biosynthetic pathway

    PubMed Central

    Sartor, Lauren; Ibarra, Charmaine; Al-Mestarihi, Ahmad; Bachmann, Brian O.; Vey, Jessica L.

    2015-01-01

    The anthracyclines are a class of highly effective natural product chemotherapeutics and are used to treat a range of cancers, including leukemia. The toxicity of the anthracyclines has stimulated efforts to further diversify the scaffold of the natural product, which has led to renewed interest in the biosynthetic pathway responsible for the formation and modification of this family of molecules. DnmZ is an N-hydroxylating flavin monooxygenase (a nitrososynthase) that catalyzes the oxidation of the exocyclic amine of the sugar nucleotide dTDP-l-epi-vancosamine to its nitroso form. Its specific role in the anthracycline biosynthetic pathway involves the synthesis of the seven-carbon acetal moiety attached to C4 of l-daunosamine observed in the anthracycline baumycin. Here, X-ray crystallography was used to elucidate the three-dimensional structure of DnmZ. Two crystal structures of DnmZ were yielded: that of the enzyme alone, solved to 3.00 Å resolution, and that of the enzyme in complex with thymidine diphosphate, the nucleotide carrier portion of the substrate, solved to 2.74 Å resolution. These models add insights into the structural features involved in substrate specificity and conformational changes involved in thymidine diphosphate binding by the nitrososynthases. PMID:26457508

  7. Evaluation of the cardiolipin biosynthetic pathway and its interactions in the diabetic heart

    PubMed Central

    Croston, Tara L.; Shepherd, Danielle L.; Thapa, Dharendra; Nichols, Cody E.; Lewis, Sara E.; Dabkowski, Erinne R.; Jagannathan, Rajaganapathi; Baseler, Walter A.; Hollander, John M.

    2013-01-01

    Aims We have previously reported alterations in cardiolipin content and inner mitochondrial membrane (IMM) proteomic make-up specifically in interfibrillar mitochondria (IFM) in the type 1 diabetic heart; however, the mechanism underlying this alteration is unknown. The goal of this study was to determine how the cardiolipin biosynthetic pathway and cardiolipin-IMM protein interactions are impacted by type 1 diabetes mellitus. Main methods Male FVB mice were made diabetic by multiple low-dose streptozotocin injections and sacrificed five weeks post-diabetic onset. Messenger RNA was measured and cardiac mitochondrial subpopulations were isolated. Further mitochondrial functional experimentation included evaluating the protein expression of the enzymes directly responsible for cardiolipin biosynthesis, as well as ATP synthase activity. Interactions between cardiolipin and ATP synthase subunits were also examined. Key findings Western blot analysis revealed a significant decrease in cardiolipin synthase (CRLS) protein content in diabetic IFM, with a concomitant decrease in its activity. ATP synthase activity was also significantly decreased. We identified two novel direct interactions between two subunits of the ATP synthase F0 complex (ATP5F1 and ATP5H), both of which were significantly decreased in diabetic IFM. Significance Overall, these results indicate that type 1 diabetes mellitus negatively impacts the cardiolipin biosynthetic pathway specifically at CRLS, contributing to decreased cardiolipin content and loss of interactions with key ATP synthase F0 complex constituents in the IFM. PMID:23872101

  8. De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis.

    PubMed

    Hyun, Tae Kyung; Rim, Yeonggil; Jang, Hui-Jeong; Kim, Cheol Hong; Park, Jongsun; Kumar, Ritesh; Lee, Sunghoon; Kim, Byung Chul; Bhak, Jong; Nguyen-Quoc, Binh; Kim, Seon-Won; Lee, Sang Yeol; Kim, Jae-Yean

    2012-07-01

    The ripe fruit of Momordica cochinchinensis Spreng, known as gac, is featured by very high carotenoid content. Although this plant might be a good resource for carotenoid metabolic engineering, so far, the genes involved in the carotenoid metabolic pathways in gac were unidentified due to lack of genomic information in the public database. In order to expedite the process of gene discovery, we have undertaken Illumina deep sequencing of mRNA prepared from aril of gac fruit. From 51,446,670 high-quality reads, we obtained 81,404 assembled unigenes with average length of 388 base pairs. At the protein level, gac aril transcripts showed about 81.5% similarity with cucumber proteomes. In addition 17,104 unigenes have been assigned to specific metabolic pathways in Kyoto Encyclopedia of Genes and Genomes, and all of known enzymes involved in terpenoid backbones biosynthetic and carotenoid biosynthetic pathways were also identified in our library. To analyze the relationship between putative carotenoid biosynthesis genes and alteration of carotenoid content during fruit ripening, digital gene expression analysis was performed on three different ripening stages of aril. This study has revealed putative phytoene synthase, 15-cis-phytone desaturase, zeta-carotene desaturase, carotenoid isomerase and lycopene epsilon cyclase might be key factors for controlling carotenoid contents during aril ripening. Taken together, this study has also made availability of a large gene database. This unique information for gac gene discovery would be helpful to facilitate functional studies for improving carotenoid quantities.

  9. 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.

  10. Elucidating the biosynthetic pathways of volatile organic compounds in Mycobacterium tuberculosis through a computational approach.

    PubMed

    Bhatter, Purva; Raman, Karthik; Janakiraman, Vani

    2017-03-28

    Microbial volatile organic compounds (VOCs) have gained prominence in the recent past for their potential use as disease markers. The discovery of microbial VOCs has benefited 'difficult to detect' diseases such as tuberculosis (TB). Few of the identified VOCs of Mycobacterium tuberculosis (Mtb) are currently being explored for their diagnostic potential. However, very little is known about the biosynthesis of these small lipophilic molecules. Here, we propose putative biosynthetic pathways in Mycobacterium tuberculosis for three VOCs, namely methyl nicotinate, methyl phenylacetate and methyl p-anisate, using computational approaches. In particular, we identify S-adenosyl methionine (SAM) transferases that play a crucial role in esterification of the acids to the final product. Our results provide important insights into the specificity of these pathways to Mtb species.

  11. The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii

    PubMed Central

    Itkin, Maxim; Davidovich-Rikanati, Rachel; Cohen, Shahar; Portnoy, Vitaly; Doron-Faigenboim, Adi; Oren, Elad; Freilich, Shiri; Tzuri, Galil; Baranes, Nadine; Shen, Shmuel; Petreikov, Marina; Sertchook, Rotem; Ben-Dor, Shifra; Gottlieb, Hugo; Hernandez, Alvaro; Nelson, David R.; Paris, Harry S.; Tadmor, Yaakov; Burger, Yosef; Lewinsohn, Efraim; Katzir, Nurit; Schaffer, Arthur

    2016-01-01

    The consumption of sweeteners, natural as well as synthetic sugars, is implicated in an array of modern-day health problems. Therefore, natural nonsugar sweeteners are of increasing interest. We identify here the biosynthetic pathway of the sweet triterpenoid glycoside mogroside V, which has a sweetening strength of 250 times that of sucrose and is derived from mature fruit of luo-han-guo (Siraitia grosvenorii, monk fruit). A whole-genome sequencing of Siraitia, leading to a preliminary draft of the genome, was combined with an extensive transcriptomic analysis of developing fruit. A functional expression survey of nearly 200 candidate genes identified the members of the five enzyme families responsible for the synthesis of mogroside V: squalene epoxidases, triterpenoid synthases, epoxide hydrolases, cytochrome P450s, and UDP-glucosyltransferases. Protein modeling and docking studies corroborated the experimentally proven functional enzyme activities and indicated the order of the metabolic steps in the pathway. A comparison of the genomic organization and expression patterns of these Siraitia genes with the orthologs of other Cucurbitaceae implicates a strikingly coordinated expression of the pathway in the evolution of this species-specific and valuable metabolic pathway. The genomic organization of the pathway genes, syntenously preserved among the Cucurbitaceae, indicates, on the other hand, that gene clustering cannot account for this novel secondary metabolic pathway. PMID:27821754

  12. The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii.

    PubMed

    Itkin, Maxim; Davidovich-Rikanati, Rachel; Cohen, Shahar; Portnoy, Vitaly; Doron-Faigenboim, Adi; Oren, Elad; Freilich, Shiri; Tzuri, Galil; Baranes, Nadine; Shen, Shmuel; Petreikov, Marina; Sertchook, Rotem; Ben-Dor, Shifra; Gottlieb, Hugo; Hernandez, Alvaro; Nelson, David R; Paris, Harry S; Tadmor, Yaakov; Burger, Yosef; Lewinsohn, Efraim; Katzir, Nurit; Schaffer, Arthur

    2016-11-22

    The consumption of sweeteners, natural as well as synthetic sugars, is implicated in an array of modern-day health problems. Therefore, natural nonsugar sweeteners are of increasing interest. We identify here the biosynthetic pathway of the sweet triterpenoid glycoside mogroside V, which has a sweetening strength of 250 times that of sucrose and is derived from mature fruit of luo-han-guo (Siraitia grosvenorii, monk fruit). A whole-genome sequencing of Siraitia, leading to a preliminary draft of the genome, was combined with an extensive transcriptomic analysis of developing fruit. A functional expression survey of nearly 200 candidate genes identified the members of the five enzyme families responsible for the synthesis of mogroside V: squalene epoxidases, triterpenoid synthases, epoxide hydrolases, cytochrome P450s, and UDP-glucosyltransferases. Protein modeling and docking studies corroborated the experimentally proven functional enzyme activities and indicated the order of the metabolic steps in the pathway. A comparison of the genomic organization and expression patterns of these Siraitia genes with the orthologs of other Cucurbitaceae implicates a strikingly coordinated expression of the pathway in the evolution of this species-specific and valuable metabolic pathway. The genomic organization of the pathway genes, syntenously preserved among the Cucurbitaceae, indicates, on the other hand, that gene clustering cannot account for this novel secondary metabolic pathway.

  13. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera.

    PubMed

    Ono, Hajime; Rewitz, Kim F; Shinoda, Tetsuro; Itoyama, Kyo; Petryk, Anna; Rybczynski, Robert; Jarcho, Michael; Warren, James T; Marqués, Guillermo; Shimell, Mary Jane; Gilbert, Lawrence I; O'Connor, Michael B

    2006-10-15

    Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each is expressed at all stages of development and shows no closely related homolog in their respective genomes. In contrast, we show here that several dipteran genomes encode two novel, highly related, microsomal P450 enzymes, Cyp307A1 and Cyp307A2, that likely participate as stage-specific components of the ecdysone biosynthetic machinery. This hypothesis comes from the observation that Cyp307A1 is encoded by the Halloween gene spook (spo), but unlike other Halloween class genes, Dmspo is not expressed during the larval stages. In contrast, Cyp307a2, dubbed spookier (spok), is expressed primarily during larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is

  14. Probing a Coral Genome for Components of the Photoprotective Scytonemin Biosynthetic Pathway and the 2-Aminoethylphosphonate Pathway

    PubMed Central

    Shoguchi, Eiichi; Tanaka, Makiko; Takeuchi, Takeshi; Shinzato, Chuya; Satoh, Nori

    2013-01-01

    Genome sequences of the reef-building coral, Acropora digitifera, have been decoded. Acropora inhabits an environment with intense ultraviolet exposure and hosts the photosynthetic endosymbiont, Symbiodinium. Acropora homologs of all four genes necessary for biosynthesis of the photoprotective cyanobacterial compound, shinorine, are present. Among metazoans, these genes are found only in anthozoans. To gain further evolutionary insights into biosynthesis of photoprotective compounds and associated coral proteins, we surveyed the Acropora genome for 18 clustered genes involved in cyanobacterial synthesis of the anti-UV compound, scytonemin, even though it had not previously been detected in corals. We identified candidates for only 6 of the 18 genes, including tyrP, scyA, and scyB. Therefore, it does not appear that Acropora digitifera can synthesize scytonemin independently. On the other hand, molecular phylogenetic analysis showed that one tyrosinase gene is an ortholog of vertebrate tyrosinase genes and that the coral homologs, scyA and scyB, are similar to bacterial metabolic genes, phosphonopyruvate (ppyr) decarboxylase and glutamate dehydrogenase (GDH), respectively. Further genomic searches for ppyr gene-related biosynthetic components indicate that the coral possesses a metabolic pathway similar to the bacterial 2-aminoethylphosphonate (AEP) biosynthetic pathway. The results suggest that de novo synthesis of carbon-phosphorus compounds is performed in corals. PMID:23434798

  15. 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.

  16. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    SciTech Connect

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  17. 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

  18. 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.

  19. Genome Sequence of Thermofilum pendens Reveals an Exceptional Loss of Biosynthetic Pathways without Genome Reduction

    SciTech Connect

    Anderson, Iain; Rodriquez, Jason; Susanti, Dwi; Porat, I.; Reich, Claudia; Ulrich, Luke; Elkins, James G; Mavromatis, K; Lykidis, A; Kim, Edwin; Thompson, Linda S; Nolan, Matt; Land, Miriam L; Copeland, A; Lapidus, Alla L.; Lucas, Susan; Detter, J C; Zhulin, Igor B; Olsen, Gary; Whitman, W. B.; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos C

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching member of class Thermoproteales of Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first Crenarchaeote and only the second archaeon found to have transporters of the phosphotransferase system. T. pendens is known to require an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. T. pendens has fewer biosynthetic enzymes than any other free-living organism. In addition to heterotrophy, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein from a new subfamily. Predicted highly expressed proteins include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins, suggesting that defense against viruses is a high priority.

  20. Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.

    PubMed

    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.

  1. Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase.

    PubMed

    Modis, Y; Wierenga, R K

    2000-04-14

    Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in

  2. The carnitine biosynthetic pathway in Arabidopsis thaliana shares similar features with the pathway of mammals and fungi.

    PubMed

    Rippa, Sonia; Zhao, Yingjuan; Merlier, Franck; Charrier, Aurélie; Perrin, Yolande

    2012-11-01

    Carnitine is an essential quaternary ammonium amino acid that occurs in the microbial, plant and animal kingdoms. The role and synthesis of this compound are very well documented in bacteria, fungi and mammals. On the contrary, although the presence of carnitine in plant tissue has been reported four decades ago and information about its biological implication are available, nothing is known about its synthesis in plants. We designed experiments to determine if the carnitine biosynthetic pathway in Arabidopsis thaliana is similar to the pathway in mammals and in the fungi Neurospora crassa and Candida albicans. We first checked for the presence of trimetyllysine (TML) and γ-butyrobetaine (γ-BB), two precursors of carnitine in fungi and in mammals, using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Both compounds were shown to be present in plant extracts at concentrations in the picomole range per mg of dry weight. We next synthesized deuterium-labeled TML and transferred A. thaliana seedlings on growth medium supplemented with 1 mM of the deuterated precursor. LC-ESI-MS/MS analysis of plant extracts clearly highlighted the synthesis of deuterium labeled γ-BB and labeled carnitine in deuterated-TML fed plants. The similarities between plant, fungal and mammalian pathways provide very useful information to search homologies between genomes. As a matter of fact the analysis of A. thaliana protein database provides homology for several enzymes responsible for carnitine synthesis in fungi and mammals. The study of mutants affected in the corresponding genes would be very useful to elucidate the plant carnitine biosynthetic pathway and to investigate further the role of carnitine in plant physiology. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  3. Sex pheromone biosynthetic pathway for disparlure in the gypsy moth, Lymantria dispar

    PubMed Central

    Jurenka, Russell A.; Subchev, Mitko; Abad, José-Luis; Choi, Man-Yeon; Fabriàs, Gemma

    2003-01-01

    The pheromone biosynthetic pathway for production of the sex pheromone disparlure, 2-methyl-7R,8S-epoxy-octadecane, was determined for the gypsy moth. Each step in the pathway was followed by using deuterium-labeled compounds that could be identified by using GC/MS. This approach provides unequivocal determination of specific reactions in the pathway. It was shown that the alkene precursor, 2-methyl-Z7-octadecene, is most likely made in oenocyte cells associated with abdominal epidermal cells. The pathway begins with valine contributing carbons for chain initiation, including the methyl-branched carbon, followed by chain elongation to 19 carbons. The double bond is introduced with an unusual Δ12 desaturase that utilizes a methyl-branched substrate. The resulting 18-methyl-Z12-nonadecenoate is decarboxylated to the hydrocarbon, 2-methyl-Z7-octadecene. The alkene is then transported to the pheromone gland through the hemolymph, most probably by lipophorin. At the pheromone gland, the alkene is unloaded and transformed into the epoxide disparlure for release into the environment. A chiral HPLC column was used to demonstrate that the (R,S)-stereoisomer of the epoxide, (+)-disparlure is found in pheromone glands. PMID:12533665

  4. First principles model calculations of the biosynthetic pathway in selinadiene synthase.

    PubMed

    Das, Susanta; Dixit, Mudit; Major, Dan Thomas

    2016-10-15

    Terpenes comprise the largest class of natural products currently known. These ubiquitous molecules are synthesized by terpene synthases via complex carbocationic reactions, incorporating highly reactive intermediates. In the current study, we present a mechanistic investigation of the biosynthetic pathway for the formation of selina-4(15),7(11)-diene. We employ density functional theory to study a model carbocation system in the gas-phase, and delineate the energetic feasibility of a plausible reaction path. Our results suggests that during formation of selina-4(15),7(11)-diene, the substrate is likely folded in a conformation conducive to sequential cyclizations. We propose that a required proton transfer cannot occur intramolecularly in the gas-phase due to a high free energy barrier, and that enzyme assistance is essential for this step. Hybrid quantum mechanics-molecular mechanics docking studies suggest that enzyme intervention could be realized through electrostatic guidance.

  5. Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

    SciTech Connect

    Chang, Aram; Singh, Shanteri; Helmich, Kate E.; Goff, Randal D.; Bingman, Craig A.; Thorson, Jon S.; Phillips, Jr., George N.

    2012-03-15

    Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

  6. Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity.

    PubMed

    Chang, Aram; Singh, Shanteri; Helmich, Kate E; Goff, Randal D; Bingman, Craig A; Thorson, Jon S; Phillips, George N

    2011-10-25

    Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.

  7. Complete set of glycosyltransferase structures in the calicheamicin biosynthetic pathway reveals the origin of regiospecificity

    PubMed Central

    Chang, Aram; Singh, Shanteri; Helmich, Kate E.; Goff, Randal D.; Bingman, Craig A.; Thorson, Jon S.; Phillips, George N.

    2011-01-01

    Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering. PMID:21987796

  8. 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.

  9. 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

  10. 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.

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

    PubMed

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

    2014-05-27

    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.

  12. Elongating internodes of Zea mays (maize): Early steps in the GA biosynthetic pathway

    SciTech Connect

    Suzuki, Y.; Phinney, B.O. ); Gaskin, P.; MacMillan, J. )

    1989-04-01

    The early steps in the gibberellin (GA) biosynthetic pathway have yet to be defined for tissues that show a growth response to GAs. To this end we have synthesized the ({sup 13}C,{sup 3}H)-ent-kaurenoids, ent-kaurenol, ent-kaurenal ent-kaukenoic acid. We also have double-labeled ent-kaurene and double-labeled GA{sub 12}-aldehyde. We feed 1 - 10{mu}g of each substrate, individually, to 1.0g diced internodes in the appropriate buffer plus cofactors. We have observed up to 80% metabolism. We have identified (full scan GC-MS) 7{beta}-hydroxy-ent-kaurenoic acid as the major metabolite from double-labeled ent-kaurenoic acid feeds, thus defining the step ent-kaurenoic acid to 7{beta}-hydroxy-ent-kaurenoic acid.

  13. Two different pathways of internal conversion in carotenoids depending on the length of the conjugated chain

    NASA Astrophysics Data System (ADS)

    Fujii, Ritsuko; Inaba, Toru; Watanabe, Yasutaka; Koyama, Yasushi; Zhang, Jian-Ping

    2003-02-01

    Near-infrared, subpicosecond time-resolved absorption spectroscopy of all- trans carotenoids having the number of conjugated double bonds, n=9-13, identified two different pathways of internal conversion in accordance with the energy diagram recently determined by measurements of resonance-Raman excitation profiles (RREPs) [K. Furuichi, T. Sashima, Y. Koyama, Chem. Phys. Lett. 356 (2002) 547]: the 1B u+→1B u-→2A g- internal conversion for neurosporene ( n=9) and spheroidene ( n=10), whereas the 1B u+→3A g-→2A g- internal conversion for lycopene ( n=11), anhydrorhodovibrin ( n=12) and spirilloxanthin ( n=13).

  14. Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

    PubMed Central

    Cardoso, Mariana S.; Junqueira, Caroline; Trigueiro, Ricardo C.; Shams-Eldin, Hosam; Macedo, Cristiana S.; Araújo, Patrícia R.; Gomes, Dawidson A.; Martinelli, Patrícia M.; Kimmel, Jürgen; Stahl, Philipp; Niehus, Sebastian; Schwarz, Ralph T.; Previato, José O.; Mendonça-Previato, Lucia; Gazzinelli, Ricardo T.; Teixeira, Santuza M. R.

    2013-01-01

    Background Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease. Methodology/Principal Findings In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene. Conclusions/Significance Analyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this

  15. Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

    PubMed Central

    Nathanson, David A.; Armijo, Amanda L.; Tom, Michelle; Li, Zheng; Dimitrova, Elizabeth; Austin, Wayne R.; Nomme, Julian; Campbell, Dean O.; Ta, Lisa; Le, Thuc M.; Lee, Jason T.; Darvish, Ryan; Gordin, Ari; Wei, Liu; Liao, Hsiang-I; Wilks, Moses; Martin, Colette; Sadeghi, Saman; Murphy, Jennifer M.; Boulos, Nidal; Phelps, Michael E.; Faull, Kym F.; Herschman, Harvey R.; Jung, Michael E.; Czernin, Johannes; Lavie, Arnon

    2014-01-01

    Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies. PMID:24567448

  16. Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway in Bacillus subtilis

    PubMed Central

    Bennallack, Philip R.; Bewley, Kathryn D.; Burlingame, Mark A.; Robison, Richard A.; Miller, Susan M.

    2016-01-01

    ABSTRACT Thiopeptides represent one of several families of highly modified peptide antibiotics that hold great promise for natural product engineering. These macrocyclic peptides are produced by a combination of ribosomal synthesis and extensive posttranslational modification by dedicated processing enzymes. We previously identified a compact, plasmid-borne gene cluster for the biosynthesis of micrococcin P1 (MP1), an archetypal thiopeptide antibiotic. In an effort to genetically dissect this pathway, we have reconstituted it in Bacillus subtilis. Successful MP1 production required promoter engineering and the reassembly of essential biosynthetic genes in a modular plasmid. The resulting system allows for rapid pathway manipulation, including protein tagging and gene deletion. We find that 8 processing proteins are sufficient for the production of MP1 and that the tailoring enzyme TclS catalyzes a C-terminal reduction step that distinguishes MP1 from its sister compound micrococcin P2. IMPORTANCE The emergence of antibiotic resistance is one of the most urgent human health concerns of our day. A crucial component in an integrated strategy for countering antibiotic resistance is the ability to engineer pathways for the biosynthesis of natural and derivatized antimicrobial compounds. In this study, the model organism B. subtilis was employed to reconstitute and genetically modularize a 9-gene system for the biosynthesis of micrococcin, the founding member of a growing family of thiopeptide antibiotics. PMID:27381911

  17. Carotenoids, inflammation, and oxidative stress--implications of cellular signaling pathways and relation to chronic disease prevention.

    PubMed

    Kaulmann, Anouk; Bohn, Torsten

    2014-11-01

    Several epidemiologic studies have shown that diets rich in fruits and vegetables reduce the risk of developing several chronic diseases, such as type 2 diabetes, atherosclerosis, and cancer. These diseases are linked with systemic, low-grade chronic inflammation. Although controversy persists on the bioactive ingredients, several secondary plant metabolites have been associated with these beneficial health effects. Carotenoids represent the most abundant lipid-soluble phytochemicals, and in vitro and in vivo studies have suggested that they have antioxidant, antiapoptotic, and anti-inflammatory properties. Recently, many of these properties have been linked to the effect of carotenoids on intracellular signaling cascades, thereby influencing gene expression and protein translation. By blocking the translocation of nuclear factor κB to the nucleus, carotenoids are able to interact with the nuclear factor κB pathway and thus inhibit the downstream production of inflammatory cytokines, such as interleukin-8 or prostaglandin E2. Carotenoids can also block oxidative stress by interacting with the nuclear factor erythroid 2-related factor 2 pathway, enhancing its translocation into the nucleus, and activating phase II enzymes and antioxidants, such as glutathione-S-transferases. In this review, which is organized into in vitro, animal, and human investigations, we summarized current knowledge on carotenoids and metabolites with respect to their ability to modulate inflammatory and oxidative stress pathways and discuss potential dose-health relations. Although many pathways involved in the bioactivity of carotenoids have been revealed, future research should be directed toward dose-response relations of carotenoids, their metabolites, and their effect on transcription factors and metabolism.

  18. Genomics of iron acquisition in the plant pathogen Erwinia amylovora: insights in the biosynthetic pathway of the siderophore desferrioxamine E.

    PubMed

    Smits, Theo H M; Duffy, Brion

    2011-10-01

    Genomics has clarified the biosynthetic pathway for desferrioxamine E critical for iron acquisition in the enterobacterial fire blight pathogen Erwinia amylovora. Evidence for each of the individual steps and the role of desferrioxamine E biosynthesis in pathogen virulence and cell protection from host defenses is presented. Using comparative genomics, it can be concluded that desferrioxamine biosynthesis is ancestral within the genera Erwinia and Pantoea.

  19. Carotenoid accumulation in durian (Durio zibethinus) fruit is affected by ethylene via modulation of carotenoid pathway gene expression.

    PubMed

    Wisutiamonkul, Apinya; Ampomah-Dwamena, Charles; Allan, Andrew C; Ketsa, Saichol

    2017-06-01

    Carotenoid content in durian (Durio zibethinus) fruit is an important aspect of fruit quality, with different cultivars distinguished by differing pigmentation. We have studied the dependence of carotenogenesis on ethylene. Fruit of the cultivar 'Chanee' harvested at the mature stage were either left untreated (controls), treated with 1-methylcyclopropene (1-MCP) for 12 h, or treated with application of an aqueous ethephon solution to the stem end, or treated for 12 h with 1-MCP followed by ethephon application. Fruit were then stored for 9 d at 25 °C. Pulp color of durian became steadily yellowish as a result of accumulation of carotenoids, which were mainly beta-carotene, and alpha-carotene, with a minor amount of zeaxanthin and lutein. 1-MCP delayed the increase in the accumulation of beta-carotene, alpha-carotene, and zeaxanthin, but not lutein. In contrast, ethephon had no significant effect on carotenoid accumulation. The expression of zeta-carotene desaturase (ZDS), lycopene beta-cyclase (LCYB), chromoplast specific lycopene beta-cyclase (CYCB) and beta-carotene hydroxylase (BCH) genes was highly correlated with carotenoid content and pulp color.1-MCP resulted in significant down-regulation of ZDS, LCYB, CYCB and BCH expression. The accumulation of beta-carotene and alpha-carotene appears to be controlled by the level of expression of LCYB gene, whose function was tested in bacteria to show conversion of lycopene and delta-carotene to beta-carotene and alpha-carotene, respectively. These results suggest that ripening-induced carotenoid accumulation is regulated by endogenous ethylene controlling the expression of key genes such as LCYB. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. Eliminating the isoleucine biosynthetic pathway to reduce competitive carbon outflow during isobutanol production by Saccharomyces cerevisiae.

    PubMed

    Ida, Kengo; Ishii, Jun; Matsuda, Fumio; Kondo, Takashi; Kondo, Akihiko

    2015-04-29

    Isobutanol is an important biorefinery target alcohol that can be used as a fuel, fuel additive, or commodity chemical. Baker's yeast, Saccharomyces cerevisiae, is a promising organism for the industrial manufacture of isobutanol because of its tolerance for low pH and resistance to autolysis. It has been reported that gene deletion of the pyruvate dehydrogenase complex, which is directly involved in pyruvate metabolism, improved isobutanol production by S. cerevisiae. However, the engineering strategies available for S. cerevisiae are immature compared to those available for bacterial hosts such as Escherichia coli, and several pathways in addition to pyruvate metabolism compete with isobutanol production. The isobutyrate, pantothenate or isoleucine biosynthetic pathways were deleted to reduce the outflow of carbon competing with isobutanol biosynthesis in S. cerevisiae. The judicious elimination of these competing pathways increased isobutanol production. ILV1 encodes threonine ammonia-lyase, the enzyme that converts threonine to 2-ketobutanoate, a precursor for isoleucine biosynthesis. S. cerevisiae mutants in which ILV1 had been deleted displayed 3.5-fold increased isobutanol productivity. The ΔILV1 strategy was further combined with two previously established engineering strategies (activation of two steps of the Ehrlich pathway and the transhydrogenase-like shunt), providing 11-fold higher isobutanol productivity as compared to the parent strain. The titer and yield of this engineered strain was 224 ± 5 mg/L and 12.04 ± 0.23 mg/g glucose, respectively. The deletion of competitive pathways to reduce the outflow of carbon, including ILV1 deletion, is an important strategy for increasing isobutanol production by S. cerevisiae.

  1. Genome Sequence of Thermofilum pendens Reveals an Exceptional Loss of Biosynthetic Pathways without Genome Reduction▿ †

    PubMed Central

    Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein. The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins. PMID:18263724

  2. Major QTL for Carrot Color are Associated with Carotenoid Biosynthetic Genes and Interact Epistatically in a Domesticated x Wild Carrot Cross

    USDA-ARS?s Scientific Manuscript database

    Wild carrot roots are white and do not accumulate pigments while the cultivated carrot is one of the richest sources of carotenoid pigments – mainly provitamin A alpha and beta carotenes. In this study we performed QTL analyses for pigment content on a carotenoid biosynthesis function map based on t...

