Science.gov

Sample records for carotenoid biosynthetic pathway

  1. Carotenoid Biosynthetic and Catabolic Pathways: Gene Expression and Carotenoid Content in Grains of Maize Landraces

    PubMed Central

    Messias, Rafael da Silva; Galli, Vanessa; Silva, Sérgio Delmar dos Anjos e; Rombaldi, Cesar Valmor

    2014-01-01

    Plant carotenoids have been implicated in preventing several age-related diseases, and they also provide vitamin A precursors; therefore, increasing the content of carotenoids in maize grains is of great interest. It is not well understood, however, how the carotenoid biosynthetic pathway is regulated. Fortunately, the maize germplasm exhibits a high degree of genetic diversity that can be exploited for this purpose. Here, the accumulation of carotenoids and the expression of genes from carotenoid metabolic and catabolic pathways were investigated in several maize landraces. The carotenoid content in grains varied from 10.03, in the white variety MC5, to 61.50 μg·g−1, in the yellow-to-orange variety MC3, and the major carotenoids detected were lutein and zeaxanthin. PSY1 (phythoene synthase) expression showed a positive correlation with the total carotenoid content. Additionally, the PSY1 and HYD3 (ferredoxin-dependent di-iron monooxygenase) expression levels were positively correlated with β-cryptoxanthin and zeaxanthin, while CYP97C (cytochrome P450-type monooxygenase) expression did not correlate with any of the carotenoids. In contrast, ZmCCD1 (carotenoid dioxygenase) was more highly expressed at the beginning of grain development, as well as in the white variety, and its expression was inversely correlated with the accumulation of several carotenoids, suggesting that CCD1 is also an important enzyme to be considered when attempting to improve the carotenoid content in maize. The MC27 and MC1 varieties showed the highest HYD3/CYP97C ratios, suggesting that they are promising candidates for increasing the zeaxanthin content; in contrast, MC14 and MC7 showed low HYD3/CYP97C, suggesting that they may be useful in biofortification efforts aimed at promoting the accumulation of provitamin A. The results of this study demonstrate the use of maize germplasm to provide insight into the regulation of genes involved in the carotenoid pathway, which would thus better

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  3. 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

  4. 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. PMID:20802040

  5. 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

  6. Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

    PubMed

    Harrison, Peter J; Newgas, Sophie A; Descombes, Flora; Shepherd, Sarah A; Thompson, Andrew J; Bugg, Timothy D H

    2015-10-01

    The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8.

  7. Biochemical characterization and selective inhibition of β-carotene cis-trans isomerase D27 and carotenoid cleavage dioxygenase CCD8 on the strigolactone biosynthetic pathway.

    PubMed

    Harrison, Peter J; Newgas, Sophie A; Descombes, Flora; Shepherd, Sarah A; Thompson, Andrew J; Bugg, Timothy D H

    2015-10-01

    The first three enzymatic steps of the strigolactone biosynthetic pathway catalysed by β-carotene cis-trans isomerase Dwarf27 (D27) from Oryza sativa and carotenoid cleavage dioxygenases CCD7 and CCD8 from Arabidopsis thaliana have been reconstituted in vitro, and kinetic assays have been developed for each enzyme, in order to develop selective enzyme inhibitors. Recombinant OsD27 shows a UV-visible λmax at 422 nm and is inactivated by silver(I) acetate, consistent with the presence of an iron-sulfur cluster that is used in catalysis. OsD27 and AtCCD7 are not inhibited by hydroxamic acids that cause shoot branching in planta, but OsD27 is partially inhibited by terpene-like hydroxamic acids. The reaction catalysed by AtCCD8 is shown to be a two-step kinetic mechanism using pre-steady-state kinetic analysis. Kinetic evidence is presented for acid-base catalysis in the CCD8 catalytic cycle and the existence of an essential cysteine residue in the CCD8 active site. AtCCD8 is inhibited in a time-dependent fashion by hydroxamic acids D2, D4, D5 and D6 (> 95% inhibition at 100 μm) that cause a shoot branching phenotype in A. thaliana, and selective inhibition of CCD8 is observed using hydroxamic acids D13H and D15 (82%, 71% inhibition at 10 μm). The enzyme inhibition data imply that the biochemical basis of the shoot branching phenotype is due to inhibition of CCD8. PMID:26257333

  8. Apocarotenoids: A New Carotenoid-Derived Pathway.

    PubMed

    Beltran, Juan Camilo Moreno; Stange, Claudia

    2016-01-01

    Carotenoids are precursors of carotenoid derived molecules termed apocarotenoids, which include isoprenoids with important functions in plant-environment interactions such as the attraction of pollinators and the defense against pathogens and herbivores. Apocarotenoids also include volatile aromatic compounds that act as repellents, chemoattractants, growth simulators and inhibitors, as well as the phytohormones abscisic acid and strigolactones. In plants, apocarotenoids can be found in several types of plastids (etioplast, leucoplast and chromoplast) and among different plant tissues such as flowers and roots. The structural similarity of some flower and spice isoprenoid volatile organic compounds (β-ionone and safranal) to carotenoids has led to the recent discovery of carotenoid-specific cleavage oxygenases, including carotenoid cleavage dioxygenases and 9-cis-epoxydioxygenases, which tailor and transform carotenoids into apocarotenoids. The great diversity of apocarotenoids is a consequence of the huge amount of carotenoid precursors, the variations in specific cleavage sites and the modifications after cleavage. Lycopene, β-carotene and zeaxanthin are the precursors of the main apocarotenoids described to date, which include bixin, crocin, picrocrocin, abscisic acid, strigolactone and mycorradicin.The current chapter will give rise to an overview of the biosynthesis and function of the most important apocarotenoids in plants, as well as the current knowledge about the carotenoid cleavage oxygenase enzymes involved in these biosynthetic pathways. PMID:27485225

  9. 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.

  10. Molecular characterization of carotenoid cleavage dioxygenases and the effect of gibberellin, abscisic acid, and sodium chloride on the expression of genes involved in the carotenoid biosynthetic pathway and carotenoid accumulation in the callus of Scutellaria baicalensis Georgi.

    PubMed

    Tuan, Pham Anh; Kim, Jae Kwang; Lee, Sanghyun; Chae, Soo Cheon; Park, Sang Un

    2013-06-12

    Three cDNAs encoding carotenoid cleavage dioxygenases (SbCCD1, SbCCD4, and SbNCED) were isolated from Scutellaria baicalensis , an important traditional herb in Asia and North America. Amino acid sequence alignments showed that they share high identity and similarity to their orthologs in other plant species. Quantitative real-time polymerase chain reaction analysis revealed that SbCCD1 and SbCCD4 were most strongly expressed in flowers, whereas SbNCED was expressed at the highest level in roots. The expression levels of phytoene synthase (SbPSY), phytoene desaturase (SbPDS), ξ-carotene desaturase (SbZDS), β-ring carotene hydroxylase (SbCHXB), zeaxanthin epoxidase (SbZEP), SbCCD1, SbCCD4, and SbNCED in the callus of S. baicalensis varied under different concentrations of gibberellic acid (GA3) and abscisic acid (ABA). Under NaCl treatment, expression levels of all genes increased with increasing NaCl concentrations. Except for zeaxanthin, increasing GA3, ABA, and NaCl concentrations caused higher losses in the total carotenoid content. The total carotenoid content substantially decreased with increasing GA3, ABA, and NaCl concentrations, with the biggest reductions observed in the NaCl treatment. The isolation and characterization of SbCCD1, SbCCD4, and SbNCED together with the study on the effect of GA3, ABA, and NaCl on carotenoid biosynthesis will be helpful to elucidate the carotenoid biosynthesis mechanism in S. baicalensis and may set new trends in metabolic engineering of carotenoids in plants. PMID:23683071

  11. The Biosynthetic Pathway for Synechoxanthin, an Aromatic Carotenoid Synthesized by the Euryhaline, Unicellular Cyanobacterium Synechococcus sp. Strain PCC 7002▿ †

    PubMed Central

    Graham, Joel E.; Bryant, Donald A.

    2008-01-01

    The euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (χ,χ-caroten-18,18′-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (χ,χ-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in Synechococcus sp. strain PCC 7002 to verify their function. The cruE gene (SYNPCC7002_A1248) encodes β-carotene desaturase/methyltransferase, which converts β-carotene to renierapurpurin. The cruH gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18′ methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed. PMID:18849428

  12. Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges.