  3. Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells.

    PubMed

    Dykstra, Kaitlyn M; Allen, Cheryl; Born, Ella J; Tong, Huaxiang; Holstein, Sarah A

    2015-12-08

    Multiple myeloma (MM) is characterized by the production of monoclonal protein (MP). We have shown previously that disruption of the isoprenoid biosynthetic pathway (IBP) causes a block in MP secretion through a disruption of Rab GTPase activity, leading to an enhanced unfolded protein response and subsequent apoptosis in MM cells. Autophagy is induced by cellular stressors including nutrient deprivation and ER stress. IBP inhibitors have been shown to have disparate effects on autophagy. Here we define the mechanisms underlying the differential effects of IBP inhibitors on autophagic flux in MM cells utilizing specific pharmacological inhibitors. We demonstrate that IBP inhibition induces a net increase in autophagy as a consequence of disruption of isoprenoid biosynthesis which is not recapitulated by direct geranylgeranyl transferase inhibition. IBP inhibitor-induced autophagy is a cellular defense mechanism as treatment with the autophagy inhibitor bafilomycin A1 enhances the cytotoxic effects of GGPP depletion, but not geranylgeranyl transferase inhibition. Immunofluorescence microscopy studies revealed that IBP inhibitors disrupt ER to Golgi trafficking of monoclonal light chain protein and that this protein is not a substrate for alternative degradative pathways such as aggresomes and autophagosomes. These studies support further development of specific GGTase II inhibitors as anti-myeloma agents.

  4. Calmodulin-mediated suppression of 2-ketoisovalerate reductase in Beauveria bassiana beauvericin biosynthetic pathway.

    PubMed

    Kim, Jiyoung; Yoon, Deok-Hyo; Oh, Junsang; Hyun, Min-Woo; Han, Jae-Gu; Sung, Gi-Ho

    2016-11-01

    Ketoisovalerate reductase (KIVR, E.C. 1.2.7.7) mediates the specific reduction of 2-ketoisovalerate (2-Kiv) to d-hydroxyisovalerate (d-Hiv), a precursor for beauvericin biosynthesis. Beauvericin, a famous mycotoxin produced by many fungi, is a cyclooligomer depsipeptide, which has insecticidal, antimicrobial, antiviral, and cytotoxic activities. In this report, we demonstrated that Beauveria bassiana 2-ketoisovalerate reductase (BbKIVR) acts as a typical KIVR enzyme in the entomopathogenic fungus B. bassiana. In addition, we found that BbKIVR interacts with calmodulin (CaM) in vitro and in vivo. The functional role of CaM-binding to BbKIVR was to negatively regulate the BbKIVR activity in B. bassiana. Environmental stimuli such as light and salt stress suppressed BbKIVR activity in B. bassiana. Interestingly, this negative effect of BbKIVR activity by light and salt stress was recovered by CaM inhibitors, suggesting that the inhibitory mechanism is mediated through stimulation of CaM activity. Therefore, this work suggests that BbKIVR plays an important role in the beauvericin biosynthetic pathway mediated by environmental stimuli such as light and salt stress via the CaM signaling pathway.

  5. Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer

    PubMed Central

    Kaushik, Akash K.; Shojaie, Ali; Panzitt, Katrin; Sonavane, Rajni; Venghatakrishnan, Harene; Manikkam, Mohan; Zaslavsky, Alexander; Putluri, Vasanta; Vasu, Vihas T.; Zhang, Yiqing; Khan, Ayesha S.; Lloyd, Stacy; Szafran, Adam T.; Dasgupta, Subhamoy; Bader, David A.; Stossi, Fabio; Li, Hangwen; Samanta, Susmita; Cao, Xuhong; Tsouko, Efrosini; Huang, Shixia; Frigo, Daniel E.; Chan, Lawrence; Edwards, Dean P.; Kaipparettu, Benny A.; Mitsiades, Nicholas; Weigel, Nancy L.; Mancini, Michael; McGuire, Sean E.; Mehra, Rohit; Ittmann, Michael M.; Chinnaiyan, Arul M.; Putluri, Nagireddy; Palapattu, Ganesh S.; Michailidis, George; Sreekumar, Arun

    2016-01-01

    The precise molecular alterations driving castration-resistant prostate cancer (CRPC) are not clearly understood. Using a novel network-based integrative approach, here, we show distinct alterations in the hexosamine biosynthetic pathway (HBP) to be critical for CRPC. Expression of HBP enzyme glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be significantly decreased in CRPC compared with localized prostate cancer (PCa). Genetic loss-of-function of GNPNAT1 in CRPC-like cells increases proliferation and aggressiveness, in vitro and in vivo. This is mediated by either activation of the PI3K-AKT pathway in cells expressing full-length androgen receptor (AR) or by specific protein 1 (SP1)-regulated expression of carbohydrate response element-binding protein (ChREBP) in cells containing AR-V7 variant. Strikingly, addition of the HBP metabolite UDP-N-acetylglucosamine (UDP-GlcNAc) to CRPC-like cells significantly decreases cell proliferation, both in-vitro and in animal studies, while also demonstrates additive efficacy when combined with enzalutamide in-vitro. These observations demonstrate the therapeutic value of targeting HBP in CRPC. PMID:27194471

  6. Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in Saccharomyces cerevisiae.

    PubMed

    Yuan, Jifeng; Mishra, Pranjul; Ching, Chi Bun

    2017-01-01

    Isoamyl alcohol can be used not only as a biofuel, but also as a precursor for various chemicals. Saccharomyces cerevisiae inherently produces a small amount of isoamyl alcohol via the leucine degradation pathway, but the yield is very low. In the current study, several strategies were devised to overproduce isoamyl alcohol in budding yeast. The engineered yeast cells with the cytosolic isoamyl alcohol biosynthetic pathway produced significantly higher amounts of isobutanol over isoamyl alcohol, suggesting that the majority of the metabolic flux was diverted to the isobutanol biosynthesis due to the broad substrate specificity of Ehrlich pathway enzymes. To channel the key intermediate 2-ketosiovalerate (KIV) towards α-IPM biosynthesis, we introduced an artificial protein scaffold to pull dihydroxyacid dehydratase and α-IPM synthase into the close proximity, and the resulting strain yielded more than twofold improvement of isoamyl alcohol. The best isoamyl alcohol producer yielded 522.76 ± 38.88 mg/L isoamyl alcohol, together with 540.30 ± 48.26 mg/L isobutanol and 82.56 ± 8.22 mg/L 2-methyl-1-butanol. To our best knowledge, our work represents the first study to bypass the native compartmentalized α-IPM biosynthesis pathway for the isoamyl alcohol overproduction in budding yeast. More importantly, artificial protein scaffold based on the feature of quaternary structure of enzymes would be useful in improving the catalytic efficiency and the product specificity of other enzymatic reactions.

  7. 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

  8. 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.

  9. A mathematical model for the branched chain amino acid biosynthetic pathways of Escherichia coli K12.

    PubMed

    Yang, Chin-Rang; Shapiro, Bruce E; Hung, She-Pin; Mjolsness, Eric D; Hatfield, G Wesley

    2005-03-25

    As a first step toward the elucidation of the systems biology of the model organism Escherichia coli, it was our goal to mathematically model a metabolic system of intermediate complexity, namely the well studied end product-regulated pathways for the biosynthesis of the branched chain amino acids L-isoleucine, L-valine, and L-leucine. This has been accomplished with the use of kMech (Yang, C.-R., Shapiro, B. E., Mjolsness, E. D., and Hatfield, G. W. (2005) Bioinformatics 21, in press), a Cellerator (Shapiro, B. E., Levchenko, A., Meyerowitz, E. M., Wold, B. J., and Mjolsness, E. D. (2003) Bioinformatics 19, 677-678) language extension that describes a suite of enzyme reaction mechanisms. Each enzyme mechanism is parsed by kMech into a set of fundamental association-dissociation reactions that are translated by Cellerator into ordinary differential equations. These ordinary differential equations are numerically solved by Mathematica. Any metabolic pathway can be simulated by stringing together appropriate kMech models and providing the physical and kinetic parameters for each enzyme in the pathway. Writing differential equations is not required. The mathematical model of branched chain amino acid biosynthesis in E. coli K12 presented here incorporates all of the forward and reverse enzyme reactions and regulatory circuits of the branched chain amino acid biosynthetic pathways, including single and multiple substrate (Ping Pong and Bi Bi) enzyme kinetic reactions, feedback inhibition (allosteric, competitive, and non-competitive) mechanisms, the channeling of metabolic flow through isozymes, the channeling of metabolic flow via transamination reactions, and active transport mechanisms. This model simulates the results of experimental measurements.

  10. Utility of the Biosynthetic Folate Pathway for Targets in Antimicrobial Discovery

    PubMed Central

    Bourne, Christina R.

    2014-01-01

    The need for new antimicrobials is great in face of a growing pool of resistant pathogenic organisms. This review will address the potential for antimicrobial therapy based on polypharmacological activities within the currently utilized bacterial biosynthetic folate pathway. The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a critical node, dihydrofolate reductase (DHFR), which is shared between prokaryotes and eukaryotes. The DHFR enzyme is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS), which is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors. Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. Recent work has established an off-target effect of sulfonamide antibiotics on a eukaryotic enzyme, sepiapterin reductase, causing alterations in neurotransmitter synthesis. Given that inhibitors of both DHFR and DHPS are designed to mimic their cognate substrate, which contain shared substructures, it is reasonable to expect such “off-target” effects. These inhibitors are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS enzymes and/or substrates of similar substructure. Computational studies designed to assess polypharmacology reiterate these conclusions. This leads to hypotheses exploring the vast utility of multiple members of the folate pathway for modulating cellular metabolism, and includes an appealing capacity for prokaryotic

  11. Effective Antibiofilm Polyketides against Staphylococcus aureus from the Pyranonaphthoquinone Biosynthetic Pathways of Streptomyces Species.

    PubMed

    Oja, Terhi; San Martin Galindo, Paola; Taguchi, Takaaki; Manner, Suvi; Vuorela, Pia M; Ichinose, Koji; Metsä-Ketelä, Mikko; Fallarero, Adyary

    2015-10-01

    Streptomyces bacteria are renowned for their ability to produce bioactive secondary metabolites. Recently, synthetic biology has enabled the production of intermediates and shunt products, which may have altered biological activities compared to the end products of the pathways. Here, we have evaluated the potential of recently isolated alnumycins and other closely related pyranonaphthoquinone (PNQ) polyketides against Staphylococcus aureus biofilms. The antimicrobial potency of the compounds against planktonic cells and biofilms was determined by redox dye-based viability staining, and the antibiofilm efficacy of the compounds was confirmed by viable counting. A novel antistaphylococcal polyketide, alnumycin D, was identified. Unexpectedly, the C-ribosylated pathway shunt product alnumycin D was more active against planktonic and biofilm cells than the pathway end product alnumycin A, where a ribose unit has been converted into a dioxane moiety. The evaluation of the antibiofilm potential of other alnumycins revealed that the presence of the ribose moiety in pyranose form is essential for high activity against preformed biofilms. Furthermore, the antibiofilm potential of other closely related PNQ polyketides was examined. Based on their previously reported activity against planktonic S. aureus cells, granaticin B, kalafungin, and medermycin were also selected for testing, and among them, granaticin B was found to be the most potent against preformed biofilms. The most active antibiofilm PNQs, alnumycin D and granaticin B, share several structural features that may be important for their antibiofilm activity. They are uncharged, glycosylated, and also contain a similar oxygenation pattern of the lateral naphthoquinone ring. These findings highlight the potential of antibiotic biosynthetic pathways as a source of effective antibiofilm compounds.

  12. The Use of ClusterMine360 for the Analysis of Polyketide and Nonribosomal Peptide Biosynthetic Pathways.

    PubMed

    Tremblay, Nicolas; Hill, Patrick; Conway, Kyle R; Boddy, Christopher N

    2016-01-01

    Polyketides and nonribosomal peptides constitute two large families of microbial natural products. Over the past 20 years a broad range of microbial polyketide and nonribosomal peptide biosynthetic pathways have been characterized leading to a surfeit of genetic data on polyketide and nonribosomal peptide biosynthesis. We developed the ClusterMine360 database, which stores the antiSMASH-based annotation of gene clusters in the NCBI database, linking the structure of the natural product to the biosynthetic gene cluster. This database is searchable and enables the user to access multiple sequence files for phylogenetic analysis of polyketide and nonribosomal peptide biosynthetic genes. Herein we describe how to add compound families and gene clusters to the database and search it using key words or structures to identify specific gene clusters. We also describe how to download multiple sequence files for specific catalytic domains from polyketide and nonribosomal peptide biosynthesis.

  13. An R2R3-MYB transcription factor regulates carotenoid pigmentation in Mimulus lewisii flowers.

    PubMed

    Sagawa, Janelle M; Stanley, Lauren E; LaFountain, Amy M; Frank, Harry A; Liu, Chang; Yuan, Yao-Wu

    2016-02-01

    Carotenoids are yellow, orange, and red pigments that contribute to the beautiful colors and nutritive value of many flowers and fruits. The structural genes in the highly conserved carotenoid biosynthetic pathway have been well characterized in multiple plant systems, but little is known about the transcription factors that control the expression of these structural genes. By analyzing a chemically induced mutant of Mimulus lewisii through bulk segregant analysis and transgenic experiments, we have identified an R2R3-MYB, Reduced Carotenoid Pigmentation 1 (RCP1), as the first transcription factor that positively regulates carotenoid biosynthesis during flower development. Loss-of-function mutations in RCP1 lead to down-regulation of all carotenoid biosynthetic genes and reduced carotenoid content in M. lewisii flowers, a phenotype recapitulated by RNA interference in the wild-type background. Overexpression of this gene in the rcp1 mutant background restores carotenoid production and, unexpectedly, results in simultaneous decrease of anthocyanin production in some transgenic lines by down-regulating the expression of an activator of anthocyanin biosynthesis. Identification of transcriptional regulators of carotenoid biosynthesis provides the 'toolbox' genes for understanding the molecular basis of flower color diversification in nature and for potential enhancement of carotenoid production in crop plants via genetic engineering.

  14. ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed1[OPEN

    PubMed Central

    Magallanes-Lundback, Maria; Lipka, Alexander E.; Angelovici, Ruthie; DellaPenna, Dean

    2016-01-01

    Elucidation of the carotenoid biosynthetic pathway has enabled altering the composition and content of carotenoids in various plants, but to achieve desired nutritional impacts, the genetic components regulating carotenoid homeostasis in seed, the plant organ consumed in greatest abundance, must be elucidated. We used a combination of linkage mapping, genome-wide association studies (GWAS), and pathway-level analysis to identify nine loci that impact the natural variation of seed carotenoids in Arabidopsis (Arabidopsis thaliana). ZEAXANTHIN EPOXIDASE (ZEP) was the major contributor to carotenoid composition, with mutants lacking ZEP activity showing a remarkable 6-fold increase in total seed carotenoids relative to the wild type. Natural variation in ZEP gene expression during seed development was identified as the underlying mechanism for fine-tuning carotenoid composition, stability, and ultimately content in Arabidopsis seed. We previously showed that two CAROTENOID CLEAVAGE DIOXYGENASE enzymes, CCD1 and CCD4, are the primary mediators of seed carotenoid degradation, and here we demonstrate that ZEP acts as an upstream control point of carotenoid homeostasis, with ZEP-mediated epoxidation targeting carotenoids for degradation by CCD enzymes. Finally, four of the nine loci/enzymatic activities identified as underlying natural variation in Arabidopsis seed carotenoids also were identified in a recent GWAS of maize (Zea mays) kernel carotenoid variation. This first comparison of the natural variation in seed carotenoids in monocots and dicots suggests a surprising overlap in the genetic architecture of these traits between the two lineages and provides a list of likely candidates to target for selecting seed carotenoid variation in other species. PMID:27208224

  15. Independent sources of condition dependency and multiple pathways determine a composite trait: lessons from carotenoid-based plumage colouration.

    PubMed

    Romero-Diaz, C; Richner, H; Granado-Lorencio, F; Tschirren, B; Fitze, P S

    2013-03-01

    Many colour ornaments are composite traits consisting of at least four components, which themselves may be more complex, determined by independent evolutionary pathways, and potentially being under different environmental control. To date, little evidence exists that several different components of colour elaboration are condition dependent and no direct evidence exists that different ornamental components are affected by different sources of variation. For example, in carotenoid-based plumage colouration, one of the best-known condition-dependent ornaments, colour elaboration stems from both condition-dependent pigment concentration and structural components. Some environmental flexibility of these components has been suggested, but specifically which and how they are affected remains unknown. Here, we tested whether multiple colour components may be condition dependent, by using a comprehensive 3 × 2 experimental design, in which we carotenoid supplemented and immune challenged great tit nestlings (Parus major) and quantified effects on different components of colouration. Plumage colouration was affected by an interaction between carotenoid availability and immune challenge. Path analyses showed that carotenoid supplementation increased plumage saturation via feather carotenoid concentration and via mechanisms unrelated to carotenoid deposition, while immune challenge affected feather length, but not carotenoid concentration. Thus, independent condition-dependent pathways, affected by different sources of variation, determine colour elaboration. This provides opportunities for the evolution of multiple signals within components of ornamental traits. This finding indicates that the selective forces shaping the evolution of different components of a composite trait and the trait's signal content may be more complex than believed so far, and that holistic approaches are required for drawing comprehensive evolutionary conclusions.

  16. Comparative SNP diversity among four Eucalyptus species for genes from secondary metabolite biosynthetic pathways

    PubMed Central

    Külheim, Carsten; Hui Yeoh, Suat; Maintz, Jens; Foley, William J; Moran, Gavin F

    2009-01-01

    Background There is little information about the DNA sequence variation within and between closely related plant species. The combination of re-sequencing technologies, large-scale DNA pools and availability of reference gene sequences allowed the extensive characterisation of single nucleotide polymorphisms (SNPs) in genes of four biosynthetic pathways leading to the formation of ecologically relevant secondary metabolites in Eucalyptus. With this approach the occurrence and patterns of SNP variation for a set of genes can be compared across different species from the same genus. Results In a single GS-FLX run, we sequenced over 103 Mbp and assembled them to approximately 50 kbp of reference sequences. An average sequencing depth of 315 reads per nucleotide site was achieved for all four eucalypt species, Eucalyptus globulus, E. nitens, E. camaldulensis and E. loxophleba. We sequenced 23 genes from 1,764 individuals and discovered 8,631 SNPs across the species, with about 1.5 times as many SNPs per kbp in the introns compared to exons. The exons of the two closely related species (E. globulus and E. nitens) had similar numbers of SNPs at synonymous and non-synonymous sites. These species also had similar levels of SNP diversity, whereas E. camaldulensis and E. loxophleba had much higher SNP diversity. Neither the pathway nor the position in the pathway influenced gene diversity. The four species share between 20 and 43% of the SNPs in these genes. Conclusion By using conservative statistical detection methods, we were confident about the validity of each SNP. With numerous individuals sampled over the geographical range of each species, we discovered one SNP in every 33 bp for E. nitens and one in every 31 bp in E. globulus. In contrast, the more distantly related species contained more SNPs: one in every 16 bp for E. camaldulensis and one in 17 bp for E. loxophleba, which is, to the best of our knowledge, the highest frequency of SNPs described in woody plant

  17. Novel expression patterns of carotenoid pathway-related gene in citrus leaves and maturing fruits

    USDA-ARS?s Scientific Manuscript database

    Carotenoids are abundant in citrus fruits and vary among cultivars and species. In the present study, HPLC and real-time PCR were used to investigate the expression patterns of 23 carotenoid biosynthesis gene family members and their possible relation with carotenoid accumulation in flavedo, juice s...

  18. Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway.

    PubMed

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-04-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.

  19. 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.

  20. Discovery of cahuitamycins as biofilm inhibitors derived from a convergent biosynthetic pathway

    PubMed Central

    Park, Sung Ryeol; Tripathi, Ashootosh; Wu, Jianfeng; Schultz, Pamela J.; Yim, Isaiah; McQuade, Thomas J.; Yu, Fengan; Arevang, Carl-Johan; Mensah, Abraham Y.; Tamayo-Castillo, Giselle; Xi, Chuanwu; Sherman, David H.

    2016-01-01

    Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes. Analysis of extracts derived from Streptomyces gandocaensis results in the discovery of three peptidic metabolites (cahuitamycins A–C), with cahuitamycin C being the most effective inhibitor (IC50=14.5 μM). Biosynthesis of cahuitamycin C proceeds via a convergent biosynthetic pathway, with one of the steps apparently being catalysed by an unlinked gene encoding a 6-methylsalicylate synthase. Efforts to assess starter unit diversification through selective mutasynthesis lead to production of unnatural analogues cahuitamycins D and E of increased potency (IC50=8.4 and 10.5 μM). PMID:26880271

  1. Discovery of cahuitamycins as biofilm inhibitors derived from a convergent biosynthetic pathway.

    PubMed

    Park, Sung Ryeol; Tripathi, Ashootosh; Wu, Jianfeng; Schultz, Pamela J; Yim, Isaiah; McQuade, Thomas J; Yu, Fengan; Arevang, Carl-Johan; Mensah, Abraham Y; Tamayo-Castillo, Giselle; Xi, Chuanwu; Sherman, David H

    2016-02-16

    Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes. Analysis of extracts derived from Streptomyces gandocaensis results in the discovery of three peptidic metabolites (cahuitamycins A-C), with cahuitamycin C being the most effective inhibitor (IC50=14.5 μM). Biosynthesis of cahuitamycin C proceeds via a convergent biosynthetic pathway, with one of the steps apparently being catalysed by an unlinked gene encoding a 6-methylsalicylate synthase. Efforts to assess starter unit diversification through selective mutasynthesis lead to production of unnatural analogues cahuitamycins D and E of increased potency (IC50=8.4 and 10.5 μM).

  2. Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis.

    PubMed

    Winterberg, Britta; Uhlmann, Stefanie; Linne, Uwe; Lessing, Franziska; Marahiel, Mohamed A; Eichhorn, Heiko; Kahmann, Regine; Schirawski, Jan

    2010-03-01

    Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron-chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non-ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC-MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl-coenzyme A (CoA)-hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)-CoA synthase Hcs1 in U. maydis that HMG-CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.

  3. 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

  4. Enzymes of the taurine biosynthetic pathway are expressed in rat mammary gland.

    PubMed

    Ueki, Iori; Stipanuk, Martha H

    2007-08-01

    Taurine is the most abundant free amino acid in the body and is present at high concentrations during development and in the early milk. It is synthesized from cysteine via oxidation of cysteine to cysteinesulfinate by the enzyme cysteine dioxygenase (CDO), followed by the decarboxylation of cysteinesulfinate to hypotaurine, catalyzed by cysteine sulfinic acid decarboxylase (CSAD). To determine whether the taurine biosynthetic pathway is present in mammary gland and whether it is differentially expressed during pregnancy and lactation, and also to further explore the possible regulation of hepatic taurine synthesis during pregnancy and lactation, we measured mammary and hepatic CDO and CSAD mRNA and protein concentrations and tissue, plasma and milk taurine concentrations. CDO and CSAD mRNA and protein were expressed in mammary gland and liver regardless of physiological state. Immunohistochemistry demonstrated the expression of CDO in ductal cells of pregnant rats, but not in other mammary epithelial cells or in ductal cells of nonpregnant rats. CDO was also present in stromal adipocytes in mammary glands of both pregnant and nonpregnant rats. Our findings support an upregulation of taurine synthetic capacity in the mammary gland of pregnant rats, based on mammary taurine and hypotaurine concentrations and the intense immunohistochemical staining for CDO in ductal cells of pregnant rats. Hepatic taurine synthetic capacity, particularly CSAD, and taurine concentrations were highest in rats during the early stages of lactation, suggesting the liver may also play a role in the synthesis of taurine to support lactation or repletion of maternal reserves.

  5. 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

  6. MoeH5: a natural glycorandomizer from the moenomycin biosynthetic pathway

    PubMed Central

    Ostash, Bohdan; Campbell, Jennifer; Luzhetskyy, Andriy; Walker, Suzanne

    2013-01-01

    Summary The biosynthesis of the phosphoglycolipid antibiotic moenomycin A attracts the attention of researchers hoping to develop new moenomycin-based antibiotics against multidrug resistant Gram-positive infections. There is detailed understanding of most steps of this biosynthetic pathway in Streptomyces ghanaensis (ATCC14672), except for the ultimate stage, where a single pentasaccharide intermediate is converted into a set of unusually modified final products. Here we report that only one gene, moeH5, encoding a homologue of the glutamine amidotransferase (GAT) enzyme superfamily, is responsible for the observed diversity of terminally decorated moenomycins. Genetic and biochemical evidence support the idea that MoeH5 is a novel member of the GAT superfamily, whose homologues are involved in the synthesis of various secondary metabolites as well as K and O antigens of bacterial lipopolysaccharide. Our results provide insights into the mechanism of MoeH5 and its counterparts, and give us a new tool for the diversification of phosphoglycolipid antibiotics. PMID:24164498

  7. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    PubMed

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

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

    SciTech Connect

    Gallo, A.; Bruno, K. S.; Solfrizzo, M.; Perrone, G.; Mule, G.; Visconti, A.; Baker, S. E.

    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 the 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.

  9. Biochemical and Structural Characterization of MycCI, a Versatile P450 Biocatalyst from the Mycinamicin Biosynthetic Pathway.

    PubMed

    DeMars, Matthew D; Sheng, Fang; Park, Sung Ryeol; Lowell, Andrew N; Podust, Larissa M; Sherman, David H

    2016-09-16

    Cytochrome P450 monooxygenases (P450s) are some of nature's most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize inert C-H bonds has led to their increasing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present study, we investigated the reactivity of the P450 MycCI from the mycinamicin biosynthetic pathway toward a variety of macrocyclic compounds and discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding experiments, steady-state kinetics studies, and X-ray crystallographic analysis of MycCI bound to its native substrate mycinamicin VIII. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared to MycCI. Thus, the ability of the latter to hydroxylate both macrocyclic aglycones and macrolides sets it apart from related biosynthetic P450s and highlights its potential for developing novel P450 biocatalysts with broad substrate scope and high regioselectivity.

  10. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

    PubMed

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-03-14

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes.

  11. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway

    PubMed Central

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes. PMID:26971881

  12. Hexosamine biosynthetic pathway activity in leptin resistant sucrose-drinking rats

    PubMed Central

    Harris, Ruth B.S.; Apolzan, John W.

    2014-01-01

    Rats offered 30% sucrose solution in addition to chow and water become leptin resistant therefore we investigated the effect of sucrose solution consumption on leptin signaling. In Experiment 1 rats were resistant to 3rd ventricle injections of 1 μg leptin after 36 days of sucrose and western blot indicated that resistance was associated with increased basal levels of signal transducer and activator of transcription 3 phosphorylation (pSTAT3). In Experiment 2 rats were resistant to a peripheral injection of 2 mg leptin/kg after 26 days of sucrose. Immunohistochemistry indicated that increased basal pSTAT3 was limited to the medial and lateral arcuate nucleus of the hypothalamus. Increased availability of glucose and fructose can stimulate the hexosamine biosynthetic pathway (HBP) which O-GlcNAc-modifies proteins. This has the potential to change protein bioactivity. We tested whether this pathway could account for the leptin resistance. There was no increase in the expression of HBP enzymes in tissues from sucrose rats in Experiment 1, however, direct activation of the HBP with a 3 hour intravenous infusion of 30 μmol/kg/min glucosamine significantly increased hypothalamic pSTAT3. Although sucrose consumption and activation of the HBP both increase hypothalamic pSTAT3 experiments described here did not provide evidence of a direct link between sucrose consumption, HBP activity and leptin resistance. Unexpectedly, we found that the HBP enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) in liver and O-GlcNAcase in hypothalamus were increased 30 minutes after leptin injection in leptin responsive animals, implying a complex interaction between activity of the HBP and leptin responsiveness. PMID:25446204

  13. Hexosamine biosynthetic pathway activity in leptin resistant sucrose-drinking rats.

    PubMed

    Harris, Ruth B S; Apolzan, John W

    2015-01-01

    Rats offered 30% sucrose solution in addition to chow and water become leptin resistant therefore we investigated the effect of sucrose solution consumption on leptin signaling. In Experiment 1 rats were resistant to 3rd ventricle injections of1.5 μg leptin after 36 days of sucrose and western blot indicated that resistance was associated with increased basal levels of signal transducer and activator of transcription 3 phosphorylation (pSTAT3). In Experiment 2 rats were resistant to a peripheral injection of 2mg leptin/kg after 26 days of sucrose. Immunohistochemistry indicated that increased basal pSTAT3 was limited to the medial and lateral arcuate nucleus of the hypothalamus. Increased availability of glucose and fructose can stimulate the hexosamine biosynthetic pathway (HBP) which O-GlcNAc-modifies proteins. This has the potential to change protein bioactivity. We tested whether this pathway could account for the leptin resistance. There was no increase in the expression of HBP enzymes in tissues from sucrose rats in Experiment 1, however, direct activation of the HBP with a 3h intravenous infusion of 30 μmol/kg/min glucosamine significantly increased hypothalamic pSTAT3. Although sucrose consumption and activation of the HBP both increase hypothalamic pSTAT3 experiments described here did not provide evidence of a direct link between sucrose consumption, HBP activity and leptin resistance. Unexpectedly, we found that the HBP enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) in liver and O-GlcNAcase in hypothalamus were increased 30min after leptin injection in leptin responsive animals, implying a complex interaction between activity of the HBP and leptin responsiveness. Copyright © 2014. Published by Elsevier Inc.