    PubMed

    Wei, Xu; Chen, Chunxian; Yu, Qibin; Gady, Antoine; Yu, Yuan; Liang, Guolu; Gmitter, Frederick G

    2014-10-01

    Carotenoid accumulation and biosynthetic gene expression levels during fruit maturation were compared between ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV). The two cultivars exhibited different carotenoid profiles and regulatory mechanisms in flavedo and juice sacs, respectively. In flavedo, there was uncoordinated carotenoid accumulation and gene expression in RRV during green stages, which might be related to the expression of certain gene(s) in the MEP (methylerythritol phosphate) pathway. The carotenoid biosynthesis pathway shifting from α,β-xanthophylls to β,β-xanthophylls synthesis occurred in RRV earlier than VAL during orange stages. In juice sacs, the low carotenoid content in both cultivars coincided with low expression of LCYE-Contig03 and LCYE-Contig24 during green stages, suggesting LCYE might be a limiting step for carotenoid accumulation. VAL mainly accumulated violaxanthin, but RRV accumulated β-cryptoxanthin and violaxanthin during orange stages, which corresponded to differences in juice color. Several upstream genes (PDS-Contig17, LCYB-Contig19, and ZDS members) and a downstream gene (ZEP) were expressed at higher levels in RRV than VAL, which might be responsible for greater accumulation of β-cryptoxanthin and violaxanthin in RRV, respectively.

  13. Molecular characterization of carotenoid biosynthetic genes and carotenoid accumulation in Lycium chinense.

    PubMed

    Zhao, Shicheng; Tuan, Pham Anh; Kim, Jae Kwang; Park, Woo Tae; Kim, Yeon Bok; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Yang, Jingli; Li, Cheng Hao; Park, Sang Un

    2014-01-01

    Lycium chinense is a shrub that has health benefits and is used as a source of medicines in Asia. In this study, a full-length cDNA clone encoding β-ring carotene hydroxylase (LcCHXB) and partial-length cDNA clones encoding phytoene synthase (LcPSY), phytoene desaturase (LcPDS), ξ-carotene desaturase (LcZDS), lycopene β-cyclase (LcLCYB), lycopene ε-cyclase (LcLCYE), ε-ring carotene hydroxylase (LcCHXE), zeaxanthin epoxidase (LcZEP), carotenoid cleavage dioxygenase (LcCCD1), and 9-cis epoxycarotenoid dioxygenase (LcNCED) were identified in L. chinense. The transcripts were constitutively expressed at high levels in leaves, flowers and red fruits, where the carotenoids are mostly distributed. In contrast, most of the carotenoid biosynthetic genes were weakly expressed in the roots and stems, which contained only small amounts of carotenoids. The level of LcLCYE transcripts was very high in leaves and correlated with the abundance of lutein in this plant tissue. During maturation, the levels of lutein and zeaxanthin in L. chinense fruits dramatically increased, concomitant with a rise in the level of β-cryptoxanthin. LcPSY, LcPDS, LcZDS, LcLCYB, and LcCHXE were highly expressed in red fruits, leading to their substantially higher total carotenoid content compared to that in green fruits. Total carotenoid content was high in both the leaves and red fruits of L. chinense. Our findings on the biosynthesis of carotenoids in L. chinense provide insights into the molecular mechanisms involved in carotenoid biosynthesis and may facilitate the optimization of carotenoid production in L. chinense. PMID:25090116

  14. 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

  15. 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. PMID:23144136

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. Molecular characterization of carotenoid biosynthetic genes and carotenoid accumulation in Scutellaria baicalensis Georgi

    PubMed Central

    Tuan, Pham Anh; Kim, Yeon Bok; Kim, Jae Kwang; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Sang Un

    2014-01-01

    Scutellaria baicalensis has a wide range of biological activities and has been considered as an important traditional drug in Asia and North America for centuries. A partial-length cDNA clone encoding phytoene synthase (SbPSY) and full-length cDNA clonesencoding phytoene desaturase (SbPDS), ξ-carotene desaturase (SbZDS), β-ring carotene hydroxylase (SbCHXB), and zeaxanthin epoxidase (SbZEP)were identifiedin S. baicalensis. Sequence analyses revealed that these proteins share high identity and conserved domains with their orthologous genes. SbPSY, SbPDS, SbZDS, SbCHXB, and SbZEP were constitutively expressed in the roots, stems, leaves, and flowers of S.baicalensis. SbPSY, SbPDS, and SbZDS were highly expressed in the stems, leaves, and flowers and showed low expression in the roots, where only trace amounts of carotenoids were detected. SbCHXB and SbZEP transcripts were expressed at relatively high levels in the roots, stems, and flowers and were expressed at low levels in the leaves, where carotenoids were mostly distributed. The predominant carotenoids in S.baicalensiswere lutein and β-carotene, with abundant amounts found in the leaves (517.19 and 228.37 μg g-1 dry weight, respectively). Our study on the biosynthesis of carotenoids in S. baicalensis will provide basic data for elucidating the contribution of carotenoids to the considerable medicinal properties of S. baicalensis. PMID:26417348

  2. Biosynthetic route towards saxitoxin and shunt pathway.

    PubMed

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

    2016-02-04

    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 (15)N-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.

  3. 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

  4. 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.

  5. Evolution-guided optimization of biosynthetic pathways

    PubMed Central

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

    2014-01-01

    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 ∼109 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. PMID:25453111

  6. 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

  7. The biosynthetic pathway for myxol-2' fucoside (myxoxanthophyll) in the cyanobacterium Synechococcus sp. strain PCC 7002.

    PubMed

    Graham, Joel E; Bryant, Donald A

    2009-05-01

    Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic beta-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2' fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2'-OH rather than on the 1'-OH position of the psi end of the molecule. In this study, the genes encoding two enzymes that modify the psi end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1',2'-hydroxylase [corrected] and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2'-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.

  8. The Neurospora crassa carotenoid biosynthetic gene (albino 3) reveals highly conserved regions among prenyltransferases.

    PubMed

    Carattoli, A; Romano, N; Ballario, P; Morelli, G; Macino, G

    1991-03-25

    In the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light. Here we report the characterization of the albino-3 (al-3) gene of N. crassa, which encodes the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthetase. This is the first geranylgeranyl-pyrophosphate synthetase gene isolated. Nucleotide sequence comparison of al-3 genomic and cDNA clones revealed that the al-3 gene is not interrupted by introns. Transcription of the al-3 gene has been examined in dark-grown and light-induced mycelia. The analysis revealed that the al-3 gene is not expressed in the dark and that its transcription is induced by blue light (Nelson, M. A., Morelli, G., Carattoli, A., Romano, N., and Macino, G. (1989) Mol. Cell. Biol. 9, 1271-1276). The al-3 gene encodes a polypeptide of 428 amino acids. Comparison of the deduced amino acid sequence of al-3 with the sequences of prenyltransferases of other species, from bacteria to humans, showed three highly conserved homologous regions. These homologous regions may be involved in the formation of the catalytic site of the prenyltransferases.

  9. Coordinated transcriptional regulation of isopentenyl diphosphate biosynthetic pathway enzymes in plastids by phytochrome-interacting factor 5.

    PubMed

    Mannen, Kazuto; Matsumoto, Takuro; Takahashi, Seiji; Yamaguchi, Yuta; Tsukagoshi, Masanori; Sano, Ryosuke; Suzuki, Hideyuki; Sakurai, Nozomu; Shibata, Daisuke; Koyama, Tanetoshi; Nakayama, Toru

    2014-01-10

    All isoprenoids are derived from a common C5 unit, isopentenyl diphosphate (IPP). In plants, IPP is synthesized via two distinct pathways; the cytosolic mevalonate pathway and the plastidial non-mevalonate (MEP) pathway. In this study, we used a co-expression analysis to identify transcription factors that coordinately regulate the expression of multiple genes encoding enzymes in the IPP biosynthetic pathway. Some candidates showed especially strong correlations with multiple genes encoding MEP-pathway enzymes. We report here that phytochrome-interacting factor 5 (PIF5), a basic-helix-loop-helix type transcription factor, functions as a positive regulator of the MEP pathway. Its overexpression in T87 suspension cultured cells resulted in increased accumulation of chlorophylls and carotenoids. Detailed analyses of carotenoids by HPLC indicated that some carotenoid biosynthetic pathways were concomitantly up-regulated, possibly as a result of enhanced IPP metabolic flow. Our results also revealed other PIF family proteins that play different roles from that of PIF5 in IPP metabolism.