  14. UDP-sugar biosynthetic pathway: contribution to cyanidin 3-galactoside biosynthesis in apple skin.

    PubMed

    Ban, Yusuke; Kondo, Satoru; Ubi, Benjamin Ewa; Honda, Chikako; Bessho, Hideo; Moriguchi, Takaya

    2009-10-01

    UDP-galactose:flavonoid 3-O-galactosyltransferase (UFGalT) is responsible for cyanidin 3-galactoside (cy3-gal) synthesis from cyanidin (cy) and UDP-galactose (UDP-gal) which are, respectively, catalyzed by anthocyanidin synthase (ANS) and UDP-glucose 4-epimerase (UGE). To clarify the contribution of UDP-galactose pathway to cy3-gal accumulation in apple skin, we analyzed the contents of UDP-gal and UDP-glucose (UDP-glu), cy, and, cy3-gal contents along with UGE activity. We confirmed that transcript levels for apple ANS and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) coincided with anthocyanin accumulation in three apple cultivars differing in their skin colors. During fruit development, changes in level of cy coincided with that of cy3-gal, whereas UDP-gal and UGE activity showed no similar trend with cy3-gal. Significant correlation was not observed between the changes in UGE activity and UDP-sugar contents. The effect of temperature and UV-B radiation (different environmental conditions) on the accumulation of UDP-sugars, cy and cy3-gal, and UGE activity were also investigated in a pale-red cultivar. High temperature tended to depress the accumulation of both UDP-sugars and cy concomitant with the decrease in cy3-gal content irrespective of UV-B radiation. Although there was no high inhibition of both cy and UDP-sugars at low-temperature without UV-B, cy3-gal accumulation was highly depressed. UGE activity was highest at low temperature with UV-B, but not much different under other conditions. Most of the parameters under different environmental conditions were significantly correlated with each other. Based on these results, contribution of UDP-sugar biosynthetic pathway to anthocyanin biosynthesis under different environmental conditions as well as during fruit development is discussed.

  15. Characterization of the biosynthetic pathway of glucosylglycerate in the archaeon Methanococcoides burtonii.

    PubMed

    Costa, Joana; Empadinhas, Nuno; Gonçalves, Luís; Lamosa, Pedro; Santos, Helena; da Costa, Milton S

    2006-02-01

    The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50 degrees C. The maximal activity of GpgP was at 50 degrees C with GPG as the substrate and at 60 degrees C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.

  16. Characterization of the Biosynthetic Pathway of Glucosylglycerate in the Archaeon Methanococcoides burtonii

    PubMed Central

    Costa, Joana; Empadinhas, Nuno; Gonçalves, Luís; Lamosa, Pedro; Santos, Helena; da Costa, Milton S.

    2006-01-01

    The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and “Dehalococcoides ethenogenes.” GpgS had maximal activity at 50°C. The maximal activity of GpgP was at 50°C with GPG as the substrate and at 60°C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate. PMID:16428406

  17. Carotenoids in marine animals.

    PubMed

    Maoka, Takashi

    2011-02-22

    Marine animals contain various carotenoids that show structural diversity. These marine animals accumulate carotenoids from foods such as algae and other animals and modify them through metabolic reactions. Many of the carotenoids present in marine animals are metabolites of β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxanthin, and astaxanthin, etc. Carotenoids found in these animals provide the food chain as well as metabolic pathways. In the present review, I will describe marine animal carotenoids from natural product chemistry, metabolism, food chain, and chemosystematic viewpoints, and also describe new structural carotenoids isolated from marine animals over the last decade.

  18. Carotenoids in Marine Animals

    PubMed Central

    Maoka, Takashi

    2011-01-01

    Marine animals contain various carotenoids that show structural diversity. These marine animals accumulate carotenoids from foods such as algae and other animals and modify them through metabolic reactions. Many of the carotenoids present in marine animals are metabolites of β-carotene, fucoxanthin, peridinin, diatoxanthin, alloxanthin, and astaxanthin, etc. Carotenoids found in these animals provide the food chain as well as metabolic pathways. In the present review, I will describe marine animal carotenoids from natural product chemistry, metabolism, food chain, and chemosystematic viewpoints, and also describe new structural carotenoids isolated from marine animals over the last decade. PMID:21566799

  19. The hedgehog Pathway Gene shifted Functions together with the hmgcr-Dependent Isoprenoid Biosynthetic Pathway to Orchestrate Germ Cell Migration

    PubMed Central

    Deshpande, Girish; Zhou, Keren; Wan, Joy Y.; Friedrich, Jana; Jourjine, Nicholas; Smith, Daniel; Schedl, Paul

    2013-01-01

    The Drosophila embryonic gonad is assembled from two distinct cell types, the Primordial Germ Cells (PGCs) and the Somatic Gonadal Precursor cells (SGPs). The PGCs form at the posterior of blastoderm stage embryos and are subsequently carried inside the embryo during gastrulation. To reach the SGPs, the PGCs must traverse the midgut wall and then migrate through the mesoderm. A combination of local repulsive cues and attractive signals emanating from the SGPs guide migration. We have investigated the role of the hedgehog (hh) pathway gene shifted (shf) in directing PGC migration. shf encodes a secreted protein that facilitates the long distance transmission of Hh through the proteoglycan matrix after it is released from basolateral membranes of Hh expressing cells in the wing imaginal disc. shf is expressed in the gonadal mesoderm, and loss- and gain-of-function experiments demonstrate that it is required for PGC migration. Previous studies have established that the hmgcr-dependent isoprenoid biosynthetic pathway plays a pivotal role in generating the PGC attractant both by the SGPs and by other tissues when hmgcr is ectopically expressed. We show that production of this PGC attractant depends upon shf as well as a second hh pathway gene gγ1. Further linking the PGC attractant to Hh, we present evidence indicating that ectopic expression of hmgcr in the nervous system promotes the release/transmission of the Hh ligand from these cells into and through the underlying mesodermal cell layer, where Hh can contact migrating PGCs. Finally, potentiation of Hh by hmgcr appears to depend upon cholesterol modification. PMID:24068944

  20. Biosynthetic pathways of plastid-derived organelles as potential drug targets against parasitic apicomplexa.

    PubMed

    Seeber, Frank

    2003-06-01

    Apicomplexan parasites are a large phylum of unicellular and obligate intracellular organisms of great medical importance. They include the human pathogens Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease, together affecting several hundred million people worldwide. The search for new and effective drugs against these pathogens has been boosted during the last years by an unexpected finding. Through molecular and cell biological analysis it was realized that probably most members of this phylum harbor a plastid-like organelle, called the apicoplast, which probably is derived from the engulfment of a red alga in ancient times. Although the apicoplast itself contains a small circular genome, most of the proteome of this organelle is encoded in the nuclear genome, and the proteins are subsequently transported to the apicoplast. It is assumed to contain a number of unique metabolic pathways not found in the vertebrate host, making it an ideal "playground" for those interested in drug targets. Recent reports have shown that the rationale of this approach is valid and that new drugs which are urgently needed especially for plasmodial infections, might be developed in the near future based on these targets. Amongst them are three enzymes of the plant-like fatty acid synthesis machinery and enzymes of the non-mevalonat isoprenoid biosynthesis pathway. From their presence in the apicoplast it can be concluded that fatty acid and lipid biosynthesis seems to be a major function of the apicoplast. Another recently described apicoplast enzyme, ferredoxin-NADP(+)-reductase and its redox partner, ferredoxin, points to another interesting organelle-specific biosynthetic pathway, namely [Fe-S] cluster biosynthesis. In the present review, the fundamental aspects of the apicoplast as drug target will be described, together with the specific pathways and their

  1. Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance.

    PubMed

    Lerksuthirat, Tassanee; Sangcakul, Areeporn; Lohnoo, Tassanee; Yingyong, Wanta; Rujirawat, Thidarat; Krajaejun, Theerapong

    2017-04-01

    Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

  2. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota)

    PubMed Central

    Simpson, Kevin; Quiroz, Luis F.; Rodriguez-Concepción, Manuel; Stange, Claudia R.

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products. PMID:27630663

  3. Regulation of the Omega-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes

    PubMed Central

    Ruyter, Bente; Berge, Gerd Marit; Sun, Yajing; Østbye, Tone-Kari Knutsdatter

    2016-01-01

    Limited availability of the n-3 fatty acids EPA and DHA have led to an interest in better understanding of the n-3 biosynthetic pathway and its regulation. The biosynthesis of alpha-linolenic acid to EPA and DHA involves several complex reaction steps including desaturation-, elongation- and peroxisomal beta-oxidation enzymes. The aims of the present experiments were to gain more knowledge on how this biosynthesis is regulated over time by different doses and fatty acid combinations. Hepatocytes isolated from salmon were incubated with various levels and combinations of oleic acid, EPA and DHA. Oleic acid led to a higher expression of the Δ6 fatty acid desaturase (fad) genes Δ6fad_a, Δ6fad_b, Δ6fad_c and the elongase genes elovl2 compared with cells cultured in medium enriched with DHA. Further, the study showed rhythmic variations in expression over time. Levels were reached where a further increase in specific fatty acids given to the cells not stimulated the conversion further. The gene expression of Δ6fad_a_and Δ6fad_b responded similar to fatty acid treatment, suggesting a co-regulation of these genes, whereas Δ5fad and Δ6fad_c showed a different regulation pattern. EPA and DHA induced different gene expression patterns, especially of Δ6fad_a. Addition of radiolabelled alpha-linolenic acid to the hepatocytes confirmed a higher degree of elongation and desaturation in cells treated with oleic acid compared to cells treated with DHA. This study suggests a complex regulation of the conversion process of n-3 fatty acids. Several factors, such as that the various gene copies are differently regulated, the gene expression show rhythmic variations and gene expression only affected to a certain level, determines when you get the maximum conversion of the beneficial n-3 fatty acids. PMID:27973547

  4. Discovery of alkenones with variable methylene-interrupted double bonds: implications for the biosynthetic pathway.

    PubMed

    Zheng, Yinsui; Dillon, James T; Zhang, Yifan; Huang, Yongsong

    2016-12-01

    Alkenones (C37 -C40 ) are highly specific biomarkers produced by certain haptophyte algae in ocean and lacustrine environments and have been widely used for paleoclimate studies. Unusual shorter-chain alkenones (SCA; e.g., C35 and C36 ) have been found in environmental and culture samples, but the origin and structure of these compounds are much less understood. The marine alkenone producer, Emiliania huxleyi CCMP2758 strain, was reported with abundant C35:2 Me (∆(12, 19) ) alkenones when cultured at 15°C (Prahl et al. 2006). Here we show, when this strain is cultured at 4°C-10°C, that CCMP2758 produces abundant C35:3 Me, C36:3 Me, and small amounts of C36:3 Et alkenones with unusual double-bond positions of ∆(7, 12, 19) . We determine the double-bond positions of the C35:3 Me and C36:3 Me alkenones by GC-MS analysis of the dimethyl disulfide and cyclobutylamine derivatives, and we provide the first temperature calibrations based on the unsaturation ratios of the C35 and C36 alkenones. Previous studies have found C35:2 Me (∆(14, 19) ) and C36:2 Et (∆(14, 19) ) alkenones with three-methylene interruption in the Black Sea sediments, but this is the first reported instance of alkenones with a mixed three- and five-methylene interruption configuration in the double-bond positions. The discovery of these alkenones allows us to propose a novel biosynthetic scheme, termed the SCA biosynthesis pathway, that simultaneously rationalizes the formation of both the C35:3 Me (∆(7, 12, 19) ) alkenone in our culture and the ∆(14, 19) Black Sea type alkenones without invoking new desaturases for the unusual double-bond positions.

  5. Dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic pathway.

    PubMed

    Gniazdowska, Agnieszka; Krasuska, Urszula; Bogatek, Renata

    2010-11-01

    The connection between classical phytohormone-ethylene and two signaling molecules, nitric oxide (NO) and hydrogen cyanide (HCN), was investigated in dormancy removal and germination "sensu stricto" of apple (Malus domestica Borkh.) embryos. Deep dormancy of apple embryos was removed by short-term (3-6 h) pre-treatment with NO or HCN. NO- or HCN-mediated stimulation of germination was associated with enhanced emission of ethylene by the embryos, coupled with transient increase in ROS concentration in embryos. Ethylene vapors stimulated germination of dormant apple embryos and eliminated morphological anomalies characteristic for young seedlings developed from dormant embryos. Inhibitors of ethylene receptors completely impeded beneficial effect of NO and HCN on embryo germination. NO- and HCN-induced ethylene emission by apple embryo was only slightly reduced by inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity during first 4 days of germination. Short-term pre-treatment of the embryos with NO and HCN modified activity of both key enzymes of ethylene biosynthetic pathway: ACC synthase and ACC oxidase. Activity of ACC synthase declined during first 4 days of germination, while activity of ACC oxidase increased markedly at that time. Additional experiments point to non-enzymatic conversion of ACC to ethylene in the presence of ROS (H(2)O(2)). The results indicate that NO and HCN may alleviate dormancy of apple embryos "via" transient accumulation of ROS, leading to enhanced ethylene emission which is required to terminate germination "sensu stricto". Therefore, ethylene seems to be a trigger factor in control of apple embryo dormancy removal and germination.

  6. Carotenoids play a positive role in the degradation of heterocycles by Sphingobium yanoikuyae.

    PubMed

    Liu, Xiaorui; Gai, Zhonghui; Tao, Fei; Tang, Hongzhi; Xu, Ping

    2012-01-01

    Microbial oxidative degradation is a potential way of removing pollutants such as heterocycles from the environment. During this process, reactive oxygen species or other oxidants are inevitably produced, and may cause damage to DNA, proteins, and membranes, thereby decreasing the degradation rate. Carotenoids can serve as membrane-integrated antioxidants, protecting cells from oxidative stress. Several genes involved in the carotenoid biosynthetic pathway were cloned and characterized from a carbazole-degrading bacterium Sphingobium yanoikuyae XLDN2-5. In addition, a yellow-pigmented carotenoid synthesized by strain XLDN2-5 was identified as zeaxanthin that was synthesized from β-carotene through β-cryptoxanthin. The amounts of zeaxanthin and hydrogen peroxide produced were significantly and simultaneously enhanced during the biodegradation of heterocycles (carbazole < carbazole + benzothiophene < carbazole + dibenzothiophene). These higher production levels were consistent with the transcriptional increase of the gene encoding phytoene desaturase, one of the key enzymes for carotenoid biosynthesis. Sphingobium yanoikuyae XLDN2-5 can enhance the synthesis of zeaxanthin, one of the carotenoids, which may modulate membrane fluidity and defense against intracellular oxidative stress. To our knowledge, this is the first report on the positive role of carotenoids in the biodegradation of heterocycles, while elucidating the carotenoid biosynthetic pathway in the Sphingobium genus.

  7. The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers.

    PubMed

    Li, Li; Yang, Yong; Xu, Qiang; Owsiany, Katherine; Welsch, Ralf; Chitchumroonchokchai, Chureeporn; Lu, Shan; Van Eck, Joyce; Deng, Xiu-Xin; Failla, Mark; Thannhauser, Theodore W

    2012-03-01

    Provitamin A carotenoids in staple crops are not very stable during storage and their loss compromises nutritional quality. To elucidate the fundamental mechanisms underlying carotenoid accumulation and stability, we investigated transgenic potato tubers that expressed the cauliflower Orange (Or) gene. We found that the Or transgene not only promoted retention of β-carotene level, but also continuously stimulated its accumulation during 5 months of cold storage. In contrast, no increased levels of carotenoids were observed in the tubers of vector-only controls or a yellow-flesh variety during the same period of storage. The increased carotenoid accumulation was found to be associated with the formation of lipoprotein-carotenoid sequestering structures, as well as with the enhanced abundance of phytoene synthase, a key enzyme in the carotenoid biosynthetic pathway. Furthermore, the provitamin A carotenoids stored were shown to be stable during simulated digestion and accessible for uptake by human intestinal absorptive cells. Proteomic analysis identified three major functional groups of proteins (i.e. heat shock proteins, glutathione-S-transferases, and carbohydrate metabolic proteins) that are potentially important in the Or-regulated carotenoid accumulation. Our results show that regulation of carotenoid sequestration capacity is an important mechanism by which carotenoid stability is regulated. Our findings suggest that induction of a proper sink structure formation in staple crops may provide the crops with a unique ability to promote and/or stabilize provitamin A accumulation during plant growth and post-harvest storage.

  8. An unusual pathway of excitation energy deactivation in carotenoids: singlet-to-triplet conversion on an ultrafast timescale in a photosynthetic antenna.

    PubMed

    Gradinaru, C C; Kennis, J T; Papagiannakis, E; van Stokkum, I H; Cogdell, R J; Fleming, G R; Niederman, R A; van Grondelle, R

    2001-02-27

    Carotenoids are important biomolecules that are ubiquitous in nature and find widespread application in medicine. In photosynthesis, they have a large role in light harvesting (LH) and photoprotection. They exert their LH function by donating their excited singlet state to nearby (bacterio)chlorophyll molecules. In photosynthetic bacteria, the efficiency of this energy transfer process can be as low as 30%. Here, we present evidence that an unusual pathway of excited state relaxation in carotenoids underlies this poor LH function, by which carotenoid triplet states are generated directly from carotenoid singlet states. This pathway, operative on a femtosecond and picosecond timescale, involves an intermediate state, which we identify as a new, hitherto uncharacterized carotenoid singlet excited state. In LH complex-bound carotenoids, this state is the precursor on the reaction pathway to the triplet state, whereas in extracted carotenoids in solution, this state returns to the singlet ground state without forming any triplets. We discuss the possible identity of this excited state and argue that fission of the singlet state into a pair of triplet states on individual carotenoid molecules constitutes the mechanism by which the triplets are generated. This is, to our knowledge, the first ever direct observation of a singlet-to-triplet conversion process on an ultrafast timescale in a photosynthetic antenna.

  9. Identification of a dTDP-rhamnose biosynthetic pathway that oscillates with the molting cycle in Caenorhabditis elegans.

    PubMed

    Feng, Likui; Shou, Qingyao; Butcher, Rebecca A

    2016-06-01

    L-Rhamnose is a common component of cell-wall polysaccharides, glycoproteins and some natural products in bacteria and plants, but is rare in fungi and animals. In the present study, we identify and characterize a biosynthetic pathway for dTDP-rhamnose in Caenorhabditis elegans that is highly conserved across nematode species. We show that RML-1 activates glucose 1-phosphate (Glc-1-P) in the presence of either dTTP or UTP to yield dTDP-glucose or UDP-glucose, respectively. RML-2 is a dTDP-glucose 4,6-dehydratase, converting dTDP-glucose into dTDP-4-keto-6-deoxyglucose. Using mass spectrometry and NMR spectroscopy, we demonstrate that coincubation of dTDP-4-keto-6-deoxyglucose with RML-3 (3,5-epimerase) and RML-4 (4-keto-reductase) produces dTDP-rhamnose. RML-4 could only be expressed and purified in an active form through co-expression with a co-regulated protein, RML-5, which forms a complex with RML-4. Analysis of the sugar nucleotide pool in C. elegans established the presence of dTDP-rhamnose in vivo Targeting the expression of the rhamnose biosynthetic genes by RNAi resulted in significant reductions in dTDP-rhamnose, but had no effect on the biosynthesis of a closely related sugar, ascarylose, found in the ascaroside pheromones. Therefore, the rhamnose and ascarylose biosynthetic pathways are distinct. We also show that transcriptional reporters for the rhamnose biosynthetic genes are expressed highly in the embryo, in the hypodermis during molting cycles and in the hypodermal seam cells specifically before the molt to the stress-resistant dauer larval stage. These expression patterns suggest that rhamnose biosynthesis may play an important role in hypodermal development or the production of the cuticle or surface coat during molting.

  10. Identification of a dTDP-rhamnose biosynthetic pathway that oscillates with the molting cycle in Caenorhabditis elegans

    PubMed Central

    Feng, Likui; Shou, Qingyao; Butcher, Rebecca A.

    2016-01-01

    L-Rhamnose is a common component of cell-wall polysaccharides, glycoproteins and some natural products in bacteria and plants, but is rare in fungi and animals. In the present study, we identify and characterize a biosynthetic pathway for dTDP-rhamnose in Caenorhabditis elegans that is highly conserved across nematode species. We show that RML-1 activates glucose 1-phosphate (Glc-1-P) in the presence of either dTTP or UTP to yield dTDP-glucose or UDP-glucose, respectively. RML-2 is a dTDP-glucose 4,6-dehydratase, converting dTDP-glucose into dTDP-4-keto-6-deoxyglucose. Using mass spectrometry and NMR spectroscopy, we demonstrate that coincubation of dTDP-4-keto-6-deoxyglucose with RML-3 (3,5-epimerase) and RML-4 (4-keto-reductase) produces dTDP-rhamnose. RML-4 could only be expressed and purified in an active form through co-expression with a co-regulated protein, RML-5, which forms a complex with RML-4. Analysis of the sugar nucleotide pool in C. elegans established the presence of dTDP-rhamnose in vivo. Targeting the expression of the rhamnose biosynthetic genes by RNAi resulted in significant reductions in dTDP-rhamnose, but had no effect on the biosynthesis of a closely related sugar, ascarylose, found in the ascaroside pheromones. Therefore, the rhamnose and ascarylose biosynthetic pathways are distinct. We also show that transcriptional reporters for the rhamnose biosynthetic genes are expressed highly in the embryo, in the hypodermis during molting cycles and in the hypodermal seam cells specifically before the molt to the stress-resistant dauer larval stage. These expression patterns suggest that rhamnose biosynthesis may play an important role in hypodermal development or the production of the cuticle or surface coat during molting. PMID:27009306

  11. Structure-Based Design of Inhibitors of the Crucial Cysteine Biosynthetic Pathway Enzyme O-Acetyl Serine Sulfhydrylase.

    PubMed

    Mazumder, Mohit; Gourinath, Samudrala

    2016-01-01

    The cysteine biosynthetic pathway is of fundamental importance for the growth, survival, and pathogenicity of the many pathogens. This pathway is present in many species but is absent in mammals. The ability of pathogens to counteract the oxidative defences of a host is critical for the survival of these pathogens during their long latent phases, especially in anaerobic pathogens such as Entamoeba histolytica, Leishmania donovani, Trichomonas vaginalis, and Salmonella typhimurium. All of these organisms rely on the de novo cysteine biosynthetic pathway to assimilate sulphur and maintain a ready supply of cysteine. The de novo cysteine biosynthetic pathway, on account of its being important for the survival of pathogens and at the same time being absent in mammals, is an important drug target for diseases such as amoebiasis, trichomoniasis & tuberculosis. Cysteine biosynthesis is catalysed by two enzymes: serine acetyl transferase (SAT) followed by O-acetylserine sulfhydrylase (OASS). OASS is well studied, and with the availability of crystal structures of this enzyme in different conformations, it is a suitable template for structure-based inhibitor development. Moreover, OASS is highly conserved, both structurally and sequence-wise, among the above-mentioned organisms. There have been several reports of inhibitor screening and development against this enzyme from different organisms such as Salmonella typhimurium, Mycobacterium tuberculosis and Entamoeba histolytica. All of these inhibitors have been reported to display micromolar to nanomolar binding affinities for the open conformation of the enzyme. In this review, we highlight the structural similarities of this enzyme in different organisms and the attempts for inhibitor development so far. We also propose that the intermediate state of the enzyme may be the ideal target for the design of effective highaffinity inhibitors.

  12. RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid-based metabolites such as lipids.

    PubMed

    Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A; Gobel, Verena

    2015-05-01

    The field of metabolomics continues to catalog new compounds, but their functional analysis remains technically challenging, and roles beyond metabolism are largely unknown. Unbiased genetic/RNAi screens are powerful tools to identify the in vivo functions of protein-encoding genes, but not of nonproteinaceous compounds such as lipids. They can, however, identify the biosynthetic enzymes of these compounds-findings that are usually dismissed, as these typically synthesize multiple products. Here, we provide a method using follow-on biosynthetic pathway screens to identify the endpoint biosynthetic enzyme and thus the compound through which they act. The approach is based on the principle that all subsequently identified downstream biosynthetic enzymes contribute to the synthesis of at least this one end product. We describe how to systematically target lipid biosynthetic pathways; optimize targeting conditions; take advantage of pathway branchpoints; and validate results by genetic assays and biochemical analyses. This approach extends the power of unbiased genetic/RNAi screens to identify in vivo functions of non-nucleic acid-based metabolites beyond their metabolic roles. It will typically require several months to identify a metabolic end product by biosynthetic pathway screens, but this time will vary widely depending, among other factors, on the end product's location in the pathway, which determines the number of screens required for its identification.

  13. Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway.

    PubMed Central

    Braus, G H

    1991-01-01

    This review focuses on the gene-enzyme relationships and the regulation of different levels of the aromatic amino acid biosynthetic pathway in a simple eukaryotic system, the unicellular yeast Saccharomyces cerevisiae. Most reactions of this branched pathway are common to all organisms which are able to synthesize tryptophan, phenylalanine, and tyrosine. The current knowledge about the two main control mechanisms of the yeast aromatic amino acid biosynthesis is reviewed. (i) At the transcriptional level, most structural genes are regulated by the transcriptional activator GCN4, the regulator of the general amino acid control network, which couples transcriptional derepression to amino acid starvation of numerous structural genes in multiple amino acid biosynthetic pathways. (ii) At the enzyme level, the carbon flow is controlled mainly by modulating the enzyme activities at the first step of the pathway and at the branch points by feedback action of the three aromatic amino acid end products. Implications of these findings for the relationship of S. cerevisiae to prokaryotic as well as to higher eukaryotic organisms and for general regulatory mechanisms occurring in a living cell such as initiation of transcription, enzyme regulation, and the regulation of a metabolic branch point are discussed. PMID:1943992

  14. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

    PubMed Central

    2012-01-01

    Background Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Results Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation. Conclusions The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization. PMID:23092390

  15. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine.

    PubMed

    Peifer, Susanne; Barduhn, Tobias; Zimmet, Sarah; Volmer, Dietrich A; Heinzle, Elmar; Schneider, Konstantin

    2012-10-24

    Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol g(CDW)⁻¹. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol g(CDW)⁻¹). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol g(CDW)⁻¹) derived from IMP degradation. The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization.

  16. The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily†

    PubMed Central

    De Laurentis, Walter; Khim, Leang; Anderson, J.L. Ross; Adam, Ariane; Johnson, Kenneth A.; Phillips, Robert S.; Chapman, Stephen K.; van Pee, Karl-Heinz; Naismith, James H.

    2012-01-01

    Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mole of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with D- and L-tryptophan and D- and L-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-L-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO

  17. The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily.

    PubMed

    De Laurentis, Walter; Khim, Leang; Anderson, J L Ross; Adam, Ariane; Johnson, Kenneth A; Phillips, Robert S; Chapman, Stephen K; van Pee, Karl-Heinz; Naismith, James H

    2007-10-30

    Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO

  18. Modification of targets related to the Entner-Doudoroff/pentose phosphate pathway route for methyl-D-erythritol 4-phosphate-dependent carotenoid biosynthesis in Escherichia coli.

    PubMed

    Li, Chun; Ying, Lan-Qing; Zhang, Sha-Sha; Chen, Nan; Liu, Wei-Feng; Tao, Yong

    2015-08-12

    In engineered strains of Escherichia coli, bioconversion efficiency is determined by not only metabolic flux but also the turnover efficiency of relevant pathways. Methyl-D-erythritol 4-phosphate (MEP)-dependent carotenoid biosynthesis in E. coli requires efficient turnover of precursors and balanced flux among precursors, cofactors, and cellular energy. However, the imbalanced supply of glyceraldehyde 3-phosphate (G3P) and pyruvate precursors remains the major metabolic bottleneck. To address this problem, we manipulated various genetic targets related to the Entner-Doudoroff (ED)/pentose phosphate (PP) pathways. Systematic target modification was conducted to improve G3P and pyruvate use and rebalance the precursor and redox fluxes. Carotenoid production was improved to different degrees by modifying various targets in the Embden-Meyerhof-Parnas (EMP) and ED pathways, which directed metabolic flux from the EMP pathway towards the ED pathway. The improvements in yield were much greater when the MEP pathway was enhanced. The coordinated modification of ED and MEP pathway targets using gene expression enhancement and protein coupling strategies in the pgi deletion background further improved carotenoid synthesis. The fine-tuning of flux at the branch point between the ED and PP pathways was important for carotenoid biosynthesis. Deletion of pfkAB instead of pgi reduced the carotenoid yield. This suggested that anaplerotic flux of G3P and pyruvate might be necessary for carotenoid biosynthesis. Improved carotenoid yields were accompanied by increased biomass and decreased acetate overflow. Therefore, efficient use of G3P and pyruvate precursors resulted in a balance among carotenoid biosynthesis, cell growth, and by-product metabolism. An efficient and balanced MEP-dependent carotenoid bioconversion strategy involving both the ED and PP pathways was implemented by the coordinated modification of diverse central metabolic pathway targets. In this strategy, enhancement

  19. Physiological and molecular responses of the isoprenoid biosynthetic pathway in a drought-resistant Mediterranean shrub, Cistus creticus exposed to water deficit.