  10. 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.

  11. 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

    2016-03-01

    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

  12. 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

  13. Dysfunction of the cholesterol biosynthetic pathway in Huntington's disease.

    PubMed

    Valenza, Marta; Rigamonti, Dorotea; Goffredo, Donato; Zuccato, Chiara; Fenu, Simone; Jamot, Laure; Strand, Andrew; Tarditi, Alessia; Woodman, Ben; Racchi, Marco; Mariotti, Caterina; Di Donato, Stefano; Corsini, Alberto; Bates, Gillian; Pruss, Rebecca; Olson, James M; Sipione, Simonetta; Tartari, Marzia; Cattaneo, Elena

    2005-10-26

    The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntington's disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an approximately 50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dose-dependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker. PMID:16251441

  14. 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. PMID:17576425

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

    PubMed

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

    2016-01-01

    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. PMID:27548127

  16. The biosynthetic pathway of vitamin C in higher plants.

    PubMed

    Wheeler, G L; Jones, M A; Smirnoff, N

    1998-05-28

    Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

  17. 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

  18. Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast.

    PubMed

    Tang, Man-Cheng; Lin, Hsiao-Ching; Li, Dehai; Zou, Yi; Li, Jian; Xu, Wei; Cacho, Ralph A; Hillenmeyer, Maureen E; Garg, Neil K; Tang, Yi

    2015-11-01

    The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here, using Saccharomyces cerevisiae as a heterologous host and a phylogenetically guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologues identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavinine and anominine and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.

  19. Carotenoids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carotenoids are lipophilic plant pigments with polyisoprenoid structures that occur naturally in plants and other photosynthetic organisms. There are over 600 known carotenoids with chemical structures characterized by a large (35-40 carbon atoms) conjugated polyene chain, sometimes terminated by ri...

  20. 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.

  1. Evolution of the Isoprene Biosynthetic Pathway in Kudzu1[w

    PubMed Central

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

    2005-01-01

    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. PMID:15653811

  2. 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.

  3. Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a.

    PubMed

    Ye, Rick W; Yao, Henry; Stead, Kristen; Wang, Tao; Tao, Luan; Cheng, Qiong; Sharpe, Pamela L; Suh, Wonchul; Nagel, Eva; Arcilla, Dennis; Dragotta, Dominic; Miller, Edward S

    2007-04-01

    Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the beta-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g(-1) of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.

  4. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

    PubMed Central

    Marx, Hans; Mattanovich, Diethard; Sauer, Michael

    2008-01-01

    Background High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. Results Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1) is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) increases the riboflavin accumulation significantly. Conclusion The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established. PMID:18664246

  5. 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.

  6. 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

  7. 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. PMID:24389954

  8. 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

  9. 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. PMID:22488437

  10. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. Heterologous expression of natural product biosynthetic gene clusters in Streptomyces coelicolor: from genome mining to manipulation of biosynthetic pathways.

    PubMed

    Gomez-Escribano, Juan Pablo; Bibb, Mervyn J

    2014-02-01

    Heterologous gene expression is one of the main strategies used to access the full biosynthetic potential of actinomycetes, as well as to study the metabolic pathways of natural product biosynthesis and to create unnatural pathways. Streptomyces coelicolor A3(2) is the most studied member of the actinomycetes, bacteria renowned for their prolific capacity to synthesize a wide range of biologically active specialized metabolites. We review here the use of strains of this species for the heterologous production of structurally diverse actinomycete natural products.

  13. Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts.

    PubMed

    Sato, Naoki; Awai, Koichiro

    2016-01-01

    Cyanobacteria and chloroplasts perform oxygenic photosynthesis, and share a common origin. Galactolipids are present in the photosynthetic membranes of both cyanobacteria and chloroplasts, but the biosynthetic pathways of the galactolipids are significantly different in the two systems. In this minireview, we explain the history of the discovery of the cyanobacterial pathway, and present a probable scenario of the evolution of the two pathways.