    PubMed

    Munné-Bosch, Sergi; Falara, Vasiliki; Pateraki, Irene; López-Carbonell, Marta; Cela, Jana; Kanellis, Angelos K

    2009-01-30

    The goal of the present research was to obtain new insights into the mechanisms underlying drought stress resistance in plants. Specifically, we evaluated changes in the expression of genes encoding enzymes involved in isoprenoid biosynthesis, together with the levels of the corresponding metabolites (chlorophylls, carotenoids, tocopherols and abscisic acid), in a drought-resistant Mediterranean shrub, Cistus creticus grown under Mediterranean field conditions. Summer drought led to reductions in the relative leaf water content (RWC) by 25%, but did not alter the maximum efficiency of PSII, indicating the absence of damage to the photosynthetic apparatus. While the expression of genes encoding C. creticus chlorophyll a oxygenase/chlorophyll b synthase (CAO) and phytoene synthase (PSY) were not affected by water deficit, the genes encoding homogentisate phytyl-transferase (HPT) and 9-cis-epoxycarotenoid dioxygenase (NCED) were induced in water-stressed (WS) plants. Drought-induced changes in gene expression were observed at early stages of drought and were strongly correlated with levels of the corresponding metabolites, with simultaneous increases in abscisic acid and alpha-tocopherol levels of up to 4-fold and 62%, respectively. Furthermore, alpha-tocopherol levels were strongly positively correlated with abscisic acid contents, but not with the levels of jasmonic acid and salicylic acid. We conclude that the abscisic acid and tocopherol biosynthetic pathway may be regulated at the transcript level in WS C. creticus plants, and that the genes encoding HPT and NCED may play a key role in the drought stress resistance of this Mediterranean shrub by modulating abscisic acid and tocopherol biosynthesis.

  20. Decoding Biosynthetic Pathways in Plants by Pulse-Chase Strategies Using 13CO2 as a Universal Tracer †

    PubMed Central

    Bacher, Adelbert; Chen, Fan; Eisenreich, Wolfgang

    2016-01-01

    13CO2 pulse-chase experiments monitored by high-resolution NMR spectroscopy and mass spectrometry can provide 13C-isotopologue compositions in biosynthetic products. Experiments with a variety of plant species have documented that the isotopologue profiles generated with 13CO2 pulse-chase labeling are directly comparable to those that can be generated by the application of [U-13C6]glucose to aseptically growing plants. However, the application of the 13CO2 labeling technology is not subject to the experimental limitations that one has to take into account for experiments with [U-13C6]glucose and can be applied to plants growing under physiological conditions, even in the field. In practical terms, the results of biosynthetic studies with 13CO2 consist of the detection of pairs, triples and occasionally quadruples of 13C atoms that have been jointly contributed to the target metabolite, at an abundance that is well above the stochastic occurrence of such multiples. Notably, the connectivities of jointly transferred 13C multiples can have undergone modification by skeletal rearrangements that can be diagnosed from the isotopologue data. As shown by the examples presented in this review article, the approach turns out to be powerful in decoding the carbon topology of even complex biosynthetic pathways. PMID:27429012

  1. Construction of a Chimeric Biosynthetic Pathway for the de novo Biosynthesis of Rosmarinic Acid in Escherichia coli

    PubMed Central

    Bloch, Sarah E.; Schmidt-Dannert, Claudia

    2014-01-01

    Hydroxycinnamic acid esters (HCEs) are widely-distributed phenylpropanoid-derived plant natural products. Rosmarinic acid (RA 7), the most well-known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, a microbial production platform for HCEs could provide a sustainable, controlled means of production. Through the overexpression of a six-enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA 7 and the related HCE isorinic acid (IA 8) in E. coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We show HCE biosynthesis using three plant RAS orthologs exhibiting different levels of HCE biosynthesis, but the same ratio of IA 8 to RA 7 produced. This work serves as a proof of concept for a microbial production platform for HCEs using a modular biosynthetic approach to access diverse natural and non-natural HCEs. PMID:25205019

  2. Laccase‐catalysed oxidations of naturally occurring phenols: from in vivo biosynthetic pathways to green synthetic applications

    PubMed Central

    Jeon, Jong‐Rok; Baldrian, Petr; Murugesan, Kumarasamy; Chang, Yoon‐Seok

    2012-01-01

    Summary Laccases are oxidases that contain several copper atoms, and catalyse single‐electron oxidations of phenolic compounds with concomitant reduction of oxygen to water. The enzymes are particularly widespread in ligninolytic basidiomycetes, but also occur in certain prokaryotes, insects and plants. Depending on the species, laccases are involved in various biosynthetic processes contributing to carbon recycling in land ecosystems and the morphogenesis of biomatrices, wherein low‐molecular‐weight naturally occurring phenols serve as key enzyme substrates. Studies of these in vivo synthetic pathways have afforded new insights into fungal laccase applicability in green synthetic chemistry. Thus, we here review fungal laccase‐catalysed oxidations of naturally occurring phenols that are particularly relevant to the synthesis of fine organic chemicals, and we discuss how the discovered synthetic strategies mimic laccase‐involved in vivo pathways, thus enhancing the green nature of such reactions. Laccase‐catalysed in vivo processes yield several types of biopolymers, including those of cuticles, lignin, polyflavonoids, humus and the melanin pigments, using natural mono‐ or poly‐phenols as building blocks. The in vivo synthetic pathways involve either phenoxyl radical‐mediated coupling or cross‐linking reactions, and can be adapted to the design of in vitro oxidative processes involving fungal laccases in organic synthesis; the laccase substrates and the synthetic mechanisms reflect in vivo processes. Notably, such in vitro synthetic pathways can also reproduce physicochemical properties (e.g. those of chromophores, and radical‐scavenging, hydration and antimicrobial activities) found in natural biomaterials. Careful study of laccase‐associated in vivo metabolic pathways has been rewarded by the discovery of novel green applications for fungal laccases. This review comprehensively summarizes the available data on laccase

  3. Carotenoid responses to environmental stimuli: integrating redox and carbon controls into a fruit model.

    PubMed

    Fanciullino, A L; Bidel, L P R; Urban, L

    2014-02-01

    Carotenoids play an important role in plant adaptation to fluctuating environments as well as in the human diet by contributing to the prevention of chronic diseases. Insights have been gained recently into the way individual factors, genetic, environmental or developmental, control the carotenoid biosynthetic pathway at the molecular level. The identification of the rate-limiting steps of carotenogenesis has paved the way for programmes of breeding, and metabolic engineering, aimed at increasing the concentration of carotenoids in different crop species. However, the complexity that arises from the interactions between the different factors as well as from the coordination between organs remains poorly understood. This review focuses on recent advances in carotenoid responses to environmental stimuli and discusses how the interactions between the modulation factors and between organs affect carotenoid build-up. We develop the idea that reactive oxygen species/redox status and sugars/carbon status can be considered as integrated factors that account for most effects of the major environmental factors influencing carotenoid biosynthesis. The discussion highlights the concept of carotenoids or carotenoid-derivatives as stress signals that may be involved in feedback controls. We propose a conceptual model of the effects of environmental and developmental factors on carotenoid build-up in fruits.

  4. Biochemistry and Molecular Biology of Carotenoid Biosynthesis in Chili Peppers (Capsicum spp.)

    PubMed Central

    del Rocío Gómez-García, María; Ochoa-Alejo, Neftalí

    2013-01-01

    Capsicum species produce fruits that synthesize and accumulate carotenoid pigments, which are responsible for the fruits’ yellow, orange and red colors. Chili peppers have been used as an experimental model for studying the biochemical and molecular aspects of carotenoid biosynthesis. Most reports refer to the characterization of carotenoids and content determination in chili pepper fruits from different species, cultivars, varieties or genotypes. The types and levels of carotenoids differ between different chili pepper fruits, and they are also influenced by environmental conditions. Yellow-orange colors of chili pepper fruits are mainly due to the accumulation of α- and β-carotene, zeaxanthin, lutein and β-cryptoxanthin. Carotenoids such as capsanthin, capsorubin and capsanthin-5,6-epoxide confer the red colors. Chromoplasts are the sites of carotenoid pigment synthesis and storage. According to the most accepted theory, the synthesis of carotenoids in chili peppers is controlled by three loci: c1, c2 and y. Several enzymes participating in carotenoid biosynthesis in chili pepper fruits have been isolated and characterized, and the corresponding gene sequences have been reported. However, there is currently limited information on the molecular mechanisms that regulate this biosynthetic pathway. Approaches to gain more knowledge of the regulation of carotenoid biosynthesis are discussed. PMID:24065101

  5. Biochemistry and molecular biology of carotenoid biosynthesis in chili peppers (Capsicum spp.).

    PubMed

    Gómez-García, María del Rocío; Ochoa-Alejo, Neftalí

    2013-09-16

    Capsicum species produce fruits that synthesize and accumulate carotenoid pigments, which are responsible for the fruits' yellow, orange and red colors. Chili peppers have been used as an experimental model for studying the biochemical and molecular aspects of carotenoid biosynthesis. Most reports refer to the characterization of carotenoids and content determination in chili pepper fruits from different species, cultivars, varieties or genotypes. The types and levels of carotenoids differ between different chili pepper fruits, and they are also influenced by environmental conditions. Yellow-orange colors of chili pepper fruits are mainly due to the accumulation of α- and β-carotene, zeaxanthin, lutein and β-cryptoxanthin. Carotenoids such as capsanthin, capsorubin and capsanthin-5,6-epoxide confer the red colors. Chromoplasts are the sites of carotenoid pigment synthesis and storage. According to the most accepted theory, the synthesis of carotenoids in chili peppers is controlled by three loci: c1, c2 and y. Several enzymes participating in carotenoid biosynthesis in chili pepper fruits have been isolated and characterized, and the corresponding gene sequences have been reported. However, there is currently limited information on the molecular mechanisms that regulate this biosynthetic pathway. Approaches to gain more knowledge of the regulation of carotenoid biosynthesis are discussed.

  6. Complex Sexual Deception in an Orchid Is Achieved by Co-opting Two Independent Biosynthetic Pathways for Pollinator Attraction.

    PubMed

    Xu, Haiyang; Bohman, Björn; Wong, Darren C J; Rodriguez-Delgado, Claudia; Scaffidi, Adrian; Flematti, Gavin R; Phillips, Ryan D; Pichersky, Eran; Peakall, Rod

    2017-07-10

    Sexually deceptive orchids lure their specific male pollinators using volatile semiochemicals that mimic female sex pheromones. To date, the semiochemicals known to be involved consist of blends of chemically and biosynthetically related compounds. In contrast, we report that (S)-β-citronellol and 2-hydroxy-6-methylacetophenone, two biosynthetically distinct compounds, are the active semiochemicals in Caladenia plicata, which is pollinated by male Zeleboria sp. thynnine wasps. They are also sex pheromone components of the female Zeleboria. A 1:4 blend elicits a high rate of attempted copulation (∼70%) in bioassays, equivalent to rates observed at orchid flowers. Whereas β-citronellol is well known, 2-hydroxy-6-methylacetophenone appears to be previously unknown as a floral volatile. Production of the two compounds is restricted to glandular sepal tips; thus, differential expression analysis of contrasting floral tissue transcriptomes was employed to illuminate the biosynthesis. As expected, production of (S)-β-citronellol commences with the terpene synthase GES1 catalyzing the irreversible conversion of geranyl diphosphate (GPP) to geraniol. Contrary to prediction, biosynthesis subsequently proceeds in three steps, commencing with the oxidation of geraniol to geranial by alcohol dehydrogenase ADH3, followed by the enantioselective reduction of a double bond in geranial by geranial reductase GER1 to give (S)-β-citronellal. Finally, ADH3-catalyzed reduction of (S)-β-citronellal results in (S)-β-citronellol. In line with previous work on insects showing that 2-hydroxy-6-methylacetophenone is derived from a polyketide pathway, we report a differentially expressed polyketide synthase (PKS) gene candidate. Thus, in this unique example of sexual deception, pollination is achieved by co-opting and regulating two independent biosynthetic pathways of floral volatile compounds. VIDEO ABSTRACT. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    SciTech Connect

    Geiger, Jim

    2013-11-30

    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.

  8. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway1[OPEN

    PubMed Central

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  9. Genetic engineering, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy elucidate the bikaverin biosynthetic pathway in Fusarium fujikuroi.

    PubMed

    Arndt, Birgit; Studt, Lena; Wiemann, Philipp; Osmanov, Helena; Kleigrewe, Karin; Köhler, Jens; Krug, Isabel; Tudzynski, Bettina; Humpf, Hans-Ulrich

    2015-11-01

    Secondary metabolites of filamentous fungi can be highly bioactive, ranging from antibiotic to cancerogenic properties. In this study we were able to identify a new, yet unknown metabolite produced by Fusarium fujikuroi, an ascomycetous rice pathogen. With the help of genomic engineering and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS) followed by isolation and detailed structure elucidation, the new substance could be designated as an unknown bikaverin precursor, missing two methyl- and one hydroxy group, hence named oxo-pre-bikaverin. Though the bikaverin gene cluster has been extensively studied in the past, elucidation of the biosynthetic pathway remained elusive due to a negative feedback loop that regulates the genes within the cluster. To decipher the bikaverin biosynthetic pathway and to overcome these negative regulation circuits, the structural cluster genes BIK2 and BIK3 were overexpressed independently in the ΔΔBIK2/BIK3+OE::BIK1 mutant background by using strong constitutive promoters. Using the software tool MZmine 2, the metabolite profile of the generated mutants obtained by HPLC-HRMS was compared, revealing further intermediates.

  10. Identification and characterization of genes involved in the jasmonate biosynthetic and signaling pathways in mulberry (Morus notabilis).

    PubMed

    Wang, Qing; Ma, Bi; Qi, Xiwu; Guo, Qing; Wang, Xuwei; Zeng, Qiwei; He, Ningjia

    2014-07-01

    Jasmonate (JA) is an important phytohormone regulating growth, development, and environmental response in plants, particularly defense response against herbivorous insects. Recently, completion of the draft genome of the mulberry (Morus notabilis) in conjunction with genome sequencing of silkworm (Bombyx mori) provides an opportunity to study this unique plant-herbivore interaction. Here, we identified genes involved in JA biosynthetic and signaling pathways in the genome of mulberry for the first time, with the majority of samples showing a tissue-biased expression pattern. The analysis of the representative genes 12-oxophytodienoic acid reductase (OPRs) and jasmonate ZIM-domain (JAZs) was performed and the results indicated that the mulberry genome contains a relatively small number of JA biosynthetic and signaling pathway genes. A gene encoding an important repressor, MnNINJA, was identified as an alternative splicing variant lacking an ethylene-responsive element binding factor-associated amphiphilic repression motif. Having this fundamental information will facilitate future functional study of JA-related genes pertaining to mulberry-silkworm interactions. © 2014 Institute of Botany, Chinese Academy of Sciences.

  11. Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids, austinol and dehydroaustinol in Aspergillus nidulans

    PubMed Central

    Lo, Hsien-Chun; Entwistle, Ruth; Guo, Chun-Jun; Ahuja, Manmeet; Szewczyk, Edyta; Hung, Jui-Hsiang; Chiang, Yi-Ming; Oakley, Berl R.; Wang, Clay C. C.

    2012-01-01

    Meroterpenoids are a class of fungal natural products that are produced from polyketide and terpenoid precursors. An understanding of meroterpenoid biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has previously been found to produce two meroterpenoids, austinol and dehydroaustinol. Using targeted deletions that we created, we have determined that, surprisingly, two separate gene clusters are required for meroterpenoid biosynthesis. One is a cluster of four genes including a polyketide synthase gene, ausA. The second is a cluster of ten additional genes including a prenyltransferase gene, ausN, located on a separate chromosome. Chemical analysis of mutant extracts enabled us to isolate 3,5-dimethylorsellinic acid and ten additional meroterpenoids that are either intermediates or shunt products from the biosynthetic pathway. Six of them were identified as novel meroterpenoids in this study. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans meroterpenoids. PMID:22329759

  12. 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.

  13. Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures

    PubMed Central

    Patil, Rohan A.; Kolewe, Martin E.; Normanly, Jennifer; Walker, Elsbeth L.; Roberts, Susan C.

    2012-01-01

    Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In this work, cultures with different aggregation profiles were established to create predictable differences in paclitaxel accumulation upon MeJA elicitation. Expression of known paclitaxel biosynthetic genes in MeJA-elicited cultures exhibiting both substantial (15-fold) and moderate (2-fold) differences in paclitaxel accumulation was analyzed using qRT-PCR. Each population exhibited the characteristic large increase in paclitaxel pathway gene expression following MeJA elicitation; however, differences in expression between populations were minor, and only observed for the cultures with the 15-fold variation in paclitaxel content. These data suggest that although upregulation of biosynthetic pathway gene expression contributes to observed increases in paclitaxel synthesis upon elicitation with MeJA, there are additional factors that need to be uncovered before paclitaxel productivity can be fully optimized. PMID:22095859

  14. 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

  15. Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids.

    PubMed

    Petroutsos, Dimitris; Amiar, Souad; Abida, Heni; Dolch, Lina-Juana; Bastien, Olivier; Rébeillé, Fabrice; Jouhet, Juliette; Falconet, Denis; Block, Maryse A; McFadden, Geoffrey I; Bowler, Chris; Botté, Cyrille; Maréchal, Eric

    2014-04-01

    Photosynthetic membranes have a unique lipid composition that has been remarkably well conserved from cyanobacteria to chloroplasts. These membranes are characterized by a very high content in galactoglycerolipids, i.e., mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Galactoglycerolipids make up the bulk of the lipid matrix in which photosynthetic complexes are embedded. They are also known to fulfill specific functions, such as stabilizing photosystems, being a source of polyunsaturated fatty acids for various purposes and, in some eukaryotes, being exported to other subcellular compartments. The conservation of MGDG and DGDG suggests that selection pressures might have conserved the enzymes involved in their biosynthesis, but this does not appear to be the case. Important evolutionary transitions comprise primary endosymbiosis (from a symbiotic cyanobacterium to a primary chloroplast) and secondary endosymbiosis (from a symbiotic unicellular algal eukaryote to a secondary plastid). In this review, we compare biosynthetic pathways based on available molecular and biochemical data, highlighting enzymatic reactions that have been conserved and others that have diverged or been lost, as well as the emergence of parallel and alternative biosynthetic systems originating from other metabolic pathways. Questions for future research are highlighted.

  16. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway.

    PubMed

    Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Hunter, C Neil; Bocian, David F; Holten, Dewey; Niedzwiedzki, Dariusz M

    2016-06-23

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC═C + NC═O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids.

  17. Quenching Capabilities of Long-Chain Carotenoids in Light-Harvesting-2 Complexes from Rhodobacter sphaeroides with an Engineered Carotenoid Synthesis Pathway

    PubMed Central

    2016-01-01

    Six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC=C + NC=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excited state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a light-harvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (∼2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids. PMID:27285777

  18. Carotenoid 3',4'-desaturase is involved in carotenoid biosynthesis in the radioresistant bacterium Deinococcus radiodurans.

    PubMed

    Tian, Bing; Sun, Zongtao; Xu, Zhenjian; Shen, Shaochuan; Wang, Hu; Hua, Yuejin

    2008-12-01

    Deinococcus radiodurans strain R1 synthesizes deinoxanthin, a unique carotenoid product, which contributes to cell resistance following various stresses. The biosynthetic pathway of deinoxanthin is unclear, although several enzymes are presumed to be involved. The gene (dr2250) predicted by gene homologue analysis to encode carotenoid 3',4'-desaturase (CrtD) was deleted to investigate its function. A mutant deficient in the gene homologue of crtLm (dr0801) was also constructed to verify the catalytic function of the gene product in the native host. Carotenoid analysis of the resultant mutants verified that DR2250 encodes carotenoid 3',4'-desaturase, which catalyses the C-3',4'-desaturation of the monocyclic precursor of deinoxanthin but not acyclic carotenoids. Mutation of the gene homologue of crtLm (dr0801) resulted in accumulation of lycopene, confirming that it encodes the lycopene cyclase in the native host. The lack of CrtD decreased the antioxidant capacity of the mutant deficient in dr2250 compared with the wild-type, indicating that the C-3',4'-desaturation step contributes to the antioxidant capacity of deinoxanthin in D. radiodurans.

  19. 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

  20. Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers

    PubMed Central

    Uitdewilligen, Jan G. A. M. L.; Kloosterman, Bjorn A.; Hutten, Ronald C. B.; Visser, Richard G. F.; van Eck, Herman J.

    2010-01-01

    We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (Lcye) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (Zep) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin. Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9647-y) contains supplementary material, which is available to authorized users. PMID:20490894

  1. Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces.

    PubMed

    Rezanka, Tomáš; Siristova, Lucie; Schreiberová, Olga; Rezanka, Michal; Masák, Jan; Melzoch, Karel; Sigler, Karel

    2011-06-01

    A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

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

    SciTech Connect

    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. - 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.

  3. 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.

  4. Enhancing a Pathway-Genome Database (PGDB) to Capture Subcellular Localization of Metabolites and Enzymes: The Nucleotide-Sugar Biosynthetic Pathways of Populus trichocarpa

    SciTech Connect

    Nag, A.; Karpinets, T. V.; Chang, C. H.; Bar-Peled, M.

    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).

  5. 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.

  6. Rapid determination of biosynthetic pathways using fractional isotope enrichment and high-resolution dynamic nuclear polarization enhanced NMR.

    PubMed

    Bowen, Sean; Sekar, Giridhar; Hilty, Christian

    2011-10-01

    Carbon-13 NMR has traditionally been a method of choice for the determination of metabolic pathways. Through fractional labeling, (13)C spectra allow the identification of fragments incorporated as a unit into a biosynthesized molecule. The low sensitivity of (13)C spectroscopy is an impediment to such studies, especially if compounded with an often limited availability of biosynthesized molecules. Dynamic nuclear polarization (DNP) can increase the signal-to-noise ratio in magnetic resonance by several orders of magnitude, and in combination with high-resolution spectroscopy has the potential to increase the reach of this technique for metabolic profiling. Here, we present an application of high-resolution DNP enhanced NMR to the study of the biosynthetic pathways for membrane lipids. We show that fatty acid methyl esters are readily hyperpolarized in organic solvent. The resulting spectra resolve the various structural features of the chains, including atoms near the termini, as well as unsaturated and cyclopropyl groups. Peak patterns observed in fractionally labeled samples are explained by the way feed molecules are incorporated into fatty acid chains during synthesis. Differences in multiplet intensity between samples made from glucose and acetate feedstock mixtures further reveal metabolic preferences for these precursors in the biosynthesis of the product. In addition to the present study of lipid biosynthesis, high-resolution DNP-NMR of fractionally (13)C-labeled metabolites may present itself for the rapid determination of biosynthetic pathways in various biomedical applications, especially in cases of limited availability of the products of interest. Copyright © 2011 John Wiley & Sons, Ltd.

  7. 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

  8. The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway.

    PubMed

    Spray, C R; Kobayashi, M; Suzuki, Y; Phinney, B O; Gaskin, P; MacMillan, J

    1996-09-17

    In plants, gibberellin (GA)-responding mutants have been used as tools to identify the genes that control specific steps in the GA-biosynthetic pathway. They have also been used to determine which native GAs are active per se, i.e., further metabolism is not necessary for bioactivity. We present metabolic evidence that the D1 gene of maize (Zea mays L.) controls the three biosynthetic steps: GA20 to GA1, Ga20 to GA5, and GA5 to GA3. We also present evidence that three gibberellins, GA1, GA5, and GA3, have per se activity in stimulating shoot elongation in maize. The metabolic evidence comes from the injection of [17-13C,3H]GA20 and [17-13C,3H]GA5 into seedlings of d1 and controls (normal and d5), followed by isolation and identification of the 13C-labeled metabolites by full-scan GC-MS and Kovats retention index. For the controls, GA20 was metabolized to GA1,GA3, and GA5; GA5 was metabolized to GA3. For the d1 mutant, GA20 was not metabolized to GA1, GA3, or to GA5, and GA5 was not metabolized to GA3. The bioassay evidence is based on dosage response curves using d1 seedlings for assay. GA1, GA3, and GA5 had similar bioactivities, and they were 10-times more active than GA20.

  9. A Root Specific Induction of Carotenoid Biosynthesis Contributes to ABA Production upon Salt Stress in Arabidopsis

    PubMed Central

    Ruiz-Sola, M. Águila; Arbona, Vicent; Gómez-Cadenas, Aurelio; Rodríguez-Concepción, Manuel; Rodríguez-Villalón, Antía

    2014-01-01

    Abscisic acid (ABA) is a hormone that plays a vital role in mediating abiotic stress responses in plants. Salt exposure induces the synthesis of ABA through the cleavage of carotenoid precursors (xanthophylls), which are found at very low levels in roots. Here we show that de novo ABA biosynthesis in salt-treated Arabidopsis thaliana roots involves an organ-specific induction of the carotenoid biosynthetic pathway. Upregulation of the genes encoding phytoene synthase (PSY) and other enzymes of the pathway producing ABA precursors was observed in roots but not in shoots after salt exposure. A pharmacological block of the carotenoid pathway substantially reduced ABA levels in stressed roots, confirming that an increase in carotenoid accumulation contributes to fuel hormone production after salt exposure. Treatment with exogenous ABA was also found to upregulate PSY expression only in roots, suggesting an organ-specific feedback regulation of the carotenoid pathway by ABA. Taken together, our results show that the presence of high concentrations of salt in the growth medium rapidly triggers a root-specific activation of the carotenoid pathway, probably to ensure a proper supply of ABA precursors required for a sustained production of the hormone. PMID:24595399

  10. Biochemical Analysis of the Biosynthetic Pathway of an Anticancer Tetracycline SF2575

    PubMed Central

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

    2009-01-01

    SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity towards 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 analogs. 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 groups addition is C-9 glycosylation, C-4 salicylation and O-4′ angelycylation. 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 towards 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 towards structural-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

  11. RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic-acid-based metabolites such as lipids

    PubMed Central

    Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A.; Gobel, Verena

    2017-01-01

    The field of metabolomics continues to catalog new compounds, but their functional analysis remains technically challenging, and roles beyond metabolism are largely unknown. Unbiased genetic/RNAi screens are powerful tools to identify the in vivo functions of protein-encoding genes, but not of non-proteinaceous compounds such as lipids. They can, however, identify the biosynthetic enzymes – of these compounds- findings that are usually dismissed, as these typically synthesize multiple products. Here, we provide a method using follow-on biosynthetic-pathway screens to identify the endpoint biosynthetic enzyme and thus the compound through which they act. The approach is based on the principle that all subsequently identified downstream biosynthetic enzymes contribute to the synthesis of at least this one end product. We describe how to: systematically target lipid biosynthetic pathways; optimize targeting conditions; take advantage of pathway branchpoints; and validate results by genetic assays and biochemical analyses. This approach extends the power of unbiased genetic/RNAi screens to identify in vivo functions of non-nucleic-acid-based metabolites beyond their metabolic roles. PMID:25837419

  12. Rewiring carotenoid biosynthesis in plants using a viral vector

    PubMed Central

    Majer, Eszter; Llorente, Briardo; Rodríguez-Concepción, Manuel; Daròs, José-Antonio

    2017-01-01

    Plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. This is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. Here we describe the use of a viral vector derived from Tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. The pathway consisted in three enzymes from the soil bacteria Pantoea ananatis. Lycopene is present at undetectable levels in chloroplasts of non-infected leaves. In tissues infected with the viral vector, however, lycopene comprised approximately 10% of the total carotenoid content. Our research further showed that plant viruses that express P. ananatis phytoene synthase (crtB), one of the three enzymes of the heterologous pathway, trigger an accumulation of endogenous carotenoids, which together with a reduction in chlorophylls eventually result in a bright yellow pigmentation of infected tissues in various host-virus combinations. So, besides illustrating the potential of viral vectors for engineering complex metabolic pathways, we also show a yellow carotenoid-based reporter that can be used to visually track infection dynamics of plant viruses either alone or in combination with other visual markers. PMID:28139696

  13. Rewiring carotenoid biosynthesis in plants using a viral vector.

    PubMed

    Majer, Eszter; Llorente, Briardo; Rodríguez-Concepción, Manuel; Daròs, José-Antonio

    2017-01-31

    Plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. This is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. Here we describe the use of a viral vector derived from Tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. The pathway consisted in three enzymes from the soil bacteria Pantoea ananatis. Lycopene is present at undetectable levels in chloroplasts of non-infected leaves. In tissues infected with the viral vector, however, lycopene comprised approximately 10% of the total carotenoid content. Our research further showed that plant viruses that express P. ananatis phytoene synthase (crtB), one of the three enzymes of the heterologous pathway, trigger an accumulation of endogenous carotenoids, which together with a reduction in chlorophylls eventually result in a bright yellow pigmentation of infected tissues in various host-virus combinations. So, besides illustrating the potential of viral vectors for engineering complex metabolic pathways, we also show a yellow carotenoid-based reporter that can be used to visually track infection dynamics of plant viruses either alone or in combination with other visual markers.

  14. First Biosynthetic pathway of 1-hepten-3-one in Iporangaia pustulosa (Opiliones)

    PubMed Central

    Rocha, Daniele F. O.; Wouters, Felipe C.; Machado, Glauco; Marsaioli, Anita J.

    2013-01-01

    Arthropods produce a great variety of natural compounds, many of which have unexplored biosynthesis. Among the armored harvestmen (Arachnida: Opiliones) of the suborder Laniatores, the defensive gland exudates contain vinyl ketones and other constituents of supposed polyketide origin. We have studied the biosynthesis of 1-hepten-3-one in the Neotropical harvestman Iporangaia pustulosa by feeding individuals with 13C-labeled precursors, demonstrating its mixed acetate/propionate origin. 13C NMR spectroscopy showed an unusual labeling pattern suggesting different propionate sources for starting and extender units. Our analysis also indicates the presence of methylmalonyl-CoA mutase, converting acetate into propionyl-CoA via succinyl-CoA, together with other C3 unit routes. This is the first biosynthetic study of alkyl vinyl ketones in arthropods. Our results shed light on the origin and diversification of chemical compounds in a major arthropod group. PMID:24193576

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

    PubMed Central

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

    2015-01-01

    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. PMID:26598135

  16. First Biosynthetic pathway of 1-hepten-3-one in Iporangaia pustulosa (Opiliones)

    NASA Astrophysics Data System (ADS)

    Rocha, Daniele F. O.; Wouters, Felipe C.; Machado, Glauco; Marsaioli, Anita J.