  14. 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.

  15. Nutrient shortage triggers the hexosamine biosynthetic pathway via the GCN2-ATF4 signalling pathway.

    PubMed

    Chaveroux, Cédric; Sarcinelli, Carmen; Barbet, Virginie; Belfeki, Sofiane; Barthelaix, Audrey; Ferraro-Peyret, Carole; Lebecque, Serge; Renno, Toufic; Bruhat, Alain; Fafournoux, Pierre; Manié, Serge N

    2016-06-03

    The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked β-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling.

  16. Nutrient shortage triggers the hexosamine biosynthetic pathway via the GCN2-ATF4 signalling pathway

    PubMed Central

    Chaveroux, Cédric; Sarcinelli, Carmen; Barbet, Virginie; Belfeki, Sofiane; Barthelaix, Audrey; Ferraro-Peyret, Carole; Lebecque, Serge; Renno, Toufic; Bruhat, Alain; Fafournoux, Pierre; Manié, Serge N.

    2016-01-01

    The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked β-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling. PMID:27255611

  17. 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.

  18. 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].

  19. 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

  20. Aerobic conditions increase isoprenoid biosynthesis pathway gene expression levels for carotenoid production in Enterococcus gilvus.

    PubMed

    Hagi, Tatsuro; Kobayashi, Miho; Nomura, Masaru

    2015-06-01

    Some lactic acid bacteria that harbour carotenoid biosynthesis genes (crtNM) can produce carotenoids. Although aerobic conditions can increase carotenoid production and crtNM expression levels, their effects on the pathways that synthesize carotenoid precursors such as mevalonate and isoprene are not completely understood. In this study, we investigated whether aerobic conditions affected gene expression levels involved in the isoprenoid biosynthesis pathway that includes the mevalonate and isoprene biosynthesis pathways in Enterococcus gilvus using real-time quantitative reverse transcription PCR. NADH oxidase (nox) and superoxide dismutase (sod) gene expression levels were investigated as controls for aerobic conditions. The expression levels of nox and sod under aerobic conditions were 7.2- and 8.0-fold higher, respectively, than those under anaerobic conditions. Aerobic conditions concomitantly increased the expression levels of crtNM carotenoid biosynthesis genes. HMG-CoA synthase gene expression levels in the mevalonate pathway were only slightly increased under aerobic conditions, whereas the expression levels of HMG-CoA reductase and five other genes in the isoprene biosynthesis pathways were 1.2-2.3-fold higher than those under anaerobic conditions. These results demonstrated that aerobic conditions could increase the expression levels of genes involved in the isoprenoid biosynthesis pathway via mevalonate in E. gilvus.

  1. Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

    PubMed Central

    Magarvey, Nathan A.; Haltli, Brad; He, Min; Greenstein, Michael; Hucul, John A.

    2006-01-01

    The mannopeptimycins are a novel class of lipoglycopeptide antibiotics active against multidrug-resistant pathogens with potential as clinically useful antibacterials. This report is the first to describe the biosynthesis of this novel class of mannosylated lipoglycopeptides. Included here are the cloning, sequencing, annotation, and manipulation of the mannopeptimycin biosynthetic gene cluster from Streptomyces hygroscopicus NRRL 30439. Encoded by genes within the mannopeptimycin biosynthetic gene cluster are enzymes responsible for the generation of the hexapeptide core (nonribosomal peptide synthetases [NRPS]) and tailoring reactions (mannosylation, isovalerylation, hydroxylation, and methylation). The NRPS system is noncanonical in that it has six modules utilizing only five amino acid-specific adenylation domains and it lacks a prototypical NRPS macrocyclizing thioesterase domain. Analysis of the mannopeptimycin gene cluster and its engineering has elucidated the mannopeptimycin biosynthetic pathway and provides the framework to make new and improved mannopeptimycins biosynthetically. PMID:16723579

  2. BCDO2 acts as a carotenoid scavenger and gatekeeper for the mitochondrial apoptotic pathway

    PubMed Central

    Lobo, Glenn P.; Isken, Andrea; Hoff, Sylvia; Babino, Darwin; von Lintig, Johannes