    2013-11-01

    Arthropods produce a great variety of natural compounds, many of which have unexplored biosynthesis. Among the armored harvestmen (Arachnida: Opiliones) of the suborder Laniatores, the defensive gland exudates contain vinyl ketones and other constituents of supposed polyketide origin. We have studied the biosynthesis of 1-hepten-3-one in the Neotropical harvestman Iporangaia pustulosa by feeding individuals with 13C-labeled precursors, demonstrating its mixed acetate/propionate origin. 13C NMR spectroscopy showed an unusual labeling pattern suggesting different propionate sources for starting and extender units. Our analysis also indicates the presence of methylmalonyl-CoA mutase, converting acetate into propionyl-CoA via succinyl-CoA, together with other C3 unit routes. This is the first biosynthetic study of alkyl vinyl ketones in arthropods. Our results shed light on the origin and diversification of chemical compounds in a major arthropod group.

  17. Investigation of biosynthetic pathways to hydroxycoumarins during post-harvest physiological deterioration in Cassava roots by using stable isotope labelling.

    PubMed

    Bayoumi, Soad A L; Rowan, Michael G; Beeching, John R; Blagbrough, Ian S

    2008-12-15

    Cassava (Manihot esculenta Crantz) is an important starch-rich crop, but the storage roots only have a short shelf-life due to post-harvest physiological deterioration (PPD), which includes the over-production and polymerisation of hydroxycoumarins. Key aspects of coumarin secondary-metabolite biosynthesis remain unresolved. Here we exploit the accumulation of hydroxycoumarins to test alternative pathways for their biosynthesis. Using isotopically labelled intermediates (p-coumarate-2-(13)C, caffeate-2-(13)C, ferulate-2-(13)C, umbelliferone-2-(18)O and esculetin-2-(18)O), we show that the major biosynthetic pathway to scopoletin and its glucoside, scopolin, in cassava roots during PPD is through p-coumaric, caffeic and then ferulic acids. An alternate pathway through 2',4'-dihydroxycinnamate and umbelliferone leads to esculetin and esculin. We have used C(18)O(2)-carboxylate-labelled cinnamic and ferulic acids, and feeding experiments under an atmosphere of (18)O(2), to investigate the o-hydroxylation and cyclisation steps. We demonstrate that the major pathway is through o-hydroxylation and not via a proposed spirolactone-dienone intermediate.

  18. Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

    PubMed

    Kopečná, Jana; Cabeza de Vaca, Israel; Adams, Nathan B P; Davison, Paul A; Brindley, Amanda A; Hunter, C Neil; Guallar, Victor; Sobotka, Roman

    2015-11-20

    In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

  19. Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway*

    PubMed Central

    Kopečná, Jana; Cabeza de Vaca, Israel; Adams, Nathan B. P.; Davison, Paul A.; Brindley, Amanda A.; Hunter, C. Neil; Guallar, Victor; Sobotka, Roman

    2015-01-01

    In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway. PMID:26446792

  20. Quenching capabilities of long-chain carotenoids in light-harvesting-2 complexes from Rhodobacter sphaeroides with an engineered carotenoid synthesis pathway

    DOE PAGES

    Dilbeck, Preston L.; Tang, Qun; Mothersole, David J.; ...

    2016-06-10

    Here, six light-harvesting-2 complexes (LH2) from genetically modified strains of the purple photosynthetic bacterium Rhodobacter (Rb.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. These strains were engineered to incorporate carotenoids for which the number of conjugated groups (N = NC=C + NC=O) varies from 9 to 15. The Rb. sphaeroides strains incorporate their native carotenoids spheroidene (N = 10) and spheroidenone (N = 11), as well as longer-chain analogues including spirilloxanthin (N = 13) and diketospirilloxantion (N = 15) normally found in Rhodospirillum rubrum. Measurements of the properties of the carotenoid first singlet excitedmore » state (S1) in antennas from the Rb. sphaeroides set show that carotenoid-bacteriochlorophyll a (BChl a) interactions are similar to those in LH2 complexes from various other bacterial species and thus are not significantly impacted by differences in polypeptide composition. Instead, variations in carotenoid-to-BChl a energy transfer are primarily regulated by the N-determined energy of the carotenoid S1 excited state, which for long-chain (N ≥ 13) carotenoids is not involved in energy transfer. Furthermore, the role of the long-chain carotenoids switches from a lightharvesting supporter (via energy transfer to BChl a) to a quencher of the BChl a S1 excited state B850*. This quenching is manifested as a substantial (~2-fold) reduction of the B850* lifetime and the B850* fluorescence quantum yield for LH2 housing the longest carotenoids« less

  1. Plastid-localized amino acid biosynthetic pathways of Plantae are predominantly composed of non-cyanobacterial enzymes

    PubMed Central

    Reyes-Prieto, Adrian; Moustafa, Ahmed

    2012-01-01

    Studies of photosynthetic eukaryotes have revealed that the evolution of plastids from cyanobacteria involved the recruitment of non-cyanobacterial proteins. Our phylogenetic survey of >100 Arabidopsis nuclear-encoded plastid enzymes involved in amino acid biosynthesis identified only 21 unambiguous cyanobacterial-derived proteins. Some of the several non-cyanobacterial plastid enzymes have a shared phylogenetic origin in the three Plantae lineages. We hypothesize that during the evolution of plastids some enzymes encoded in the host nuclear genome were mistargeted into the plastid. Then, the activity of those foreign enzymes was sustained by both the plastid metabolites and interactions with the native cyanobacterial enzymes. Some of the novel enzymatic activities were favored by selective compartmentation of additional complementary enzymes. The mosaic phylogenetic composition of the plastid amino acid biosynthetic pathways and the reduced number of plastid-encoded proteins of non-cyanobacterial origin suggest that enzyme recruitment underlies the recompartmentation of metabolic routes during the evolution of plastids. PMID:23233874

  2. Exploitation of the Streptomyces coelicolor A3(2) genome sequence for discovery of new natural products and biosynthetic pathways.

    PubMed

    Challis, Gregory L

    2014-02-01

    Streptomyces, and related genera of Actinobacteria, are renowned for their ability to produce antibiotics and other bioactive natural products with a wide range of applications in medicine and agriculture. Streptomyces coelicolor A3(2) is a model organism that has been used for more than five decades to study the genetic and biochemical basis for the production of bioactive metabolites. In 2002, the complete genome sequence of S. coelicolor was published. This greatly accelerated progress in understanding the biosynthesis of metabolites known or suspected to be produced by S. coelicolor and revealed that streptomycetes have far greater potential to produce bioactive natural products than suggested by classical bioassay-guided isolation studies. In this article, efforts to exploit the S. coelicolor genome sequence for the discovery of novel natural products and biosynthetic pathways are summarized.

  3. Formation of norisoprenoid flavor compounds in carrot (Daucus carota L.) roots: characterization of a cyclic-specific carotenoid cleavage dioxygenase 1 gene

    USDA-ARS?s Scientific Manuscript database

    Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural produce. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still widely unknown. We found that geranial i...

  4. Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

    PubMed Central

    Ménard, Robert; Schoenhofen, Ian C.; Tao, Limei; Aubry, Annie; Bouchard, Patrice; Reid, Christopher W.; Lachance, Paule; Twine, Susan M.; Fulton, Kelly M.; Cui, Qizhi; Hogues, Hervé; Purisima, Enrico O.

    2014-01-01

    Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 μM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 μM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 μM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella. PMID:25267679

  5. Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers.

    PubMed

    Lopez, Alex B; Van Eck, Joyce; Conlin, Brian J; Paolillo, Dominick J; O'Neill, Jennifer; Li, Li

    2008-01-01

    Transgenic plants have facilitated our understanding of the functional roles of genes and the metabolic processes affected in plants. Recently, the Or gene was isolated from an orange cauliflower mutant and it was shown that the Or gene could serve as a novel genetic tool to enrich carotenoid content in transgenic potato tubers. An in-depth characterization of these Or transgenic lines is presented here. It was found that the Or transgene may facilitate the identification of potential rate-limiting step(s) of the carotenoid biosynthetic pathway. The Or transgenic tubers accumulated not only increased levels of carotenoids that normally are present in the controls, but also three additional metabolite intermediates of phytoene, phytofluene, and zeta-carotene, indicating that the desaturation steps became limiting following the expression of the Or transgene. Moreover, we observed that long-term cold storage greatly enhanced carotenoid content in the Or transgenic tubers to a level of 10-fold over controls. Expression of the Or transgene in the transgenic plants caused no dramatic changes in the transcript levels of the endogenous carotenoid biosynthetic genes, which is in agreement with the Or gene not directly controlling carotenoid biosynthesis. Microscope analysis revealed that the Or transgene conferred the formation of chromoplasts containing carotenoid sequestering structures in a heterologous system. Such structures were not observed in tubers of potato cultivars that accumulate high levels of carotenoids. Collectively, these results provide direct evidence demonstrating that the Or gene indeed controls chromoplast differentiation and that regulation of chromoplast formation can have a profound effect on carotenoid accumulation in plants.

  6. 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.

  7. Mutations in apbC (mrp) prevent function of the alternative pyrimidine biosynthetic pathway in Salmonella typhimurium.

    PubMed

    Petersen, L; Downs, D M

    1996-10-01

    The alternative pyrimidine biosynthetic (APB) pathway can synthesize the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine in Salmonella typhimurium independently of de novo purine biosynthesis. When mutants defective in function of the APB pathway were isolated, the predominant class (40%) were defective in a single locus we have designated apbC. Mutations in apbC block function of the APB pathway since they prevent growth of a purF mutant in the absence of thiamine. Lesions in apbC also cause a thiamine auxotrophy in strains proficient in purine biosynthesis when fructose is provided as the sole carbon and energy source. Results presented here are consistent with ApbC being involved in the conversion of aminoimidazole ribonucleotide to HMP, and we suggest that ApbC performs a redundant step in thiamine synthesis. Sequence analysis demonstrated that apbC mutations were alleles of mrp, a locus previously reported in Escherichia coli as a metG-related protein. We propose that this locus in S. typhimurium be designated apbC to reflect its involvement in thiamine synthesis.

  8. Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways.

    PubMed

    Michener, Joshua K; Thodey, Kate; Liang, Joe C; Smolke, Christina D

    2012-05-01

    Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.

  9. Biosynthetic pathways in Methanospirillum hungatei as determined by 13C nuclear magnetic resonance.

    PubMed Central

    Ekiel, I; Smith, I C; Sprott, G D

    1983-01-01

    The main metabolic pathways in Methanospirillum hungatei GP1 were followed by using 13C nuclear magnetic resonance, with 13C-labeled acetate and CO2 as carbon sources. The labeling patterns found in carbohydrates, amino acids, lipids, and nucleosides were consistent with the formation of pyruvate from acetate and CO2 as the first step in biosynthesis. Carbohydrates are formed by the glucogenic pathway, and no scrambling of label was observed, indicating that the oxidative or reductive pentose phosphate pathways are not functioning at significant rates. The pathways for amino acid biosynthesis are the usual ones, with the exception of that for isoleucine. The tricarboxylic acid pathway is incomplete and operates in a reductive direction to form alpha-ketoglutarate. The phytanyl chains of lipids are synthesized from acetate via mevalonic acid. PMID:6619097

  10. Increase in transcript accumulation of Psy1 and e-Lcy genes in grain development is associated with differences in seed carotenoid content between durum wheat and tritordeum.

    PubMed

    Rodríguez-Suárez, Cristina; Mellado-Ortega, Elena; Hornero-Méndez, Dámaso; Atienza, Sergio G

    2014-04-01

    Carotenoid rich diets have been associated with lower risk of certain diseases. The great importance of cereals in human diet has directed breeding programs towards carotenoid enhancement to alleviate these deficiencies in developing countries and to offer new functional foods in the developed ones. The new cereal tritordeum (×Tritordeum Ascherson et Graebener) derived from durum wheat (Triticum turgidum ssp. durum) and the wild barley Hordeum chilense, naturally presents carotenoid levels 5-8 times higher than those of durum wheat. The improvement of tritordeum properties as a new functional food requires the elucidation of biosynthetic steps for carotenoid accumulation in seeds that differ from durum wheat. In this work expression patterns of nine genes from the isoprenoid and carotenoid biosynthetic pathways were monitored during grain development in durum wheat and tritordeum. Additionally, a fine identification and quantification of pigments (chlorophylls and carotenoids) during grain development and in mature seeds has been addressed. Transcript levels of Psy1, Psy2, Zds, e-Lcy and b-Lcy were found to correlate to carotenoid content in mature grains. The specific activation of the homeologous genes Psy1, e-Lcy from H. chilense and the high lutein esterification found in tritordeum may serve to explain the differences with durum wheat in carotenoid accumulation.

  11. 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

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

    PubMed

    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

    2017-01-04

    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.

  13. A symbiotic mutant of Sinorhizobium meliloti reveals a novel genetic pathway involving succinoglycan biosynthetic functions.

    PubMed

    Griffitts, Joel S; Long, Sharon R

    2008-03-01

    A large-scale screen for symbiotic mutants was carried out using the model root nodulating bacterium Sinorhizobium meliloti. Several mutations in the previously uncharacterized gene msbA2 were isolated. msbA2 encodes a member of the ATP-binding cassette exporter family. This protein family is known to export a wide variety of compounds from bacterial cells. S. meliloti MsbA2 is required for the invasion of nodule tissue, with msbA2 mutant cells stimulating nodule primordium morphogenesis, but failing to invade plant tissue beyond the epidermal cell layer. msbA2 mutants do not exhibit any of the free-living traits often found to correlate with symbiotic defects, suggesting that MsbA2 may take part in a specifically symbiotic function. In strains that overproduce the symbiotic signalling polysaccharide succinoglycan, loss of MsbA2 function is extremely deleterious. This synthetic lethal phenotype can be suppressed by disrupting the succinoglycan biosynthetic genes exoY or exoA. It can also be suppressed by disrupting putative glycosyltransferase-encoding genes found upstream of msbA2. Finally, the symbiotic phenotype of a msbA2 null mutant is suppressed by secondary mutations in these upstream transferase genes, indicating that the msbA2 mutant phenotype may be caused by an inhibitory accumulation of a novel polysaccharide that is synthesized from succinoglycan precursors.

  14. Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control

    PubMed Central

    Leonard, Effendi; Ajikumar, Parayil Kumaran; Thayer, Kelly; Xiao, Wen-Hai; Mo, Jeffrey D.; Tidor, Bruce; Stephanopoulos, Gregory; Prather, Kristala L. J.

    2010-01-01

    A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits high-level levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg/L was subsequently obtained by cultivation in a bench-scale bioreactor. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products. PMID:20643967

  15. Differential Selection on Carotenoid Biosynthesis Genes as a Function of Gene Position in the Metabolic Pathway: A Study on the Carrot and Dicots

    PubMed Central

    Clotault, Jérémy; Peltier, Didier; Soufflet-Freslon, Vanessa; Briard, Mathilde; Geoffriau, Emmanuel

    2012-01-01

    Background Selection of genes involved in metabolic pathways could target them differently depending on the position of genes in the pathway and on their role in controlling metabolic fluxes. This hypothesis was tested in the carotenoid biosynthesis pathway using population genetics and phylogenetics. Methodology/Principal Findings Evolutionary rates of seven genes distributed along the carotenoid biosynthesis pathway, IPI, PDS, CRTISO, LCYB, LCYE, CHXE and ZEP, were compared in seven dicot taxa. A survey of deviations from neutrality expectations at these genes was also undertaken in cultivated carrot (Daucus carota subsp. sativus), a species that has been intensely bred for carotenoid pattern diversification in its root during its cultivation history. Parts of sequences of these genes were obtained from 46 individuals representing a wide diversity of cultivated carrots. Downstream genes exhibited higher deviations from neutral expectations than upstream genes. Comparisons of synonymous and nonsynonymous substitution rates between genes among dicots revealed greater constraints on upstream genes than on downstream genes. An excess of intermediate frequency polymorphisms, high nucleotide diversity and/or high differentiation of CRTISO, LCYB1 and LCYE in cultivated carrot suggest that balancing selection may have targeted genes acting centrally in the pathway. Conclusions/Significance Our results are consistent with relaxed constraints on downstream genes and selection targeting the central enzymes of the carotenoid biosynthesis pathway during carrot breeding history. PMID:22737218

  16. Dual role of the pigmentation gene B in affecting carotenoid and vitamin E content in squash (Cucurbita pepo) mesocarp.

    PubMed

    Tadmor, Yaakov; Paris, Harry S; Meir, Ayala; Schaffer, Arthur A; Lewinsohn, Efraim

    2005-12-14

    The yellow and orange colorations of the mesocarp of pumpkins and squash of Cucurbita pepo are due to the presence of carotenoid pigments that also greatly contribute to their nutritional value. Carotenoids, as well as tocopherols (vitamin E), are isoprenoid compounds formed by branches of a common biosynthetic pathway. Photodiode array HPLC analysis was used to simultaneously determine the content and composition of fruit-flesh carotenoids and tocopherols in five pairs of near-isogenic lines differing in the allelic state of genes previously identified as having profound effects on fruit color. The dominant B allele promoted carotenoid accumulation up to 5-fold and prevented tocopherol accumulation in all genetic backgrounds. The dominant L-2 allele doubled carotenoid content and, in combination with the dominant B allele, increased carotenoid content by 10-15-fold as compared to the recessive l-2 allele. The genes D and L-1 had no significant effects on mesocarp tocopherols or carotenoids content. These results indicate that the B gene, which affects both carotenoids and tocopherols, may play a regulatory role in the flux of the isoprenoid pathway products.

  17. Novel Key Metabolites Reveal Further Branching of the Roquefortine/Meleagrin Biosynthetic Pathway*

    PubMed Central

    Ries, Marco I.; Ali, Hazrat; Lankhorst, Peter P.; Hankemeier, Thomas; Bovenberg, Roel A. L.; Driessen, Arnold J. M.; Vreeken, Rob J.

    2013-01-01

    Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches. PMID:24225953

  18. Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol.

    PubMed

    Pereira, Brian; Zhang, Haoran; De Mey, Marjan; Lim, Chin Giaw; Li, Zheng-Jun; Stephanopoulos, Gregory

    2016-02-01

    Ethylene glycol (EG) is an important commodity chemical with broad industrial applications. It is presently produced from petroleum or natural gas feedstocks in processes requiring consumption of significant quantities of non-renewable resources. Here, we report a novel pathway for biosynthesis of EG from the renewable sugar glucose in metabolically engineered Escherichia coli. Serine-to-EG conversion was first achieved through a pathway comprising serine decarboxylase, ethanolamine oxidase, and glycolaldehyde reductase. Serine provision in E. coli was then enhanced by overexpression of the serine-biosynthesis pathway. The integration of these two parts into the complete EG-biosynthesis pathway in E. coli allowed for production of 4.1 g/L EG at a cumulative yield of 0.14 g-EG/g-glucose, establishing a foundation for a promising biotechnology. © 2015 Wiley Periodicals, Inc.

  19. Novel key metabolites reveal further branching of the roquefortine/meleagrin biosynthetic pathway.

    PubMed

    Ries, Marco I; Ali, Hazrat; Lankhorst, Peter P; Hankemeier, Thomas; Bovenberg, Roel A L; Driessen, Arnold J M; Vreeken, Rob J

    2013-12-27

    Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.

  20. Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes

    PubMed Central

    Davuluri, Ganga Rao; van Tuinen, Ageeth; Fraser, Paul D; Manfredonia, Alessandro; Newman, Robert; Burgess, Diane; Brummell, David A; King, Stephen R; Palys, Joe; Uhlig, John; Bramley, Peter M; Pennings, Henk M J; Bowler, Chris

    2013-01-01

    Tomatoes are a principal dietary source of carotenoids and flavonoids, both of which are highly beneficial for human health1,2. Overexpression of genes encoding biosynthetic enzymes or transcription factors have resulted in tomatoes with improved carotenoid or flavonoid content, but never with both3–7. We attempted to increase tomato fruit nutritional value by suppressing an endogenous photomorphogenesis regulatory gene, DET1, using fruit-specific promoters combined with RNA interference (RNAi) technology. Molecular analysis indicated that DET1 transcripts were indeed specifically degraded in transgenic fruits. Both carotenoid and flavonoid contents were increased significantly, whereas other parameters of fruit quality were largely unchanged. These results demonstrate that manipulation of a plant regulatory gene can simultaneously influence the production of several phytonutrients generated from independent biosynthetic pathways, and provide a novel example of the use of organ-specific gene silencing to improve the nutritional value of plant-derived products. PMID:15951803

  1. Constructing de novo biosynthetic pathways for chemical synthesis inside living cells.

    PubMed

    Weeks, Amy M; Chang, Michelle C Y

    2011-06-21

    Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and allows the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts to understand the relationship among sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform for elucidating the in vivo specificity and function of enzymes and reconstituting complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineering synthetic pathways in microbial hosts for chemical production.

  2. Phosphorylation Mechanism of Phosphomevalonate Kinase: Implications for Rational Engineering of Isoprenoid Biosynthetic Pathway Enzymes.

    PubMed

    Huang, Meilan; Wei, Kexin; Li, Xiao; McClory, James; Hu, Guixiang; Zou, Jian-Wei; Timson, David

    2016-10-11

    The mevalonate pathway is of important clinical, pharmaceutical, and biotechnological relevance. However, lack of the understanding of the phosphorylation mechanism of the kinases in this pathway has limited rationally engineering the kinases in industry. Here the phosphorylation reaction mechanism of a representative kinase in the mevalonate pathway, phosphomevalonate kinase, was studied by using molecular dynamics and hybrid QM/MM methods. We find that a conserved residue (Ser106) is reorientated to anchor ATP via a stable H-bond interaction. In addition, Ser213 located on the α-helix at the catalytic site is repositioned to further approach the substrate, facilitating the proton transfer during the phosphorylation. Furthermore, we elucidate that Lys101 functions to neutralize the negative charge developed at the β-, γ-bridging oxygen atom of ATP during phosphoryl transfer. We demonstrate that the dissociative catalytic reaction occurs via a direct phosphorylation pathway. This is the first study on the phosphorylation mechanism of a mevalonate pathway kinase. The elucidation of the catalytic mechanism not only sheds light on the common catalytic mechanism of the GHMP kinase superfamily but also provides the structural basis for engineering the mevalonate pathway kinases to further exploit their applications in the production of a wide range of fine chemicals such as biofuels or pharmaceuticals.

  3. Constructing de novo biosynthetic pathways for chemical synthesis inside living cells†

    PubMed Central

    Weeks, Amy M.; Chang, Michelle C. Y.

    2011-01-01

    Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and enables the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts in understanding the relationship between sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform to elucidate the in vivo specificity and function of enzymes and to reconstitute complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineer synthetic pathways in microbial hosts for chemical production. PMID:21591680

  4. Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression.

    PubMed

    Moreno, J C; Cerda, A; Simpson, K; Lopez-Diaz, I; Carrera, E; Handford, M; Stange, C

    2016-04-01

    Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition.

  5. Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression

    PubMed Central

    Moreno, J.C.; Cerda, A.; Simpson, K.; Lopez-Diaz, I.; Carrera, E; Handford, M.; Stange, C.

    2016-01-01

    Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. PMID:26893492

  6. Carotenoid composition of the flowers of Mimulus lewisii and related species: Implications regarding the prevalence and origin of two unique, allenic pigments.

    PubMed

    LaFountain, Amy M; Frank, Harry A; Yuan, Yao-Wu

    2015-05-01

    The genus Mimulus has been used as a model system in a wide range of ecological and evolutionary studies and contains many species with carotenoid pigmented flowers. However, the detailed carotenoid composition of these flowers has never been reported. In this paper the floral carotenoid composition of 11 Mimulus species are characterized using high-performance liquid chromatography, mass spectrometry and chemical methods with a particular focus on the genetic model species, Mimulus lewisii. M. lewisii flowers have five major carotenoids: antheraxanthin, violaxanthin, neoxanthin, and the unique allenic carotenoids, deepoxyneoxanthin and mimulaxanthin. This carotenoid profile is consistent with the expression levels of putative carotenoid biosynthetic genes in the M. lewisii flower. The other 10 species possess the same five carotenoids or a subset of these. Comparison of the carotenoid profiles among species in a phylogenetic context provides new insights into the biosynthesis and evolution of deepoxyneoxanthin and mimulaxanthin. This work also lays the foundation for future studies regarding transcriptional control of the carotenoid biosynthesis pathway in Mimulus flowers. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. 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

  8. Triterpenoid saponin biosynthetic pathway profiling and candidate gene mining of the Ilex asprella root using RNA-Seq.

    PubMed

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

    2014-04-09

    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.

  9. 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.

  10. Time Dependency of Chemodiversity and Biosynthetic Pathways: An LC-MS Metabolomic Study of Marine-Sourced Penicillium

    PubMed Central

    Roullier, Catherine; Bertrand, Samuel; Blanchet, Elodie; Peigné, Mathilde; Robiou du Pont, Thibaut; Guitton, Yann; Pouchus, Yves François; Grovel, Olivier

    2016-01-01

    This work aimed at studying metabolome variations of marine fungal strains along their growth to highlight the importance of the parameter “time” for new natural products discovery. An untargeted time-scale metabolomic study has been performed on two different marine-derived Penicillium strains. They were cultivated for 18 days and their crude extracts were analyzed by HPLC-DAD-HRMS (High Performance Liquid Chromatography-Diode Array Detector-High Resolution Mass Spectrometry) each day. With the example of griseofulvin biosynthesis, a pathway shared by both strains, this work provides a new approach to study biosynthetic pathway regulations, which could be applied to other metabolites and more particularly new ones. Moreover, the results of this study emphasize the interest of such an approach for the discovery of new chemical entities. In particular, at every harvesting time, previously undetected features were observed in the LC-MS (Liquid Chromatography-Mass Spectrometry) data. Therefore, harvesting times for metabolite extraction should be performed at different time points to access the hidden metabolome. PMID:27213411

  11. TRIB3 Mediates Glucose-Induced Insulin Resistance via a Mechanism That Requires the Hexosamine Biosynthetic Pathway

    PubMed Central

    Zhang, Wei; Liu, Jiarong; Tian, Ling; Liu, Qinglan; Fu, Yuchang; Garvey, W. Timothy

    2013-01-01

    In the current study, we investigated the role of tribbles homolog 3 (TRIB3) in glucose-induced insulin resistance and whether the induction of TRIB3 by glucose is dependent on the nutrient-sensing hexosamine biosynthetic pathway (HBP) known to mediate glucose toxicity in diabetes. In diabetic rats, TRIB3 expression in skeletal muscle was increased after 10 days of hyperglycemia, and glycemia and muscle TRIB3 were both restored toward normal by insulin therapy. In L6 myocytes, the induction of TRIB3 by high glucose or glucosamine was reversible upon removal of these substrates. To assess the role of HBP in the induction of TRIB3, we demonstrated that the ability of high glucose to augment TRIB3 expression was prevented by azaserine, an inhibitor of glutamine: fructose-6-phosphate amidotransferase (GFAT), which is the rate-limiting enzyme in the HBP pathway. TRIB3 expression was also substantially stimulated by glucosamine, which bypasses GFAT, accompanied by a decrease in the insulin-stimulated glucose transport rate, and neither response was affected by azaserine. Further, knockdown of TRIB3 inhibited, and TRIB3 overexpression enhanced, the ability of both high glucose and glucosamine to induce insulin resistance. These data provide the mechanistic link between the HBP flux and insulin resistance and point to TRIB3 as a novel target for treatment of glucose-induced insulin resistance. PMID:23990361

  12. Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids.

    PubMed

    Kachanovsky, David E; Filler, Shdema; Isaacson, Tal; Hirschberg, Joseph

    2012-11-13

    Tomato (Solanum lycopersicum) fruit accumulate the red carotenoid pigment lycopene. The recessive mutation yellow-flesh (locus r) in tomato eliminates fruit carotenoids by disrupting the activity of the fruit-specific phytoene synthase (PSY1), the first committed step in the carotenoid biosynthesis pathway. Fruits of the recessive mutation tangerine (t) appear orange due to accumulation of 7,9,7',9'-tetra-cis-lycopene (prolycopene) as a result of a mutation in the carotenoid cis-trans isomerase. It was established 60 y ago that tangerine is epistatic to yellow-flesh. This uncharacteristic epistasis interaction defies a paradigm in biochemical genetics arguing that mutations that disrupt enzymes acting early in a biosynthetic pathway are epistatic to other mutations that block downstream steps in the same pathway. To explain this conundrum, we have investigated the interaction between tangerine and yellow-flesh at the molecular level. Results presented here indicate that allele r(2997) of yellow-flesh eliminates transcription of PSY1 in fruits. In a genetic background of tangerine, transcription of PSY1 is partially restored to a level sufficient for producing phytoene and downstream carotenoids. Our results revealed the molecular mechanism underlying the epistasis of t over r and suggest the involvement of cis-carotenoid metabolites in a feedback regulation of PSY1 gene expression.

  13. Chapter 3: Omics Advances of Biosynthetic Pathways of Isoprenoid Production in Microalgae

    SciTech Connect

    Paniagua-Michel, J.; Subramanian, Venkataramanan

    2017-01-01

    In this chapter, the current status of microalgal isoprenoids and the role of omics technologies, or otherwise specified, in bioproducts optimization and applications are reviewed. Emphasis is focused in the metabolic pathways of microalgae involved in the production of commercially important products, namely, hydrocarbons and biofuels, nutraceuticals, and pharmaceuticals.