    2012-01-01

    Carotenoids and their metabolites are widespread and exert key biological functions in living organisms. In vertebrates, the carotenoid oxygenase BCMO1 converts carotenoids such as β,β-carotene to retinoids, which are required for embryonic pattern formation and cell differentiation. Vertebrate genomes encode a structurally related protein named BCDO2 but its physiological function remains undefined. Here, we show that BCDO2 is expressed as an oxidative stress-regulated protein during zebrafish development. Targeted knockdown of this mitochondrial enzyme resulted in anemia at larval stages. Marker gene analysis and staining for hemoglobin revealed that erythropoiesis was not impaired but that erythrocytes underwent apoptosis in BCDO2-deficient larvae. To define the mechanism of this defect, we have analyzed the role of BCDO2 in human cell lines. We found that carotenoids caused oxidative stress in mitochondria that eventually led to cytochrome c release, proteolytic activation of caspase 3 and PARP1, and execution of the apoptotic pathway. Moreover, BCDO2 prevented this induction of the apoptotic pathway by carotenoids. Thus, our study identifying BCDO2 as a crucial protective component against oxidative stress establishes this enzyme as mitochondrial carotenoid scavenger and a gatekeeper of the intrinsic apoptotic pathway. PMID:22764054

  3. 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.

  4. 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

  5. Diversity in Biosynthetic Pathways of Galactolipids in the Light of Endosymbiotic Origin of Chloroplasts

    PubMed Central

    Sato, Naoki; Awai, Koichiro

    2016-01-01

    Cyanobacteria and chloroplasts perform oxygenic photosynthesis, and share a common origin. Galactolipids are present in the photosynthetic membranes of both cyanobacteria and chloroplasts, but the biosynthetic pathways of the galactolipids are significantly different in the two systems. In this minireview, we explain the history of the discovery of the cyanobacterial pathway, and present a probable scenario of the evolution of the two pathways. PMID:26904079

  6. 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

  7. Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

    PubMed

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

    2011-04-12

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

  8. Retention and loss of amino acid biosynthetic pathways based on analysis of whole-genome sequences.

    PubMed

    Payne, Samuel H; Loomis, William F

    2006-02-01

    Plants and fungi can synthesize each of the 20 amino acids by using biosynthetic pathways inherited from their bacterial ancestors. However, the ability to synthesize nine amino acids (Phe, Trp, Ile, Leu, Val, Lys, His, Thr, and Met) was lost in a wide variety of eukaryotes that evolved the ability to feed on other organisms. Since the biosynthetic pathways and their respective enzymes are well characterized, orthologs can be recognized in whole genomes to understand when in evolution pathways were lost. The pattern of pathway loss and retention was analyzed in the complete genomes of three early-diverging protist parasites, the amoeba Dictyostelium, and six animals. The nine pathways were lost independently in animals, Dictyostelium, Leishmania, Plasmodium, and Cryptosporidium. Seven additional pathways appear to have been lost in one or another parasite, demonstrating that they are dispensable in a nutrition-rich environment. Our predictions of pathways retained and pathways lost based on computational analyses of whole genomes are validated by minimal-medium studies with mammals, fish, worms, and Dictyostelium. The apparent selective advantages of retaining biosynthetic capabilities for amino acids available in the diet are considered.

  9. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk.

    PubMed

    Tsunematsu, Yuta; Ishikawa, Noriyasu; Wakana, Daigo; Goda, Yukihiro; Noguchi, Hiroshi; Moriya, Hisao; Hotta, Kinya; Watanabe, Kenji

    2013-12-01

    Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis.

  10. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk.

    PubMed

    Tsunematsu, Yuta; Ishikawa, Noriyasu; Wakana, Daigo; Goda, Yukihiro; Noguchi, Hiroshi; Moriya, Hisao; Hotta, Kinya; Watanabe, Kenji

    2013-12-01

    Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis. PMID:24121553

  11. 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

  12. 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

  13. 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.