  14. The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids in Arabidopsis thaliana

    PubMed Central

    Tzin, Vered; Galili, Gad

    2010-01-01

    The aromatic amino acids phenylalanine, tyrosine and tryptophan in plants are not only essential components of protein synthesis, but also serve as precursors for a wide range of secondary metabolites that are important for plant growth as well as for human nutrition and health. The aromatic amino acids are synthesized via the shikimate pathway followed by the branched aromatic amino acid metabolic pathway, with chorismate serving as a major branch point intermediate metabolite. Yet, the regulation of their synthesis is still far from being understood. So far, only three enzymes in this pathway, namely, chorismate mutase of phenylalanine and tyrosine synthesis, tryptophan synthase of tryptophan biosynthesis and arogenate dehydratase of phenylalanine biosynthesis, proved experimentally to be allosterically regulated. The major biosynthesis route of phenylalanine in plants occurs via arogenate. Yet, recent studies suggest that an alternative route of phynylalanine biosynthesis via phenylpyruvate may also exist in plants, similarly to many microorganisms. Several transcription factors regulating the expression of genes encoding enzymes of both the shikimate pathway and aromatic amino acid metabolism have also been recently identified in Arabidopsis and other plant species. PMID:22303258

  15. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway

    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...

  16. 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.

  17. De novo transcriptome assembly and the putative biosynthetic pathway of steroidal sapogenins of Dioscorea composita.

    PubMed

    Wang, Xia; Chen, Dijia; Wang, Yuqi; Xie, Jun

    2015-01-01

    The plant Dioscorea composita has important applications in the medical and energy industries, and can be used for the extraction of steroidal sapogenins (important raw materials for the synthesis of steroidal drugs) and bioethanol production. However, little is known at the genetic level about how sapogenins are biosynthesized in this plant. Using Illumina deep sequencing, 62,341 unigenes were obtained by assembling its transcriptome, and 27,720 unigenes were annotated. Of these, 8,022 unigenes were mapped to 243 specific pathways, and 531 unigenes were identified to be involved in 24 secondary metabolic pathways. 35 enzymes, which were encoded by 79 unigenes, were related to the biosynthesis of steroidal sapogenins in this transcriptome database, covering almost all the nodes in the steroidal pathway. The results of real-time PCR experiments on ten related transcripts (HMGR, MK, SQLE, FPPS, DXS, CAS, HMED, CYP51, DHCR7, and DHCR24) indicated that sapogenins were mainly biosynthesized by the mevalonate pathway. The expression of these ten transcripts in the tuber and leaves was found to be much higher than in the stem. Also, expression in the shoots was low. The nucleotide and protein sequences and conserved domains of four related genes (HMGR, CAS, SQS, and SMT1) were highly conserved between D. composita and D. zingiberensis; but expression of these four genes is greater in D. composita. However, there is no expression of these key enzymes in potato and no steroidal sapogenins are synthesized.

  18. The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

    PubMed

    Wu, Bo; Novelli, Jacopo; Foster, Jeremy; Vaisvila, Romualdas; Conway, Leslie; Ingram, Jessica; Ganatra, Mehul; Rao, Anita U; Hamza, Iqbal; Slatko, Barton

    2009-07-14

    Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5'-aminolevulinic acid

  19. The evolution of the starch biosynthetic pathway in cereals and other grasses.

    PubMed

    Comparot-Moss, Sylviane; Denyer, Kay

    2009-01-01

    In most species, the precursor for starch synthesis, ADPglucose, is made exclusively in the plastids by the enzyme ADPglucose pyrophosphorylase (AGPase). However, in the endosperm of grasses, including the economically important cereals, ADPglucose is also made in the cytosol via a cytosolic form of AGPase. Cytosolic ADPglucose is imported into plastids for starch synthesis via an ADPglucose/ADP antiporter (ADPglucose transporter) in the plastid envelope. The genes encoding the two subunits of cytosolic AGPase and the ADPglucose transporter are unique to grasses. In this review, the evolutionary origins of this unique endosperm pathway of ADPglucose synthesis and its functional significance are discussed. It is proposed that the genes encoding the pathway originated from a whole-genome-duplication event in an early ancestor of the grasses.

  20. 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.

  1. 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

  2. Hijacking the Hexosamine Biosynthetic Pathway to Promote EMT-Mediated Neoplastic Phenotypes

    PubMed Central

    Taparra, Kekoa; Tran, Phuoc T.; Zachara, Natasha E.

    2016-01-01

    The epithelial–mesenchymal transition (EMT) is a highly conserved program necessary for orchestrating distant cell migration during embryonic development. Multiple studies in cancer have demonstrated a critical role for EMT during the initial stages of tumorigenesis and later during tumor invasion. Transcription factors (TFs) such as SNAIL, TWIST, and ZEB are master EMT regulators that are aberrantly overexpressed in many malignancies. Recent evidence correlates EMT-related transcriptomic alterations with metabolic reprograming in cancer. Metabolic alterations may allow cancer to adapt to environmental stressors, supporting the irregular macromolecular demand of rapid proliferation. One potential metabolic pathway of increasing importance is the hexosamine biosynthesis pathway (HBP). The HBP utilizes glycolytic intermediates to generate the metabolite UDP–GlcNAc. This and other charged nucleotide sugars serve as the basis for biosynthesis of glycoproteins and other glycoconjugates. Recent reports in the field of glycobiology have cultivated great curiosity within the cancer research community. However, specific mechanistic relationships between the HBP and fundamental pathways of cancer, such as EMT, have yet to be elucidated. Altered protein glycosylation downstream of the HBP is well positioned to mediate many cellular changes associated with EMT including cell–cell adhesion, responsiveness to growth factors, immune system evasion, and signal transduction programs. Here, we outline some of the basics of the HBP and putative roles the HBP may have in driving EMT-related cancer processes. With novel appreciation of the HBP’s connection to EMT, we hope to illuminate the potential for new therapeutic targets of cancer. PMID:27148477

  3. 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

  4. Plasma Membrane Proteolipid 3 Protein Modulates Amphotericin B Resistance through Sphingolipid Biosynthetic Pathway

    PubMed Central

    Bari, Vinay K.; Sharma, Sushma; Alfatah, Md.; Mondal, Alok K.; Ganesan, K.

    2015-01-01

    Invasive opportunistic fungal infections of humans are common among those suffering from impaired immunity, and are difficult to treat resulting in high mortality. Amphotericin B (AmB) is one of the few antifungals available to treat such infections. The AmB resistance mechanisms reported so far mainly involve decrease in ergosterol content or alterations in cell wall. In contrast, depletion of sphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene, encoding plasma membrane proteolipid 3 protein, was shown to increase and its deletion to decrease, AmB resistance. Here we have explored the mechanistic basis of PMP3 effect on AmB resistance. It was found that ergosterol content and cell wall integrity are not related to modulation of AmB resistance by PMP3. A few prominent phenotypes of PMP3 delete strain, namely, defective actin polarity, impaired salt tolerance, and reduced rate of endocytosis are also not related to its AmB-sensitivity. However, PMP3 overexpression mediated increase in AmB resistance requires a functional sphingolipid pathway. Moreover, AmB sensitivity of strains deleted in PMP3 can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate, confirming the importance of this pathway in modulation of AmB resistance by PMP3. PMID:25965669

  5. Hijacking the Hexosamine Biosynthetic Pathway to Promote EMT-Mediated Neoplastic Phenotypes.

    PubMed

    Taparra, Kekoa; Tran, Phuoc T; Zachara, Natasha E

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a highly conserved program necessary for orchestrating distant cell migration during embryonic development. Multiple studies in cancer have demonstrated a critical role for EMT during the initial stages of tumorigenesis and later during tumor invasion. Transcription factors (TFs) such as SNAIL, TWIST, and ZEB are master EMT regulators that are aberrantly overexpressed in many malignancies. Recent evidence correlates EMT-related transcriptomic alterations with metabolic reprograming in cancer. Metabolic alterations may allow cancer to adapt to environmental stressors, supporting the irregular macromolecular demand of rapid proliferation. One potential metabolic pathway of increasing importance is the hexosamine biosynthesis pathway (HBP). The HBP utilizes glycolytic intermediates to generate the metabolite UDP-GlcNAc. This and other charged nucleotide sugars serve as the basis for biosynthesis of glycoproteins and other glycoconjugates. Recent reports in the field of glycobiology have cultivated great curiosity within the cancer research community. However, specific mechanistic relationships between the HBP and fundamental pathways of cancer, such as EMT, have yet to be elucidated. Altered protein glycosylation downstream of the HBP is well positioned to mediate many cellular changes associated with EMT including cell-cell adhesion, responsiveness to growth factors, immune system evasion, and signal transduction programs. Here, we outline some of the basics of the HBP and putative roles the HBP may have in driving EMT-related cancer processes. With novel appreciation of the HBP's connection to EMT, we hope to illuminate the potential for new therapeutic targets of cancer.

  6. Novel bioassay for the discovery of inhibitors of the 2-C-Methyl-D-Erythritol 4-Phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis

    USDA-ARS?s Scientific Manuscript database

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl-phosphate (IPP) in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisi...

  7. Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway[OPEN

    PubMed Central

    Yin, Cui-Cui; Ma, Biao; Collinge, Derek Phillip; Pogson, Barry James; He, Si-Jie; Xiong, Qing; Duan, Kai-Xuan; Chen, Hui; Yang, Chao; Lu, Xiang; Wang, Yi-Qin; Zhang, Wan-Ke; Chu, Cheng-Cai; Sun, Xiao-Hong; Fang, Shuang; Chu, Jin-Fang; Lu, Tie-Gang; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice. PMID:25841037

  8. 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.

  9. A foundation for provitamin A biofortification of maize: genome-wide association and genomic prediction models of carotenoid levels.

    PubMed

    Owens, Brenda F; Lipka, Alexander E; Magallanes-Lundback, Maria; Tiede, Tyler; Diepenbrock, Christine H; Kandianis, Catherine B; Kim, Eunha; Cepela, Jason; Mateos-Hernandez, Maria; Buell, C Robin; Buckler, Edward S; DellaPenna, Dean; Gore, Michael A; Rocheford, Torbert

    2014-12-01

    Efforts are underway for development of crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency. As a global staple crop with considerable variation in kernel carotenoid composition, maize (Zea mays L.) could have a widespread impact. We performed a genome-wide association study (GWAS) of quantified seed carotenoids across a panel of maize inbreds ranging from light yellow to dark orange in grain color to identify some of the key genes controlling maize grain carotenoid composition. Significant associations at the genome-wide level were detected within the coding regions of zep1 and lut1, carotenoid biosynthetic genes not previously shown to impact grain carotenoid composition in association studies, as well as within previously associated lcyE and crtRB1 genes. We leveraged existing biochemical and genomic information to identify 58 a priori candidate genes relevant to the biosynthesis and retention of carotenoids in maize to test in a pathway-level analysis. This revealed dxs2 and lut5, genes not previously associated with kernel carotenoids. In genomic prediction models, use of markers that targeted a small set of quantitative trait loci associated with carotenoid levels in prior linkage studies were as effective as genome-wide markers for predicting carotenoid traits. Based on GWAS, pathway-level analysis, and genomic prediction studies, we outline a flexible strategy involving use of a small number of genes that can be selected for rapid conversion of elite white grain germplasm, with minimal amounts of carotenoids, to orange grain versions containing high levels of provitamin A. Copyright © 2014 by the Genetics Society of America.

  10. A Foundation for Provitamin A Biofortification of Maize: Genome-Wide Association and Genomic Prediction Models of Carotenoid Levels

    PubMed Central

    Owens, Brenda F.; Lipka, Alexander E.; Magallanes-Lundback, Maria; Tiede, Tyler; Diepenbrock, Christine H.; Kandianis, Catherine B.; Kim, Eunha; Cepela, Jason; Mateos-Hernandez, Maria; Buell, C. Robin; Buckler, Edward S.; DellaPenna, Dean; Gore, Michael A.; Rocheford, Torbert

    2014-01-01

    Efforts are underway for development of crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency. As a global staple crop with considerable variation in kernel carotenoid composition, maize (Zea mays L.) could have a widespread impact. We performed a genome-wide association study (GWAS) of quantified seed carotenoids across a panel of maize inbreds ranging from light yellow to dark orange in grain color to identify some of the key genes controlling maize grain carotenoid composition. Significant associations at the genome-wide level were detected within the coding regions of zep1 and lut1, carotenoid biosynthetic genes not previously shown to impact grain carotenoid composition in association studies, as well as within previously associated lcyE and crtRB1 genes. We leveraged existing biochemical and genomic information to identify 58 a priori candidate genes relevant to the biosynthesis and retention of carotenoids in maize to test in a pathway-level analysis. This revealed dxs2 and lut5, genes not previously associated with kernel carotenoids. In genomic prediction models, use of markers that targeted a small set of quantitative trait loci associated with carotenoid levels in prior linkage studies were as effective as genome-wide markers for predicting carotenoid traits. Based on GWAS, pathway-level analysis, and genomic prediction studies, we outline a flexible strategy involving use of a small number of genes that can be selected for rapid conversion of elite white grain germplasm, with minimal amounts of carotenoids, to orange grain versions containing high levels of provitamin A. PMID:25258377

  11. Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis

    PubMed Central

    Zhou, Xiangjun; Welsch, Ralf; Yang, Yong; Álvarez, Daniel; Riediger, Matthias; Yuan, Hui; Fish, Tara; Liu, Jiping; Thannhauser, Theodore W.; Li, Li

    2015-01-01

    Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in the carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control carotenoid biosynthesis and accumulation in plants. However, little is known about the mechanisms underlying their posttranscriptional regulation. Here we report that PSY and OR family proteins [Arabidopsis thaliana OR (AtOR) and AtOR-like] physically interacted with each other in plastids. We found that alteration of OR expression in Arabidopsis exerted minimal effect on PSY transcript abundance. However, overexpression of AtOR significantly increased the amount of enzymatically active PSY, whereas an ator ator-like double mutant exhibited a dramatically reduced PSY level. The results indicate that the OR proteins serve as the major posttranscriptional regulators of PSY. The ator or ator-like single mutant had little effect on PSY protein levels, which involves a compensatory mechanism and suggests partial functional redundancy. In addition, modification of PSY expression resulted in altered AtOR protein levels, corroborating a mutual regulation of PSY and OR. Carotenoid content showed a correlated change with OR-mediated PSY level, demonstrating the function of OR in controlling carotenoid biosynthesis by regulating PSY. Our findings reveal a novel mechanism by which carotenoid biosynthesis is controlled via posttranscriptional regulation of PSY in plants. PMID:25675505

  12. 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 (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.

  13. Metabolic Regulation of Carotenoid-Enriched Golden Rice Line

    PubMed Central

    Gayen, Dipak; Ghosh, Subhrajyoti; Paul, Soumitra; Sarkar, Sailendra N.; Datta, Swapan K.; Datta, Karabi

    2016-01-01

    Vitamin A deficiency (VAD) is the leading cause of blindness among children and is associated with high risk of maternal mortality. In order to enhance the bioavailability of vitamin A, high carotenoid transgenic golden rice has been developed by manipulating enzymes, such as phytoene synthase (psy) and phytoene desaturase (crtI). In this study, proteome and metabolite analyses were carried out to comprehend metabolic regulation and adaptation of transgenic golden rice after the manipulation of endosperm specific carotenoid pathways. The main alteration was observed in carbohydrate metabolism pathways of the transgenic seeds. The 2D based proteomic studies demonstrated that carbohydrate metabolism-related enzymes, such as pullulanase, UDP-glucose pyrophosphorylase, and glucose-1-phosphate adenylyltransferase, were primarily up-regulated in transgenic rice seeds. In addition, the enzyme PPDK was also elevated in transgenic seeds thus enhancing pyruvate biosynthesis, which is the precursor in the carotenoids biosynthetic pathway. GC-MS based metabolite profiling demonstrated an increase in the levels of glyceric acid, fructo-furanose, and galactose, while decrease in galactonic acid and gentiobiose in the transgenic rice compared to WT. It is noteworthy to mention that the carotenoid content, especially β-carotene level in transgenic rice (4.3 μg/g) was significantly enhanced. The present study highlights the metabolic adaptation process of a transgenic golden rice line (homozygous T4 progeny of SKBR-244) after enhancing carotenoid biosynthesis. The presented information would be helpful in the development of crops enriched in carotenoids by expressing metabolic flux of pyruvate biosynthesis. PMID:27840631

  14. Metabolic Regulation of Carotenoid-Enriched Golden Rice Line.

    PubMed

    Gayen, Dipak; Ghosh, Subhrajyoti; Paul, Soumitra; Sarkar, Sailendra N; Datta, Swapan K; Datta, Karabi

    2016-01-01

    Vitamin A deficiency (VAD) is the leading cause of blindness among children and is associated with high risk of maternal mortality. In order to enhance the bioavailability of vitamin A, high carotenoid transgenic golden rice has been developed by manipulating enzymes, such as phytoene synthase (psy) and phytoene desaturase (crtI). In this study, proteome and metabolite analyses were carried out to comprehend metabolic regulation and adaptation of transgenic golden rice after the manipulation of endosperm specific carotenoid pathways. The main alteration was observed in carbohydrate metabolism pathways of the transgenic seeds. The 2D based proteomic studies demonstrated that carbohydrate metabolism-related enzymes, such as pullulanase, UDP-glucose pyrophosphorylase, and glucose-1-phosphate adenylyltransferase, were primarily up-regulated in transgenic rice seeds. In addition, the enzyme PPDK was also elevated in transgenic seeds thus enhancing pyruvate biosynthesis, which is the precursor in the carotenoids biosynthetic pathway. GC-MS based metabolite profiling demonstrated an increase in the levels of glyceric acid, fructo-furanose, and galactose, while decrease in galactonic acid and gentiobiose in the transgenic rice compared to WT. It is noteworthy to mention that the carotenoid content, especially β-carotene level in transgenic rice (4.3 μg/g) was significantly enhanced. The present study highlights the metabolic adaptation process of a transgenic golden rice line (homozygous T4 progeny of SKBR-244) after enhancing carotenoid biosynthesis. The presented information would be helpful in the development of crops enriched in carotenoids by expressing metabolic flux of pyruvate biosynthesis.

  15. Carotenoids in Microalgae.

    PubMed

    Henríquez, Vitalia; Escobar, Carolina; Galarza, Janeth; Gimpel, Javier

    Carotenoids are a class of isoprenoids synthesized by all photosynthetic organisms as well as by some non-photosynthetic bacteria and fungi with broad applications in food, feed and cosmetics, and also in the nutraceutical and pharmaceutical industries. Microalgae represent an important source of high-value products, which include carotenoids, among others. Carotenoids play key roles in light harvesting and energy transfer during photosynthesis and in the protection of the photosynthetic apparatus against photooxidative damage. Carotenoids are generally divided into carotenes and xanthophyls, but accumulation in microalgae can also be classified as primary (essential for survival) and secondary (by exposure to specific stimuli).In this chapter, we outline the high value carotenoids produced by commercially important microalgae, their production pathways, the improved production rates that can be achieved by genetic engineering as well as their biotechnological applications.

  16. Carotenoids of biotechnological importance.

    PubMed

    Sandmann, Gerhard

    2015-01-01

    Carotenoids are natural pigments with antioxidative functions that protect against oxidative stress. They are essential for humans and must be supplied through the diet. Carotenoids are the precursors for the visual pigment rhodopsin, and lutein and zeaxanthin must be accumulated in the yellow eye spot to protect the retina from excess light and ultraviolet damage. There is a global market for carotenoids as food colorants, animal feed, and nutraceuticals. Some carotenoids are chemically synthesized, whereas others are from natural sources. Microbial mass production systems of industrial interest for carotenoids are in use, and new ones are being developed by metabolic pathway engineering of bacteria, fungi, and plants. Several examples will be highlighted in this chapter.

  17. 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

  18. Membrane lipids in Agrobacterium tumefaciens: biosynthetic pathways and importance for pathogenesis

    PubMed Central

    Aktas, Meriyem; Danne, Linna; Möller, Philip; Narberhaus, Franz

    2014-01-01

    Many cellular processes critically depend on the membrane composition. In this review, we focus on the biosynthesis and physiological roles of membrane lipids in the plant pathogen Agrobacterium tumefaciens. The major components of A. tumefaciens membranes are the phospholipids (PLs), phosphatidylethanolamine (PE), phosphatidylglycerol, phosphatidylcholine (PC) and cardiolipin, and ornithine lipids (OLs). Under phosphate-limited conditions, the membrane composition shifts to phosphate-free lipids like glycolipids, OLs and a betaine lipid. Remarkably, PC and OLs have opposing effects on virulence of A. tumefaciens. OL-lacking A. tumefaciens mutants form tumors on the host plant earlier than the wild type suggesting a reduced host defense response in the absence of OLs. In contrast, A. tumefaciens is compromised in tumor formation in the absence of PC. In general, PC is a rare component of bacterial membranes but amount to ~22% of all PLs in A. tumefaciens. PC biosynthesis occurs via two pathways. The phospholipid N-methyltransferase PmtA methylates PE via the intermediates monomethyl-PE and dimethyl-PE to PC. In the second pathway, the membrane-integral enzyme PC synthase (Pcs) condenses choline with CDP-diacylglycerol to PC. Apart from the virulence defect, PC-deficient A. tumefaciens pmtA and pcs double mutants show reduced motility, enhanced biofilm formation and increased sensitivity towards detergent and thermal stress. In summary, there is cumulative evidence that the membrane lipid composition of A. tumefaciens is critical for agrobacterial physiology and tumor formation. PMID:24723930

  19. 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

  20. Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum*

    PubMed Central

    Cobbold, Simon A.; Vaughan, Ashley M.; Lewis, Ian A.; Painter, Heather J.; Camargo, Nelly; Perlman, David H.; Fishbaugher, Matthew; Healer, Julie; Cowman, Alan F.; Kappe, Stefan H. I.; Llinás, Manuel

    2013-01-01

    The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a “PDH-like” enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage. PMID:24163372

  1. Strain-Specific Proteogenomics Accelerates Discovery of Natural Products Via Their Biosynthetic Pathways

    PubMed Central

    Albright, Jessica C.; Goering, Anthony W.; Doroghazi, James R.; Metcalf, William W.; Kelleher, Neil L.

    2014-01-01

    The use of proteomics for direct detection of expressed pathways producing natural products has yielded many new compounds, even when used in a screening mode without a bacterial genome sequence available. Here we quantify the advantages of having draft DNA-sequence available for strain-specific proteomics using the latest in ultrahigh-resolution mass spectrometry (MS) for both proteins and the small molecules they generate. Using the draft sequence of Streptomyces lilacinus NRRL B-1968, we show a >10-fold increase in the number of peptide identifications vs. using publicly available databases. Detected in this strain were six expressed gene clusters with varying homology to those known. To date, we have identified three of these clusters as encoding for the production of griseobactin (known), rakicidin D (an orphan NRPS/PKS hybrid cluster), and a putative thr and DHB-containing siderophore produced by a new non-ribosomal peptide sythetase gene cluster. The remaining three clusters show lower homology to those known, and likely encode enzymes for production of novel compounds. Using an interpreted strain-specific DNA sequence enables deep proteomics for the detection of multiple pathways and their encoded natural products in a single cultured bacterium. PMID:24242000

  2. Functional analysis of the small subunit of the putative homoaconitase from Pyrococcus horikoshii in the Thermus lysine biosynthetic pathway.

    PubMed

    Lombo, Tania; Takaya, Naoki; Miyazaki, Junichi; Gotoh, Kazumi; Nishiyama, Makoto; Kosuge, Takehide; Nakamura, Akira; Hoshino, Takayuki

    2004-04-15

    An in vivo disruption-integration vector system for Thermus thermophilus was developed and used for the functional analysis of an evolutionary-related archaeal protein for lysine biosynthesis. In contrast to fungal one, the putative homoaconitase of T. thermophilus consists of two subunits and catalyzes the second and third steps of lysine biosynthesis. ORFs from hyperthermophilic archaeon Pyrococcus horikoshii, PH1726 and PH1724, share a high degree of amino acid identity with the T. thermophilus subunits LysT and LysU, respectively. In the present report, gene encoding the putative small subunit of archaeal homoaconitase, PH1724, was integrated into the lysU locus of T. thermophilus. The archaeal gene was expressed under the control of PslpA promoter and functional analyses were performed. Transformants were able to grow on minimal medium without lysine when PH1724 ORF was integrated, whereas the lysU disruption led to lysine auxotrophy. Chromosomal integration was verified by PCR analysis, and homoaconitase assay showed that the archaeal gene product functions as a small subunit of homoaconitase, possibly by forming a heterodimer with the LysT subunit of T. thermophilus. These results strongly suggest the functional relation of P. horikoshii PH1724 with LysU in the Thermus lysine biosynthetic pathway, together with functional assignment of LysU as small subunit of homoaconitase. In addition, the provided results indicate that archaeal genes products from hyperthermophiles can be studied in a thermophilic eubacterium such as T. thermophilus.

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

    PubMed

    Nara, Takeshi; Hashimoto, Muneaki; Hirawake, Hiroko; Liao, Chien-Wei; Fukai, Yoshihisa; Suzuki, Shigeo; Tsubouchi, Akiko; Morales, Jorge; Takamiya, Shinzaburo; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Fan, Chia-Kwung; Inaoka, Daniel Ken; Inoue, Masayuki; Tanaka, Akiko; Harada, Shigeharu; Kita, Kiyoshi; Aoki, Takashi

    2012-02-03

    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 fusion 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Studies on the nonmevalonate terpene biosynthetic pathway: Metabolic role of IspH (LytB) protein

    PubMed Central

    Rohdich, Felix; Hecht, Stefan; Gärtner, Katrin; Adam, Petra; Krieger, Cornelia; Amslinger, Sabine; Arigoni, Duilio; Bacher, Adelbert; Eisenreich, Wolfgang

    2002-01-01

    Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-d-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy-d-xylulose can be converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U-13C5]1-deoxy-d-xylulose into a 5:1 mixture of [U-13C5]isopentenyl diphosphate and [U-13C5]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes. PMID:11818558

  5. Genetic manipulation of the Fusarium fujikuroi fusarin gene cluster yields insight into the complex regulation and fusarin biosynthetic pathway.

    PubMed

    Niehaus, Eva-Maria; Kleigrewe, Karin; Wiemann, Philipp; Studt, Lena; Sieber, Christian M K; Connolly, Lanelle R; Freitag, Michael; Güldener, Ulrich; Tudzynski, Bettina; Humpf, Hans-Ulrich

    2013-08-22

    In this work, the biosynthesis and regulation of the polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS)-derived mutagenic mycotoxin fusarin C was studied in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes (fus1-fus9) that are coexpressed under high-nitrogen and acidic pH conditions. Chromatin immunoprecipitation revealed a correlation between high expression and enrichment of activating H3K9-acetylation marks under inducing conditions. We provide evidence that only four genes are sufficient for the biosynthesis. The combination of genetic engineering with nuclear magnetic resonance and mass-spectrometry-based structure elucidation allowed the discovery of the putative fusarin biosynthetic pathway. Surprisingly, we indicate that PKS/NRPS releases its product with an open ring structure, probably as an alcohol. Our data indicate that 2-pyrrolidone ring closure, oxidation at C-20, and, finally, methylation at C-20 are catalyzed by Fus2, Fus8, and Fus9, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Two Catechol Siderophores, Acinetobactin and Amonabactin, Are Simultaneously Produced by Aeromonas salmonicida subsp. salmonicida Sharing Part of the Biosynthetic Pathway.

    PubMed

    Balado, Miguel; Souto, Alba; Vences, Ana; Careaga, Valeria P; Valderrama, Katherine; Segade, Yuri; Rodríguez, Jaime; Osorio, Carlos R; Jiménez, Carlos; Lemos, Manuel L

    2015-12-18

    The iron uptake mechanisms based on siderophore synthesis used by the fish pathogen Aeromonas salmonicida subsp. salmonicida are still not completely understood, and the precise structure of the siderophore(s) is unknown. The analysis of genome sequences revealed that this bacterium possesses two gene clusters putatively involved in the synthesis of siderophores. One cluster is a candidate to encode the synthesis of acinetobactin, the siderophore of the human pathogen Acinetobacter baumannii, while the second cluster shows high similarity to the genes encoding amonabactin synthesis in Aeromonas hydrophila. Using a combination of genomic analysis, mutagenesis, biological assays, chemical purification, and structural determination procedures, here we demonstrate that most A. salmonicida subsp. salmonicida strains produce simultaneously the two siderophores, acinetobactin and amonabactin. Interestingly, the synthesis of both siderophores relies on a single copy of the genes encoding the synthesis of the catechol moiety (2,3-dihydroxybenzoic acid) and on one encoding a phosphopantetheinyl transferase. These genes are present only in the amonabactin cluster, and a single mutation in any of them abolishes production of both siderophores. We could also demonstrate that some strains, isolated from fish raised in seawater, produce only acinetobactin since they present a deletion in the amonabactin biosynthesis gene amoG. Our study represents the first evidence of simultaneous production of acinetobactin and amonabactin by a bacterial pathogen and reveals the plasticity of bacterial genomes and biosynthetic pathways. The fact that the same siderophore is produced by unrelated pathogens highlights the importance of these systems and their interchangeability between different bacteria.