  14. TREX: a universal tool for the transfer and expression of biosynthetic pathways in bacteria.

    PubMed

    Loeschcke, Anita; Markert, Annette; Wilhelm, Susanne; Wirtz, Astrid; Rosenau, Frank; Jaeger, Karl-Erich; Drepper, Thomas

    2013-01-18

    Secondary metabolites represent a virtually inexhaustible source of natural molecules exhibiting a high potential as pharmaceuticals or chemical building blocks. To gain broad access to these compounds, sophisticated expression systems are needed that facilitate the transfer and expression of large chromosomal regions, whose genes encode complex metabolic pathways. Here, we report on the development of the novel system for the transfer and expression of biosynthetic pathways (TREX), which comprises all functional elements necessary for the delivery and concerted expression of clustered pathway genes in different bacteria. TREX employs (i) conjugation for DNA transfer, (ii) randomized transposition for its chromosomal insertion, and (iii) T7 RNA polymerase for unimpeded bidirectional gene expression. The applicability of the TREX system was demonstrated by establishing the biosynthetic pathways of two pigmented secondary metabolites, zeaxanthin and prodigiosin, in bacteria with different metabolic capacities. Thus, TREX represents a valuable tool for accessing natural products by allowing comparative expression studies with clustered genes. PMID:23656323

  15. The Biosynthetic Pathways of Tanshinones and Phenolic Acids in Salvia miltiorrhiza.

    PubMed

    Ma, Xiao-Hui; Ma, Ying; Tang, Jin-Fu; He, Ya-Li; Liu, Yu-Chen; Ma, Xiao-Jing; Shen, Ye; Cui, Guang-Hong; Lin, Hui-Xin; Rong, Qi-Xian; Guo, Juan; Huang, Lu-Qi

    2015-01-01

    Secondary metabolites from plants play key roles in human medicine and chemical industries. Due to limited accumulation of secondary metabolites in plants and their important roles, characterization of key enzymes involved in biosynthetic pathway will enable metabolic engineering or synthetic biology to improve or produce the compounds in plants or microorganisms, which provides an alternative for production of these valuable compounds. Salvia miltiorrhiza, containing tanshinones and phenolic acids as its active compounds, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. The biosynthetic analysis of secondary metabolites in S. miltiorrhiza has made great progress due to the successful genetic transformation system, simplified hairy roots system, and high-throughput sequencing. The cloned genes in S. miltiorrhiza had provided references for functional characterization of the post-modification steps involved in biosynthesis of tanshinones and phenolic acids, and further utilization of these steps in metabolic engineering. The strategies used in these studies could provide solid foundation for elucidation of biosynthetic pathways of diterpenoids and phenolic acids in other species. The present review systematically summarizes recent advances in biosynthetic pathway analysis of tanshinones and phenolic acids as well as synthetic biology and metabolic engineering applications of the rate-limiting genes involved in the secondary metabolism in S. miltiorrhiza. PMID:26370949

  16. 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.

  17. Localization and interactions between Arabidopsis auxin biosynthetic enzymes in the TAA/YUC-dependent pathway.

    PubMed

    Kriechbaumer, Verena; Botchway, Stanley W; Hawes, Chris

    2016-07-01

    The growth regulator auxin is involved in all key developmental processes in plants. A complex network of a multiplicity of potential biosynthetic pathways as well as transport, signalling plus conjugation and deconjugation lead to a complex and multifaceted system system for auxin function. This raises the question how such a system can be effectively organized and controlled. Here we report that a subset of auxin biosynthetic enzymes in the TAA/YUC route of auxin biosynthesis is localized to the endoplasmic reticulum (ER). ER microsomal fractions also contain a significant percentage of auxin biosynthetic activity. This could point toward a model of auxin function using ER membrane location and subcellular compartmentation for supplementary layers of regulation. Additionally we show specific protein-protein interactions between some of the enzymes in the TAA/YUC route of auxin biosynthesis. PMID:27208541

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

    SciTech Connect

    Guo, Chun-Jun; Yeh, Hsu-Hua; Chiang, Yi Ming; Sanchez, James F.; Chang, ShuLin; Bruno, Kenneth S.; Wang, Clay C.

    2013-04-15

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

  19. 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

  20. Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes

    PubMed Central

    Bradshaw, Rosie E.; Bhatnagar, Deepak; Ganley, Rebecca J.; Gillman, Carmel J.; Monahan, Brendon J.