  7. Biosynthetic pathway of aliphatic formates via a Baeyer–Villiger oxidation in mechanism present in astigmatid mites

    PubMed Central

    Shimizu, Nobuhiro; Sakata, Daisuke; Schmelz, Eric A.; Mori, Naoki; Kuwahara, Yasumasa

    2017-01-01

    Astigmatid mites depend on bioactive glandular secretions, pheromones, and defensive agents to mediate intra- and interspecies interactions. Aliphatic formates, such as (Z,Z)-8,11-heptadecadienyl formate (8,11-F17) and (Z)-8-heptadecenyl formate (8-F17), are rarely encountered natural products that are abundant in Sancassania sp. Sasagawa (Acari: Acaridae) mite secretions. Linoleic acid and oleic acid are predicted as key intermediates in the synthesis of the closely related aliphatic formates. To gain insight in this biosynthetic pathway, acarid mite feeding experiments were conducted using 13C-labeled precursors to precisely track incorporation. Analyses using 13C NMR spectroscopy demonstrated that the 13C-labeling pattern of the precursors was detectable on formates in exocrine secretions and likewise on fatty acids in total lipid pools. Curiously, the results demonstrated that the formates were biosynthesized without the dehomologation of corresponding fatty acids. Careful examination of the mass spectra from labeling experiments revealed that the carbonyl carbon of the formates is originally derived from the C-1 position of the fatty acids. Consistent with a Baeyer–Villiger oxidation reaction, labeling studies support the insertion of an oxygen atom between the carbonyl group and carbon chain. Empirical data support the existence of a Baeyer–Villiger monooxygenase responsible for the catalyzation of the Baeyer–Villiger oxidation. The predicted existence of a Baeyer–Villiger monooxygenase capable of converting aliphatic aldehydes to formates represents an exciting opportunity to expand the enzymatic toolbox available for controlled biochemical synthesis. PMID:28223501

  8. The biosynthetic pathway of pyrimidine (deoxy)nucleotides: A sensor of oxygen tension necessary for maintaining cell proliferation

    SciTech Connect

    Loeffler, M. )

    1989-06-01

    Culture experiments on Ehrlich ascites tumor cells revealed that a low oxygen tension (about 20% in normoxic atmosphere) induced an increase in the length of the growth cycle. The relative growth of aerobic control cells after transfer to the second in vitro passage was 145% within 24 h, and reduced to 50% at 1% O{sub 2} and about 30% at 0.1% O{sub 2}. The increase in protein and DNA content of these hypoxic cultures was equally impaired. Also, the cell cycle traverse as analyzed by flow cytometry was affected predominantly at the G{sub 1}/early S stage. Uptake of labeled thymidine into acid-insoluble material of hypoxic cells was below that of controls whereas incorporation of uridine exceeded that of normoxic controls. The results are interpreted as supporting the concept that the biosynthetic pathway of pyrimidine (deoxy)nucleotides - because of two oxygen-dependent enzymes, dihydroorotate dehydrogenase and ribonucleotide reductase - is a potential transducer of environmental limitations in oxygen tension to the proliferative capacity of cells.

  9. Expression of genes associated with the biosynthetic pathways of abscisic acid, gibberellin, and ethylene during the germination of lettuce seeds.

    PubMed

    Clemente, A C S; Guimarães, R M; Martins, D C; Gomes, L A A; Caixeta, F; Reis, R G E; Rosa, S D V F

    2015-05-11

    Seed germination and dormancy are complex phenomena that are controlled by many genes and environmental factors. Such genes are indicated by phytohormones that interact with each other, and may cause dormancy or promote seed germination. The objective of this study was to investigate gene expression associated with the biosynthetic pathways of abscisic acid (ABA), gibberellic acid (GA), and ethylene (ET) in dormant and germinated lettuce seeds. The expressions of LsNCED, LsGA3ox1, and ACO-B were evaluated in germinating and dormant seeds from the cultivars Everglades, Babá de Verão, Verônica, Salinas, Colorado, and Regina 71. The expressions of LsNCED, LsGA3ox1, and ACO-B were related to the biosynthesis of ABA, GA, and ET, respectively; therefore, the presence of these substances depends on genotype. LsNCED expression only occurred in dormant seeds, and was connected to dormancy. LsGA3ox1expression only occurred in germinated seeds, and was connected to germination. The ACO-B gene was involved in ET biosynthesis, and was expressed differently in germinated and dormant seeds, depending on the genotype, indicating different functions for different characteristics. Furthermore, sensitivity to phytohormones appeared to be more important than the expression levels of LsNCED, LsGA3ox1, or ACO-B.

  10. Combinatorial biosynthesis of cyclic lipopeptide antibiotics: a model for synthetic biology to accelerate the evolution of secondary metabolite biosynthetic pathways.

    PubMed

    Baltz, Richard H

    2014-10-17

    Nonribosomal peptide synthetases (NRPSs) are giant multi-enzymes that carry out sequencial assembly line couplings of amino acids to generate linear or cyclic peptides. NRPSs are composed of repeating enzyme domains with modular organization to activate and couple specific amino acids in a particular order. From a synthetic biology perspective, they can be considered as peptide assembly machines composed of devices to couple fatty acids to l-amino acids, l-amino acids to l-amino acids, and d-amino acids to l-amino acids. The coupling devices are composed of specific parts that contain two or more enzyme domains that can be exchanged combinatorially to generate novel peptide assembly machines to produce novel peptides. The potent lipopeptide antibiotics daptomycin and A54145E have identical cyclic depsipeptide ring structures and stereochemistry but have divergent amino acid sequences. As their biosynthetic gene clusters are derived from an ancient ancestral lipopetide pathway, these lipopeptides provided an attractive model to develop combinatorial biosynthesis to generate antibiotics superior to daptomycin. These studies on combinatorial biosynthesis have helped generate guidelines for the successful assembly of NRPS parts and devices that can be used to generate novel lipopeptide structures and have established a basis for future synthetic biology studies to further develop combinatorial biosynthesis as a robust approach to natural product drug discovery.

  11. Functional characteristics of spirilloxanthin and keto-bearing Analogues in light-harvesting LH2 complexes from Rhodobacter sphaeroides with a genetically modified carotenoid synthesis pathway.

    PubMed

    Niedzwiedzki, Dariusz M; Dilbeck, Preston L; Tang, Qun; Mothersole, David J; Martin, Elizabeth C; Bocian, David F; Holten, Dewey; Hunter, C Neil

    2015-01-01

    Light-harvesting 2 (LH2) complexes from a genetically modified strain of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were studied using static and ultrafast optical methods and resonance Raman spectroscopy. Carotenoid synthesis in the Rba. sphaeroides strain was engineered to redirect carotenoid production away from spheroidene into the spirilloxanthin synthesis pathway. The strain assembles LH2 antennas with substantial amounts of spirilloxanthin (total double-bond conjugation length N=13) if grown anaerobically and of keto-bearing long-chain analogs [2-ketoanhydrorhodovibrin (N=13), 2-ketospirilloxanthin (N=14) and 2,2'-diketospirilloxanthin (N=15)] if grown semi-aerobically (with ratios that depend on growth conditions). We present the photophysical, electronic, and vibrational properties of these carotenoids, both isolated in organic media and assembled within LH2 complexes. Measurements of excited-state energy transfer to the array of excitonically coupled bacteriochlorophyll a molecules (B850) show that the mean lifetime of the first singlet excited state (S1) of the long-chain (N≥13) carotenoids does not change appreciably between organic media and the protein environment. In each case, the S1 state appears to lie lower in energy than that of B850. The energy-transfer yield is ~0.4 in LH2 (from the strain grown aerobically or semi-aerobically), which is less than half that achieved for LH2 that contains short-chain (N≤11) analogues. Collectively, the results suggest that the S1 excited state of the long-chain (N≥13) carotenoids participates little if at all in carotenoid-to-BChl a energy transfer, which occurs predominantly via the carotenoid S2 excited state in these antennas.

  12. Use of the valine biosynthetic pathway to convert glucose into isobutanol.

    PubMed

    Savrasova, Ekaterina A; Kivero, Aleksander D; Shakulov, Rustem S; Stoynova, Nataliya V

    2011-09-01

    Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259-2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also--as has been demonstrated previously (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009)--used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD

  13. 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.

  14. Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways

    PubMed Central

    Ryczko, Michael C.; Pawling, Judy; Chen, Rui; Abdel Rahman, Anas M.; Yau, Kevin; Copeland, Julia K.; Zhang, Cunjie; Surendra, Anu; Guttman, David S.; Figeys, Daniel; Dennis, James W.

    2016-01-01

    De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhibition of Golgi N-glycan branching blocked GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase enzymes of the N-glycan branching pathway (Mgat1,2,4,5) display multistep ultrasensitivity to UDP-GlcNAc, as well as branching-dependent compensation. Indeed, oral GlcNAc rescued fat accumulation in lean Mgat5−/− mice and in cultured Mgat5−/− hepatocytes, consistent with N-glycan branching compensation. Our results suggest GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the UDP-GlcNAc supply to N-glycan branching pathway. PMID:26972830

  15. Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway.

    PubMed

    Voynova, Natalya E; Rios, Sandra E; Miziorko, Henry M

    2004-01-01

    It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.

  16. Engineering Salidroside Biosynthetic Pathway in Hairy Root Cultures of Rhodiola crenulata Based on Metabolic Characterization of Tyrosine Decarboxylase

    PubMed Central

    Zeng, Lingjiang; Liu, Xiaoqiang; Qiu, Fei; Zheng, Weilie; Quan, Hong; Liao, Zhihua; Chen, Min; Huang, Wenlin; Liu, Wanhong; Wang, Qiang

    2013-01-01

    Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21–6.84, 1.50–2.19 and 1.27–3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside. PMID:24124492

  17. Engineering salidroside biosynthetic pathway in hairy root cultures of Rhodiola crenulata based on metabolic characterization of tyrosine decarboxylase.

    PubMed

    Lan, Xiaozhong; Chang, Kai; Zeng, Lingjiang; Liu, Xiaoqiang; Qiu, Fei; Zheng, Weilie; Quan, Hong; Liao, Zhihua; Chen, Min; Huang, Wenlin; Liu, Wanhong; Wang, Qiang

    2013-01-01

    Tyrosine decarboxylase initializes salidroside biosynthesis. Metabolic characterization of tyrosine decarboxylase gene from Rhodiola crenulata (RcTYDC) revealed that it played an important role in salidroside biosynthesis. Recombinant 53 kDa RcTYDC converted tyrosine into tyramine. RcTYDC gene expression was induced coordinately with the expression of RcUDPGT (the last gene involved in salidroside biosynthesis) in SA/MeJA treatment; the expression of RcTYDC and RcUDPGT was dramatically upregulated by SA, respectively 49 folds and 36 folds compared with control. MeJA also significantly increased the expression of RcTYDC and RcUDPGT in hairy root cultures. The tissue profile of RcTYDC and RcUDPGT was highly similar: highest expression levels found in stems, higher expression levels in leaves than in flowers and roots. The gene expressing levels were consistent with the salidroside accumulation levels. This strongly suggested that RcTYDC played an important role in salidroside biosynthesis in R. crenulata. Finally, RcTYDC was used to engineering salidroside biosynthetic pathway in R. crenulata hairy roots via metabolic engineering strategy of overexpression. All the transgenic lines showed much higher expression levels of RcTYDC than non-transgenic one. The transgenic lines produced tyramine, tyrosol and salidroside at higher levels, which were respectively 3.21-6.84, 1.50-2.19 and 1.27-3.47 folds compared with the corresponding compound in non-transgenic lines. In conclusion, RcTYDC overexpression promoted tyramine biosynthesis that facilitated more metabolic flux flowing toward the downstream pathway and as a result, the intermediate tyrosol was accumulated more that led to the increased production of the end-product salidroside.

  18. Nitric oxide enhances plant ultraviolet-B protection up-regulating gene expression of the phenylpropanoid biosynthetic pathway.

    PubMed

    Tossi, Vanesa; Amenta, Melina; Lamattina, Lorenzo; Cassia, Raúl

    2011-06-01

    The link between ultraviolet (UV)-B, nitric oxide (NO) and phenylpropanoid biosynthetic pathway (PPBP) was studied in maize and Arabidopsis. The transcription factor (TF) ZmP regulates PPBP in maize. A genetic approach using P-rr (ZmP+) and P-ww (ZmP⁻) maize lines demonstrate that: (1) NO protects P-rr leaves but not P-ww from UV-B-induced reactive oxygen species (ROS) and cell damage; (2) NO increases flavonoid and anthocyanin content and prevents chlorophyll loss in P-rr but not in P-ww and (3) the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) blocks the UV-B-induced expression of ZmP and their targets CHS and CHI suggesting that NO plays a key role in the UV-B-regulated PPBP. Involvement of endogenous NO was studied in Arabidopsis nitric oxide dioxygenase (NOD) plants that express a NO dioxygenase gene under the control of a dexamethasone (DEX)-inducible promoter. Expression of HY5 and MYB12, TFs involved in PPBP regulation, was induced by UV-B, reduced by DEX in NOD plants and recovered by subsequent NO treatment. C4H regulates synapate esters synthesis and is UV-B-induced in a NO-independent pathway. Data indicate that UV-B perception increases NO concentration, which protects plant against UV-B by two ways: (1) scavenging ROS; and (2) up-regulating the expression of HY5, MYB12 and ZmP, resulting in the PPBP activation.

  19. Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

    SciTech Connect

    Whicher, Jonathan R.; Florova, Galina; Sydor, Paulina K.; Singh, Renu; Alhamadsheh, Mamoun; Challis, Gregory L.; Reynolds, Kevin A.; Smith, Janet L.

    2011-08-17

    Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a {beta}-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 {angstrom} resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

  20. Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor*

    PubMed Central

    Whicher, Jonathan R.; Florova, Galina; Sydor, Paulina K.; Singh, Renu; Alhamadsheh, Mamoun; Challis, Gregory L.; Reynolds, Kevin A.; Smith, Janet L.

    2011-01-01

    Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole. PMID:21543318

  1. Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays[OPEN

    PubMed Central

    Schaff, Claudia; Zhang, Zhiwu; Lipka, Alexander E.; Preiß, Susanne; Irmisch, Sandra

    2016-01-01

    Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL. Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT. The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities. PMID:27662898

  2. Arctic mustard flower color polymorphism controlled by petal-specific downregulation at the threshold of the anthocyanin biosynthetic pathway.

    PubMed

    Dick, Cynthia A; Buenrostro, Jason; Butler, Timothy; Carlson, Matthew L; Kliebenstein, Daniel J; Whittall, Justen B

    2011-04-07

    Intra- and interspecific variation in flower color is a hallmark of angiosperm diversity. The evolutionary forces underlying the variety of flower colors can be nearly as diverse as the colors themselves. In addition to pollinator preferences, non-pollinator agents of selection can have a major influence on the evolution of flower color polymorphisms, especially when the pigments in question are also expressed in vegetative tissues. In such cases, identifying the target(s) of selection starts with determining the biochemical and molecular basis for the flower color variation and examining any pleiotropic effects manifested in vegetative tissues. Herein, we describe a widespread purple-white flower color polymorphism in the mustard Parrya nudicaulis spanning Alaska. The frequency of white-flowered individuals increases with increasing growing-season temperature, consistent with the role of anthocyanin pigments in stress tolerance. White petals fail to produce the stress responsive flavonoid intermediates in the anthocyanin biosynthetic pathway (ABP), suggesting an early pathway blockage. Petal cDNA sequences did not reveal blockages in any of the eight enzyme-coding genes in white-flowered individuals, nor any color differentiating SNPs. A qRT-PCR analysis of white petals identified a 24-fold reduction in chalcone synthase (CHS) at the threshold of the ABP, but no change in CHS expression in leaves and sepals. This arctic species has avoided the deleterious effects associated with the loss of flavonoid intermediates in vegetative tissues by decoupling CHS expression in petals and leaves, yet the correlation of flower color and climate suggests that the loss of flavonoids in the petals alone may affect the tolerance of white-flowered individuals to colder environments.

  3. The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

    PubMed Central

    de Gontijo, Fabiano Assis; Pascon, Renata C.; Fernandes, Larissa; Machado, Joel; Alspaugh, J. Andrew; Vallim, Marcelo A

    2015-01-01

    Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de novo synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. In addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30°C to 37°C, and that transcriptional regulation of de novo and salvage pyrimidine pathway are under the control of the Ura4 protein. PMID:25011011

  4. Characterization of Biosynthetic Pathways for the Production of the Volatile Homoterpenes DMNT and TMTT in Zea mays.

    PubMed

    Richter, Annett; Schaff, Claudia; Zhang, Zhiwu; Lipka, Alexander E; Tian, Feng; Köllner, Tobias G; Schnee, Christiane; Preiß, Susanne; Irmisch, Sandra; Jander, Georg; Boland, Willhelm; Gershenzon, Jonathan; Buckler, Edward S; Degenhardt, Jörg

    2016-10-01

    Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.

  5. Novel Bioassay for the Discovery of Inhibitors of the 2-C-Methyl-D-erythritol 4-Phosphate (MEP) and Terpenoid Pathways Leading to Carotenoid Biosynthesis

    PubMed Central

    Corniani, Natália; Velini, Edivaldo D.; Silva, Ferdinando M. L.; Nanayakkara, N. P. Dhammika; Witschel, Matthias; Dayan, Franck E.

    2014-01-01

    The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay. PMID:25077957

  6. Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis.

    PubMed

    Corniani, Natália; Velini, Edivaldo D; Silva, Ferdinando M L; Nanayakkara, N P Dhammika; Witschel, Matthias; Dayan, Franck E

    2014-01-01

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

  7. Progress in Understanding the Genetic Information and Biosynthetic Pathways behind Amycolatopsis Antibiotics, with Implications for the Continued Discovery of Novel Drugs.

    PubMed

    Chen, Su; Wu, Qihao; Shen, Qingqing; Wang, Hong

    2016-01-01

    Species of Amycolatopsis, well recognized as producers of both vancomycin and rifamycin, are also known for producing other secondary metabolites, with wide usage in medicine and agriculture. The molecular genetics of natural antibiotics produced by this genus have been well studied. Since the rise of antibiotic resistance, finding new drugs to fight infection has become an urgent priority. Progress in understanding the biosynthesis of metabolites greatly helps the rational manipulation of biosynthetic pathways, and thus to achieve the goal of generating novel natural antibiotics. The efforts made in exploiting Amycolatopsis genome sequences for the discovery of novel natural products and biosynthetic pathways are summarized. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. 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

  9. Novel acetylcholinesterase inhibitors from Zijuan tea and biosynthetic pathway of caffeoylated catechin in tea plant.

    PubMed

    Wang, Wei; Fu, Xi-Wen; Dai, Xin-Long; Hua, Fang; Chu, Gang-Xiu; Chu, Ming-Jie; Hu, Feng-Lin; Ling, Tie-Jun; Gao, Li-Ping; Xie, Zhong-Wen; Wan, Xiao-Chun; Bao, Guan-Hu

    2017-12-15

    Zijuan tea is a special cultivar of Yunnan broad-leaf tea (Camellia sinensis var. assamica) with purple buds, leaves, and stems. Phytochemical study on this tea led to the discovery of three hydroxycinnamoylated catechins (HCCs) (1-3), seven other catechins (4-10), three proanthocyanidins (11-13), five flavones and flavone glycosides (14-18), two alkaloids (19, 20), one steroid (21), and one phenylpropanoid glycoside (22). The isolation and structural elucidation of the caffeoylated catechin (1) by means of spectroscopic techniques were described. We also provide the first evidence that 1 is synthesized via a two-step pathway in tea plant. The three HCCs (1-3) were investigated on their bioactivity through molecular modeling simulation and biochemical experiments. Our results show that they bind acetylcholinesterase (AChE) tightly and have strong AChE inhibitory activity with IC50 value at 2.49, 11.41, 62.26μM, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. The Glutathione Biosynthetic Pathway of Plasmodium Is Essential for Mosquito Transmission

    PubMed Central

    Vega-Rodríguez, Joel; Janse, Chris J.; Pastrana-Mena, Rebecca; Waters, Andrew P.; Coppens, Isabelle; Rodríguez-Orengo, José F.; Jacobs-Lorena, Marcelo; Serrano, Adelfa E.

    2009-01-01

    Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs− parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs− parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito. PMID:19229315

  11. Recent advances in targeting the fatty acid biosynthetic pathway using fatty acid synthase inhibitors.

    PubMed

    Angeles, Thelma S; Hudkins, Robert L

    2016-12-01

    Elevated lipogenesis has been associated with a variety of diseases including obesity, cancer and nonalcoholic fatty liver disease (NAFLD). Fatty acid synthase (FASN) plays a pivotal role in de novo lipogenesis, making this multi-catalytic protein an attractive target for therapeutic intervention. Recently, the first FASN inhibitor successfully advanced through the drug development process and entered clinical evaluation in oncology. Areas covered: This review discusses the biological roles of FASN in three prominent disease areas: cancer, obesity-related disorders and NAFLD. Recent advances in drug discovery strategies and design of newer FASN inhibitors are also highlighted. Expert opinion: Despite the abundance of evidence linking the lipogenic pathway to cancer, progression of FASN-targeted molecules has been rather slow and challenging and no compounds have moved past the preclinical phase. The landscape has recently changed with the recent advancement of the first FASN inhibitor into clinical evaluation for solid tumors. Needless to say, the successful translation into the clinical setting will open opportunities for expanding the therapeutic utility of FASN inhibitors not just in oncology but in other diseases associated with elevated lipogenesis such as obesity, type 2 diabetes, and NAFLD.

  12. Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

    PubMed Central

    Siitonen, Vilja; Selvaraj, Brinda; Niiranen, Laila; Lindqvist, Ylva; Schneider, Gunter; Metsä-Ketelä, Mikko

    2016-01-01

    Nogalamycin, an aromatic polyketide displaying high cytotoxicity, has a unique structure, with one of the carbohydrate units covalently attached to the aglycone via an additional carbon–carbon bond. The underlying chemistry, which implies a particularly challenging reaction requiring activation of an aliphatic carbon atom, has remained enigmatic. Here, we show that the unusual C5′′–C2 carbocyclization is catalyzed by the non-heme iron α-ketoglutarate (α-KG)–dependent SnoK in the biosynthesis of the anthracycline nogalamycin. The data are consistent with a mechanistic proposal whereby the Fe(IV) = O center abstracts the H5′′ atom from the amino sugar of the substrate, with subsequent attack of the aromatic C2 carbon on the radical center. We further show that, in the same metabolic pathway, the homologous SnoN (38% sequence identity) catalyzes an epimerization step at the adjacent C4′′ carbon, most likely via a radical mechanism involving the Fe(IV) = O center. SnoK and SnoN have surprisingly similar active site architectures considering the markedly different chemistries catalyzed by the enzymes. Structural studies reveal that the differences are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. Our findings significantly expand the repertoire of reactions reported for this important protein family and provide an illustrative example of enzyme evolution. PMID:27114534

  13. Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways

    PubMed Central

    Werstuck, Geoff H.; Lentz, Steven R.; Dayal, Sanjana; Hossain, Gazi S.; Sood, Sudesh K.; Shi, Yuan Y.; Zhou, Ji; Maeda, Nobuyo; Krisans, Skaidrite K.; Malinow, M. Rene; Austin, Richard C.

    2001-01-01

    Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine β-synthase. However, the mechanism by which homocysteine promotes the development and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regulatory element–binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intracellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by overexpression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the activation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secretion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia. PMID:11375416

  14. 1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway.

    PubMed

    Hirata, Kazumasa; Poeaknapo, Chotima; Schmidt, Juergen; Zenk, Meinhart H

    2004-04-01

    A synthase which oxidizes (S)-reticuline to 1,2-dehydroreticuline has been found to occur in seedlings of opium poppy (Papaver somniferum L.). Due to its instability, this enzyme could only be partly purified (ca. 5-fold enrichment). Partial characterization at this stage of purification showed that it does not need a redox cofactor and accepts both (S)-reticuline and (S)-norreticuline as substrates. [1-(2)H, (13)C]-(R,S)-reticuline was enzymatically converted into [1-(13)C]-dehydroreticuline, which has been identified by mass spectrometry. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion, was exploited as a sensitive assay system for this enzyme. The enzyme has a pH optimum of 8.75, a temperature optimum of 37 degrees C and the apparent K(M) value for the substrate reticuline was shown to be 117 microM. Moreover it could be demonstrated by sucrose density gradient centrifugation that the enzyme is located in vesicles of varying size. In combination with the previously discovered strictly stereoselective and NADPH dependent 1,2-dehydroreticuline reductase the detection of this enzyme, the 1,2-dehydroreticuline synthase, provides the necessary inversion of configuration and completes the pathway from two molecules of L-tyrosine via (S)-norcoclaurine to (R)-reticuline in opium poppy involving a total number of 11 enzymes.

  15. The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission.

    PubMed

    Vega-Rodríguez, Joel; Franke-Fayard, Blandine; Dinglasan, Rhoel R; Janse, Chris J; Pastrana-Mena, Rebecca; Waters, Andrew P; Coppens, Isabelle; Rodríguez-Orengo, José F; Srinivasan, Prakash; Jacobs-Lorena, Marcelo; Serrano, Adelfa E

    2009-02-01

    Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that gamma-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs(-) parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs(-) parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

  16. The biosynthetic pathway of 2-azahypoxanthine in fairy-ring forming fungus

    PubMed Central

    Suzuki, Tomohiro; Yamamoto, Naoki; Choi, Jae-Hoon; Takano, Tomoyuki; Sasaki, Yohei; Terashima, Yurika; Ito, Akinobu; Dohra, Hideo; Hirai, Hirofumi; Nakamura, Yukino; Yano, Kentaro; Kawagishi, Hirokazu

    2016-01-01

    “Fairy rings” resulting from fungus-stimulated plant growth occur all over the world. In 2010, 2-azahypoxanthine (AHX) from a fungus Lepista sordida was identified as the “fairy” that stimulates plant growth. Furthermore, 2-aza-8-oxohypoxanthine (AOH) was isolated as a common metabolite of AHX in plants, and the endogenous existence of AHX and AOH in plants was proved. The structure of AHX allowed us to hypothesize that AHX was derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Thus, we performed a feeding experiment that supplied AICAR to L. sordida. Consumption of AICAR and accumulation of AHX were observed after feeding. The mycelia extract had enzymatic activity of adenine/5-aminoimidazole-4-carboxamide phosphoribosyltransferase (APRT). APRT gene of L. sordida revealed its structural characteristics in homology modeling and showed transcriptional enhancement after feeding. These results support that AHX was synthesized from AICAR and AHX biosynthesis was transcriptionally controlled by AICAR, indicating the presence of novel purine metabolic pathway in L. sordida. PMID:27991529

  17. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.

    PubMed

    Guo, Juan; Ma, Xiaohui; Cai, Yuan; Ma, Ying; Zhan, Zhilai; Zhou, Yongjin J; Liu, Wujun; Guan, Mengxin; Yang, Jian; Cui, Guanghong; Kang, Liping; Yang, Lei; Shen, Ye; Tang, Jinfu; Lin, Huixin; Ma, Xiaojing; Jin, Baolong; Liu, Zhenming; Peters, Reuben J; Zhao, Zongbao K; Huang, Luqi

    2016-04-01

    Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  18. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones

    PubMed Central

    Guo, Juan; Ma, Xiaohui; Cai, Yuan; Ma, Ying; Zhan, Zhilai; Zhou, Yongjin J; Liu, Wujun; Guan, Mengxin; Yang, Jian; Cui, Guanghong; Kang, Liping; Yang, Lei; Shen, Ye; Tang, Jinfu; Lin, Huixin; Ma, Xiaojing; Jin, Baolong; Liu, Zhenming; Peters, Reuben J.; Zhao, Zongbao K.; Huang, Luqi

    2016-01-01

    Summary Cytochromes P450 (CYPs) play key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products.Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, that act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen.These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrate the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation.Our results highlight the incorporation of multiple CYPs in diterpenoids metabolic engineering, and a continuing trend of CYPs promiscuity generating complex networks in terpenoid biosynthesis. PMID:26682704

  19. 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.

  20. Tissue-Specific Whole Transcriptome Sequencing in Castor, Directed at Understanding Triacylglycerol Lipid Biosynthetic Pathways

    PubMed Central

    Swarbreck, David; Febrer, Melanie; Larson, Tony R.; Graham, Ian A.; Caccamo, Mario; Slabas, Antoni R.

    2012-01-01

    Background Storage triacylglycerols in castor bean seeds are enriched in the hydroxylated fatty acid ricinoleate. Extensive tissue-specific RNA-Seq transcriptome and lipid analysis will help identify components important for its biosynthesis. Methodology/Findings Storage triacylglycerols (TAGs) in the endosperm of developing castor (Ricinus communis) seeds are highly enriched in ricinoleic acid (18:1-OH). We have analysed neutral lipid fractions from other castor tissues using TLC, GLC and mass spectrometry. Cotyledons, like the endosperm, contain high levels of 18:1-OH in TAG. Pollen and male developing flowers accumulate TAG but do not contain 18:1-OH and leaves do not contain TAG or 18:1-OH. Analysis of acyl-CoAs in developing endosperm shows that ricinoleoyl-CoA is not the dominant acyl-CoA, indicating that either metabolic channelling or enzyme substrate selectivity are important in the synthesis of tri-ricinolein in this tissue. RNA-Seq transcriptomic analysis, using Illumina sequencing by synthesis technology, has been performed on mRNA isolated from two stages of developing seeds, germinating seeds, leaf and pollen-producing male flowers in order to identify differences in lipid-metabolic pathways and enzyme isoforms which could be important in the biosynthesis of TAG enriched in 18:1-OH. This study gives comprehensive coverage of gene expression in a variety of different castor tissues. The potential role of differentially expressed genes is discussed against a background of proteins identified in the endoplasmic reticulum, which is the site of TAG biosynthesis, and transgenic studies aimed at increasing the ricinoleic acid content of TAG. Conclusions/Significance Several of the genes identified in this tissue-specific whole transcriptome study have been used in transgenic plant research aimed at increasing the level of ricinoleic acid in TAG. New candidate genes have been identified which might further improve the level of ricinoleic acid in transgenic

  1. Reconstitution of the Costunolide Biosynthetic Pathway in Yeast and Nicotiana benthamiana

    PubMed Central

    Cankar, Katarina; Goedbloed, Miriam; Charnikhova, Tatsiana; Verstappen, Francel W. A.; de Vos, Ric C. H.; Beekwilder, Jules; van der Krol, Sander; Bouwmeester, Harro J.

    2011-01-01

    The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g−1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates. PMID:21858047

  2. Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining

    PubMed Central

    Millán-Aguiñaga, Natalie; Zhang, Jia Jia; O'Neill, Ellis C.; Ugalde, Juan A.; Jensen, Paul R.; Mantovani, Simone M.; Moore, Bradley S.

    2016-01-01

    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 co-localized 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 auto-resistance. 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. PMID:26458099

  3. Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli.

    PubMed

    Rossi, Elio; Motta, Sara; Aliverti, Alessandro; Cossu, Federica; Gourlay, Louise; Mauri, Pierluigi; Landini, Paolo

    2017-09-11

    Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Elucidation of Pseurotin Biosynthetic Pathway Points to Trans-Acting C-Methyltransferase and Source of Chemical Diversity Generation**

    PubMed Central

    Tsunematsu, Yuta; Fukutomi, Manami; Saruwatari, Takayoshi; Noguchi, Hiroshi; Watanabe, Kenji; Hotta, Kinya; Tang, Yi

    2015-01-01

    Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivity. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. Here, we systematically deleted the pseurotin biosynthetic genes in A. fumigatus and performed in vivo and in vitro characterization of the tailoring enzymes to determine the biosynthetic intermediates and the gene products responsible for the formation of each intermediate. This allowed us to elucidate the main biosynthetic steps leading to the formation of pseurotin A from the predominant precursor, azaspirene. The study revealed the combinatorial nature of the biosynthesis of the pseurotin family of compounds and the intermediates. Most interestingly, we report the first identification of an epoxidase–C-methyltransferase bifunctional fusion protein PsoF that appears to methylate the nascent polyketide backbone carbon atom in trans. PMID:24939566

  5. Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress.

    PubMed

    Teves, Franco; Lamas-Maceiras, Mónica; García-Estrada, Carlos; Casqueiro, Javier; Naranjo, Leopoldo; Ullán, Ricardo V; Scervino, José-Martín; Wu, Xiaobin; Velasco-Conde, Tania; Martín, Juan F

    2009-12-01

    The lysine biosynthetic pathway has to supply large amounts of alpha-aminoadipic acid for penicillin biosynthesis in Penicillium chrysogenum. In this study, we have characterized the P. chrysogenum L2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes (lys1, lys2, lys3, lys7). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by the Aspergillus nidulans lysF gene. We have cloned a gene (named lys3) that complements the L2 mutation by transformation with a P. chrysogenum genomic library, constructed in an autonomous replicating plasmid. The lys3-encoded protein showed high identity to homoaconitases. In addition, we cloned the mutant lys3 allele from the L2 strain that showed a G(1534) to A(1534) point mutation resulting in a Gly(495) to Asp(495) substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron-sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of the lys1 gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes.

  6. The Evolution of Oxygen As a Biosynthetic Reagent

    PubMed Central

    Goldfine, Howard

    1965-01-01

    The biosynthesis of certain cell constituents: monounsaturated fatty acids, tyrosine, and nicotinic acid, is oxygen-dependent in many higher organisms. The same compounds can be synthesized by different, oxygen-independent pathways in lower organisms. The general outlines of these pathways are described and the importance of the compounds synthesized is discussed. An examination of the distribution of these pathways among living organisms reveals that oxygen-dependent pathways replaced the "anaerobic" pathways at different branch points on the evolutionary tree. Other groups of compounds are discussed, which are not distributed as widely among living organisms, but are found in all higher organisms. These compounds have specialized functions and their biosynthesis requires molecular oxygen. The oxygen-dependent portions of the biosynthetic pathways leading to porphyrins, quinone coenzymes, carotenoids, sterols, and polyunsaturated fatty acids are summarized. The distribution and functions of these compounds are also considered and an attempt is made to place them in the framework of evolution. While sterols and polyunsaturated fatty acids are found exclusively in the higher Protista and multicellular organisms, carotenoids, porphyrins, and quinones are also found in bacteria. The possibility of oxygen-independent mechanisms for their biosynthesis is discussed. PMID:5859924

  7. Strigolactones, a novel carotenoid-derived plant hormone.

    PubMed

    Al-Babili, Salim; Bouwmeester, Harro J

    2015-01-01

    Strigolactones (SLs) are carotenoid-derived plant hormones and signaling molecules. When released into the soil, SLs indicate the presence of a host to symbiotic fungi and root parasitic plants. In planta, they regulate several developmental processes that adapt plant architecture to nutrient availability. Highly branched/tillered mutants in Arabidopsis, pea, and rice have enabled the identification of four SL biosynthetic enzymes: a cis/trans-carotene isomerase, two carotenoid cleavage dioxygenases, and a cytochrome P450 (MAX1). In vitro and in vivo enzyme assays and analysis of mutants have shown that the pathway involves a combination of new reactions leading to carlactone, which is converted by a rice MAX1 homolog into an SL parent molecule with a tricyclic lactone moiety. In this review, we focus on SL biosynthesis, describe the hormonal and environmental factors that determine this process, and discuss SL transport and downstream signaling as well as the role of SLs in regulating plant development.

  8. Hypoxia decreases the expression of the two enzymes responsible for producing linear and cyclic tetrapyrroles in the heme biosynthetic pathway.

    PubMed

    Vargas, Patrick D; Furuyama, Kazumichi; Sassa, Shigeru; Shibahara, Shigeki

    2008-12-01

    Heme is synthesized in all cell types in aerobic organisms. Hydroxymethylbilane synthase (HMBS) and uroporphyrinogen III synthase (UROS) catalyze two consecutive reactions in the heme biosynthetic pathway, generating the first linear and the first cyclic tetrapyrroles, respectively. Each of the HMBS and UROS genes contains the two separate promoters that generate ubiquitous and erythroid-specific mRNAs. Despite the functional significance of HMBS and UROS, regulation of their gene expression remains to be investigated. Here, we showed that hypoxia (1% O(2)) decreased the expression of ubiquitous mRNAs for HMBS and UROS by three- and twofold, respectively, in human hepatic cells (HepG2 and Hep3B), whereas the expression of ubiquitous and erythroid HMBS and UROS mRNAs remained unchanged in erythroid cells (YN-1 and K562). Unexpectedly, hypoxia did not decrease the half-life of HMBS mRNA (8.4 h under normoxia versus 9.1 h under hypoxia) or UROS mRNA (9.0 versus 10.4 h) in hepatic cells. It is therefore unlikely that a change in mRNA stability is responsible for the hypoxia-mediated decrease in the expression levels of these mRNAs. Furthermore, expression levels of HMBS and UROS mRNAs were decreased under normoxia by treatment with deferoxamine or cobalt chloride in hepatic cells, while hypoxia-inducible factor 1alpha was accumulated. Thus, the decrease in the expression of ubiquitous HMBS and UROS mRNAs is associated with accumulation of hypoxia-inducible factor 1alpha protein. In conclusion, the expression of HMBS and UROS mRNAs may be coordinately regulated, which represents a newly identified mechanism that is important for heme homeostasis.

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

    PubMed

    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

    2014-03-01

    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.

  10. 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

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

    SciTech Connect

    Nara, Takeshi; Hashimoto, Muneaki; Hirawake, Hiroko; Liao, Chien-Wei; Fukai, Yoshihisa; Suzuki, Shigeo; Tsubouchi, Akiko; Morales, Jorge; Takamiya, Shinzaburo; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Fan, Chia-Kwung; Inaoka, Daniel Ken; Inoue, Masayuki; Tanaka, Akiko; Harada, Shigeharu; Kita, Kiyoshi; and others

    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 fusion 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.

  12. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

    PubMed Central

    2010-01-01

    Background The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. Results We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. Conclusions This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change. PMID:20302676

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

    PubMed

    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

  14. 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

  15. Constitutively High Expression of the Histidine Biosynthetic Pathway Contributes to Nickel Tolerance in Hyperaccumulator PlantsW⃞

    PubMed Central

    Ingle, Robert A.; Mugford, Sam T.; Rees, Jonathan D.; Campbell, Malcolm M.; Smith, J. Andrew C.

    2005-01-01

    Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species. PMID:15923352

  16. Crystal Structure of a Sulfur Carrier Protein Complex Found in the Cysteine Biosynthetic Pathway of Mycobacterium tuberculosis

    SciTech Connect

    Jurgenson, Christopher T.; Burns, Kristin E.; Begley, Tadhg P.; Ealick, Steven E.

    2008-10-02

    The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 {angstrom} resolution. CysM (Rv1336) is a PLP-containing {beta}-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 {angstrom} resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.

  17. Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway.

    PubMed Central

    Bartley, G E; Viitanen, P V; Pecker, I; Chamovitz, D; Hirschberg, J; Scolnik, P A

    1991-01-01

    Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins. Images PMID:1862081

  18. Engineering Bacillus subtilis for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression.

    PubMed

    Li, Shanshan; Wen, Jianping; Jia, Xiaoqiang

    2011-08-01

    In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P(43) was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.

  19. High isoprenoid flux Escherichia coli as a host for carotenoids production.

    PubMed

    Suh, Wonchul

    2012-01-01

    A noncarotenogenic microbe E. coli was engineered for high production of carotenoids. To increase the isoprenoid flux, the chromosomal native promoters of the rate-controlling steps (dxs, idi and ispDispF) in the isoprenoid pathway were replaced with a strong bacteriophage T5 promoter (P(T5)) by using the λ-Red recombinase system in combination with the Flp/FRT site-specific recombination system for marker excision and P1 transduction for gene trait stacking. The resulting high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes. In this study, the high isoprenoid flux E. coli was transformed with a plasmid carrying the β-carotene biosynthetic genes from Pantoea stewartii for β-carotene production.

  20. Engineering the central biosynthetic and secondary metabolic pathways of Pseudomonas aeruginosa strain PA1201 to improve phenazine-1-carboxylic acid production.

    PubMed

    Jin, Kaiming; Zhou, Lian; Jiang, Haixia; Sun, Shuang; Fang, Yunling; Liu, Jianhua; Zhang, Xuehong; He, Ya-Wen

    2015-11-01

    The secondary metabolite phenazine-1-carboxylic acid (PCA) is an important component of the newly registered biopesticide Shenqinmycin. We used a combined method involving gene, promoter, and protein engineering to modify the central biosynthetic and secondary metabolic pathways in the PCA-producing Pseudomonas aeruginosa strain PA1201. The PCA yield of the resulting strain PA-IV was increased 54.6-fold via the following strategies: (1) blocking PCA conversion and enhancing PCA efflux pumping; (2) increasing metabolic flux towards the PCA biosynthetic pathway through the over-production of two DAHP synthases and blocking the synthesis of 21 secondary metabolites; (3) increasing the PCA precursor supply through the engineering of five chorismate-utilizing enzymes; (4) engineering the promoters of two PCA biosynthetic gene clusters. Strain PA-IV produced 9882 mg/L PCA in fed-batch fermentation, which is twice as much as that produced by the current industrial strain. Strain PA-IV was also genetically stable and comparable to Escherichia coli in cytotoxicity. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis

    SciTech Connect

    Rock, C.D.; Zeevaart, J.A.D. )

    1991-09-01

    The three mutant alleles of the ABA locus of Arabidopsis thaliana result in plants that are deficient in the plant growth regulator abscisic acid (ABA). The authors have used {sup 18}O{sub 2} to label ABA in water-stressed leaves of mutant and wild-type Arabidopsis. Analysis by selected ion monitoring and tandem mass spectrometry of ({sup 18}O)ABA and its catabolites, phaseic acid and ABA-glucose ester ({beta}-D-glucopyranosyl abscisate), indicates that the aba genotypes are impaired in ABA biosynthesis and have a small ABA precursor pool of compounds that contain oxygens on the rings, presumably oxygenated carotenoids (xanthophylls). Quantitation of the carotenoids form mutant and wild-type leaves establishes that the aba alleles cause a deficiency of the epoxy-carotenoids violaxanthin and neoxanthin and an accumulation of their biosynthetic precursor, zeaxanthin. These results provide evidence that ABA is synthesized by oxidative cleavage of epoxy-carotenoids (the indirect pathway). Furthermore the carotenoid mutant they describe undergoes normal greening. Thus the aba alleles provide an opportunity to study the physiological roles of epoxy-carotenoids in photosynthesis in a higher plants.

  2. Association Mapping of Total Carotenoids in Diverse Soybean Genotypes Based on Leaf Extracts and High-Throughput Canopy Spectral Reflectance Measurements.

    PubMed

    Dhanapal, Arun Prabhu; Ray, Jeffery D; Singh, Shardendu K; Hoyos-Villegas, Valerio; Smith, James R; Purcell, Larry C; King, C Andy; Fritschi, Felix B

    2015-01-01

    Carotenoids are organic pigments that are produced predominantly by photosynthetic organisms and provide antioxidant activity to a wide variety of plants, animals, bacteria, and fungi. The carotenoid biosynthetic pathway is highly conserved in plants and occurs mostly in chromoplasts and chloroplasts. Leaf carotenoids play important photoprotective roles and targeted selection for leaf carotenoids may offer avenues to improve abiotic stress tolerance. A collection of 332 soybean [Glycine max (L.) Merr.] genotypes was grown in two years and total leaf carotenoid content was determined using three different methods. The first method was based on extraction and spectrophotometric determination of carotenoid content (eCaro) in leaf tissue, whereas the other two methods were derived from high-throughput canopy spectral reflectance measurements using wavelet transformed reflectance spectra (tCaro) and a spectral reflectance index (iCaro). An association mapping approach was employed using 31,253 single nucleotide polymorphisms (SNPs) to identify SNPs associated with total carotenoid content using a mixed linear model based on data from two growing seasons. A total of 28 SNPs showed a significant association with total carotenoid content in at least one of the three approaches. These 28 SNPs likely tagged 14 putative loci for carotenoid content. Six putative loci were identified using eCaro, five loci with tCaro, and nine loci with iCaro. Three of these putative loci were detected by all three carotenoid determination methods. All but four putative loci were located near a known carotenoid-related gene. These results showed that carotenoid markers can be identified in soybean using extract-based as well as by high-throughput canopy spectral reflectance-based approaches, demonstrating the utility of field-based canopy spectral reflectance phenotypes for association mapping.

  3. Association Mapping of Total Carotenoids in Diverse Soybean Genotypes Based on Leaf Extracts and High-Throughput Canopy Spectral Reflectance Measurements

    PubMed Central

    Dhanapal, Arun Prabhu; Ray, Jeffery D.; Singh, Shardendu K.; Hoyos-Villegas, Valerio; Smith, James R.; Purcell, Larry C.; King, C. Andy; Fritschi, Felix B.

    2015-01-01

    Carotenoids are organic pigments that are produced predominantly by photosynthetic organisms and provide antioxidant activity to a wide variety of plants, animals, bacteria, and fungi. The carotenoid biosynthetic pathway is highly conserved in plants and occurs mostly in chromoplasts and chloroplasts. Leaf carotenoids play important photoprotective roles and targeted selection for leaf carotenoids may offer avenues to improve abiotic stress tolerance. A collection of 332 soybean [Glycine max (L.) Merr.] genotypes was grown in two years and total leaf carotenoid content was determined using three different methods. The first method was based on extraction and spectrophotometric determination of carotenoid content (eCaro) in leaf tissue, whereas the other two methods were derived from high-throughput canopy spectral reflectance measurements using wavelet transformed reflectance spectra (tCaro) and a spectral reflectance index (iCaro). An association mapping approach was employed using 31,253 single nucleotide polymorphisms (SNPs) to identify SNPs associated with total carotenoid content using a mixed linear model based on data from two growing seasons. A total of 28 SNPs showed a significant association with total carotenoid content in at least one of the three approaches. These 28 SNPs likely tagged 14 putative loci for carotenoid content. Six putative loci were identified using eCaro, five loci with tCaro, and nine loci with iCaro. Three of these putative loci were detected by all three carotenoid determination methods. All but four putative loci were located near a known carotenoid-related gene. These results showed that carotenoid markers can be identified in soybean using extract-based as well as by high-throughput canopy spectral reflectance-based approaches, demonstrating the utility of field-based canopy spectral reflectance phenotypes for association mapping. PMID:26368323

  4. Enhanced accumulation of carotenoids in sweetpotato plants overexpressing IbOr-Ins gene in purple-fleshed sweetpotato cultivar.

    PubMed

    Park, Sung-Chul; Kim, Sun Ha; Park, Seyeon; Lee, Hyeong-Un; Lee, Joon Seol; Park, Woo Sung; Ahn, Mi-Jeong; Kim, Yun-Hee; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

    2015-01-01

    Sweetpotato [Ipomoea batatas (L.) Lam] is an important root crop that produces low molecular weight antioxidants such as carotenoids and anthocyanin. The sweetpotato orange (IbOr) protein is involved in the accumulation of carotenoids. To increase the levels of carotenoids in the storage roots of sweetpotato, we generated transgenic sweetpotato plants overexpressing IbOr-Ins under the control of the cauliflower mosaic virus (CaMV) 35S promoter in an anthocyanin-rich purple-fleshed cultivar (referred to as IbOr plants). IbOr plants exhibited increased carotenoid levels (up to 7-fold) in their storage roots compared to wild type (WT) plants, as revealed by HPLC analysis. The carotenoid contents of IbOr plants were positively correlated with IbOr transcript levels. The levels of zeaxanthin were ∼ 12 times elevated in IbOr plants, whereas β-carotene increased ∼ 1.75 times higher than those of WT. Quantitative RT-PCR analysis revealed that most carotenoid biosynthetic pathway genes were up-regulated in the IbOr plants, including PDS, ZDS, LCY-β, CHY-β, ZEP and Pftf, whereas LCY-ɛ was down-regulated. Interestingly, CCD1, CCD4 and NCED, which are related to the degradation of carotenoids, were also up-regulated in the IbOr plants. Anthocyanin contents and transcription levels of associated biosynthetic genes seemed to be altered in the IbOr plants. The yields of storage roots and aerial parts of IbOr plants and WT plants were not significantly different under field cultivation. Taken together, these results indicate that overexpression of IbOr-Ins can increase the carotenoid contents of sweetpotato storage roots.

  5. A Genomics Approach Reveals That Aroma Production in Apple Is Controlled by Ethylene Predominantly at the Final Step in Each Biosynthetic Pathway[W

    PubMed Central

    Schaffer, Robert J.; Friel, Ellen N.; Souleyre, Edwige J.F.; Bolitho, Karen; Thodey, Kate; Ledger, Susan; Bowen, Judith H.; Ma, Jun-Hong; Nain, Bhawana; Cohen, Daniel; Gleave, Andrew P.; Crowhurst, Ross N.; Janssen, Bart J.; Yao, Jia-Long; Newcomb, Richard D.

    2007-01-01

    Ethylene is the major effector of ripening in many fleshy fruits. In apples (Malus x domestica) the addition of ethylene causes a climacteric burst of respiration, an increase in aroma, and softening of the flesh. We have generated a transgenic line of ‘Royal Gala’ apple that produces no detectable levels of ethylene using antisense ACC OXIDASE, resulting in apples with no ethylene-induced ripening attributes. In response to external ethylene these antisense fruits undergo a normal climacteric burst and produced increasing concentrations of ester, polypropanoid, and terpene volatile compounds over an 8-d period. A total of 186 candidate genes that might be involved in the production of these compounds were mined from expressed sequence tags databases and full sequence obtained. Expression patterns of 179 of these were assessed using a 15,720 oligonucleotide apple microarray. Based on sequence similarity and gene expression patterns we identified 17 candidate genes that are likely to be ethylene control points for aroma production in apple. While many of the biosynthetic steps in these pathways were represented by gene families containing two or more genes, expression patterns revealed that only a single member is typically regulated by ethylene. Only certain points within the aroma biosynthesis pathways were regulated by ethylene. Often the first step, and in all pathways the last steps, contained enzymes that were ethylene regulated. This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple. PMID:17556515

  6. Response differences between Ectocarpus siliculosus populations to copper stress involve cellular exclusion and induction of the phytochelatin biosynthetic pathway.

    PubMed

    Roncarati, Francesca; Sáez, Claudio A; Greco, Maria; Gledhill, Martha; Bitonti, Maria B; Brown, Murray T

    2015-02-01

    Some populations of brown seaweed species inhabit metal-polluted environments and can develop tolerance to metal stress, but the mechanisms by which this is accomplished are still to be elucidated. To address this, the responses of two strains of the model brown alga Ectocarpus siliculosus isolated from sites with different histories of metal contamination exposed to total copper (CuT) concentrations ranging between 0 and 2.4 μM for 10 days were investigated. The synthesis of the metal-chelator phytochelatin (PCs) and relative levels of transcripts encoding the enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione synthase (GS) and phytochelatin synthase (PCS) that participate in the PC biosynthetic pathway were measured, along with the effects on growth, and adsorption and uptake of Cu. Growth of strain LIA, from a pristine site in Scotland, was inhibited to a greater extent, and at lower concentrations, than that of Es524, isolated from a Cu-contaminated site in Chile. Concentrations of intra-cellular Cu were higher and the exchangeable fraction was lower in LIA than Es524, especially at the highest exposure levels. Total glutathione concentrations increased in both strains with Cu exposure, whereas total PCs levels were higher in Es524 than LIA; PC2 and PC3 were detected in Es524 but PC2 only was found in LIA. The greater production and levels of polymerisation of PCs in Es524 can be explained by the up-regulation of genes encoding for key enzymes involved in the synthesis of PCs. In Es524 there was an increase in the transcripts of γ-GCS, GS and PCS, particularly under high Cu exposure, whereas in LIA4 transcripts of γ-GCS1 increased only slightly, γ-GCS2 and GS decreased and PCS did not change. The consequences of higher intra-cellular concentrations of Cu, lower production of PCs, and lower expression of enzymes involved in GSH-PCs synthesis may be contributing to an induced oxidative stress condition in LIA, which explains, at least in part, the

  7. Molecular Link between Leaf Coloration and Gene Expression of Flavonoid and Carotenoid Biosynthesis in Camellia sinensis Cultivar 'Huangjinya'.

    PubMed

    Song, Lubin; Ma, Qingping; Zou, Zhongwei; Sun, Kang; Yao, Yuantao; Tao, Jihan; Kaleri, Najeeb A; Li, Xinghui

    2017-01-01

    'Huangjinya' is an excellent albino tea germplasm cultivated in China because of its bright color and high amino acid content. It is light sensitive, with yellow leaves under intense light while green leaves under weak light. As well, the flavonoid and carotenoid levels increased after moderate shading treatment. However, the mechanism underlying this interesting phenomenon remains unclear. In this study, the transcriptome of 'Huangjinya' plants exposed to sunlight and shade were analyzed by high-throughput sequencing followed by de novo assembly. Shading 'Huangjinya' made its leaf color turn green. De novo assembly showed that the transcriptome of 'Huangjinya' leaves comprises of 127,253 unigenes, with an average length of 914 nt. Among the 81,128 functionally annotated unigenes, 207 differentially expressed genes were identified, including 110 up-regulated and 97 down-regulated genes under moderate shading compared to full light. Gene ontology (GO) indicated that the differentially expressed genes are mainly involved in protein and ion binding and oxidoreductase activity. Antioxidation-related pathways, including flavonoid and carotenoid biosynthesis, were highly enriched in these functions. Shading inhibited the expression of flavonoid biosynthesis-associated genes and induced carotenoid biosynthesis-related genes. This would suggest that decreased flavonoid biosynthetic gene expression coincides with increased flavonoids (e.g., catechin) content upon moderate shading, while carotenoid levels and biosynthetic gene expression are positively correlated in 'Huangjinya.' In conclusion, the leaf color changes in 'Huangjinya' are largely determined by the combined effects of flavonoid and carotenoid biosynthesis.

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26960569

  9. 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.

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

    PubMed

    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

    2016-03-10

    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.

  11. Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production.

    PubMed

    Ravanello, Monica P; Ke, Dangyang; Alvarez, Julie; Huang, Bihua; Shewmaker, Christine K

    2003-10-01

    Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with

  12. A novel carotenoid, 4-keto-α-carotene, as an unexpected by-product during genetic engineering of carotenogenesis in rice callus.

    PubMed

    Breitenbach, Jürgen; Bai, Chao; Rivera, Sol M; Canela, Ramon; Capell, Teresa; Christou, Paul; Zhu, Changfu; Sandmann, Gerhard

    2014-02-01

    Rice endosperm is devoid of carotenoids because the initial biosynthetic steps are absent. The early carotenogenesis reactions were constituted through co-transformation of endosperm-derived rice callus with phytoene synthase and phytoene desaturase transgenes. Subsequent steps in the pathway such as cyclization and hydroxylation reactions were catalyzed by endogenous rice enzymes in the endosperm. The carotenoid pathway was extended further by including a bacterial ketolase gene able to form astaxanthin, a high value carotenoid which is not a typical plant carotenoid. In addition to astaxanthin and precursors, a carotenoid accumulated in the transgenic callus which did not fit into the pathway to astaxanthin. This was subsequently identified as 4-keto-α-carotene by HPLC co-chromatography, chemical modification, mass spectrometry and the reconstruction of its biosynthesis pathway in Escherichia coli. We postulate that this keto carotenoid is formed from α-carotene which accumulates by combined reactions of the heterologous gene products and endogenous rice endosperm cyclization reactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Overexpression of halophilic serine hydroxymethyltransferase in fresh water cyanobacterium Synechococcus elongatus PCC7942 results in increased enzyme activities of serine biosynthetic pathways and enhanced salinity tolerance.

    PubMed

    Waditee-Sirisattha, Rungaroon; Kageyama, Hakuto; Tanaka, Yoshito; Fukaya, Minoru; Takabe, Teruhiro

    2017-01-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the conversion of serine to glycine and provides activated one-carbon units required for synthesis of nucleic acids, proteins and numerous biological compounds. SHMT is involved in photorespiratory pathway of oxygenic photosynthetic organisms. Accumulating evidence revealed that SHMT plays vital role for abiotic stresses such as low CO2 and high salinity in plants, but its role in cyanobacteria remains to be clarified. In this study, we examined to overexpress the SHMT from halotolerant cyanobacterium Aphanothece halophytica in freshwater cyanobacterium, Synechococcus elongatus PCC7942. The transformed cells did not show an obvious phenotype under non-stress condition, but exhibited more tolerance to salinity than the control cells harboring vector only under high salinity. Elevated levels of enzymes in phosphorylated serine biosynthetic pathway and photorespiration pathway were observed in the transformed cells. Glycine level was also increased in the transformed cells. Physiological roles of SHMT for salt tolerance were discussed.

  14. The carotenoid pathway: what is important for excitation quenching in plant antenna complexes?

    PubMed

    Fox, Kieran F; Balevičius, Vytautas; Chmeliov, Jevgenij; Valkunas, Leonas; Ruban, Alexander V; Duffy, Christopher D P

    2017-08-30

    Plant light-harvesting is regulated by the Non-Photochemical Quenching (NPQ) mechanism involving the reversible formation of excitation quenching sites in the Photosystem II (PSII) antenna in response to high light. While the major antenna complex, LHCII, is known to be a site of NPQ, the precise mechanism of excitation quenching is not clearly understood. A preliminary model of the quenched crystal structure of LHCII implied that quenching arises from slow energy capture by Car pigments. It predicted a thoroughly quenched system but offered little insight into the defining aspects of this quenching. In this work, we present a thorough theoretical investigation of this quenching, addressing the factors defining the quenching pathway and possible mechanism for its (de)activation. We show that quenching in LHCII crystals is the result of slow energy transfer from chlorophyll to the centrally-bound lutein Cars, predominantly the Lut620 associated with the chlorophyll 'terminal emitter', one of the proposed in vivo pathways. We show that this quenching is rather independent of the particular species of Car and excitation 'site' energy. The defining parameter is the resonant coupling between the pigment co-factors. Lastly, we show that these interactions must be severely suppressed for a light-harvesting state to be recovered.

  15. Structure and Functional Analysis of RifR, the Type II Thioesterase from the Rifamycin Biosynthetic Pathway

    SciTech Connect

    Claxton, Heather B.; Akey, David L.; Silver, Monica K.; Admiraal, Suzanne J.; Smith, Janet L.

    2009-03-16

    Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR.

  16. Reversible naftifine-induced carotenoid depigmentation in Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison causing onychomycosis.

    PubMed

    Moț, Augustin C; Pârvu, Marcel; Pârvu, Alina E; Roşca-Casian, Oana; Dina, Nicoleta E; Leopold, Nicolae; Silaghi-Dumitrescu, Radu; Mircea, Cristina

    2017-09-11

    Rhodotorula mucilaginosa was isolated from a patient with onychomycosis, and identification was confirmed by morphological and cultural characteristics as well as by DNA molecular analysis. Antifungal agents naftifine (10 mg/mL, active substance in Exoderil) and bifonazole (10 mg/mL, active substance in Canespor) were tested in different concentrations to assess in vitro effects on fungal growth and carotenoid synthesis. The antifungal mechanisms of action of naftifine and bifonazole against R. mucilaginosa isolates were similar and affected the biosynthetic pathway of ergosterol. For the first time, this research demonstrates that naftifine affects the carotenoid biosynthetic pathway, producing depigmentation of R. mucilaginosa in solid and liquid media. Furthermore, depigmentation was a reversible process; naftifine-treated yeast cells that were depigmented resumed carotenoid production upon transfer to fresh media. Raman and UV-vis spectrophotometry in conjunction with chromatographic analysis detected changes in carotenoids in yeast cells, with torulene decreasing and B-carotene increasing after repigmentation. Transmission electron micrographs revealed critical ultrastructural modifications in the depigmented cells after naftifine treatment, i.e., a low-electron-density cell wall without visible mucilage or lamellate structure.

  17. Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels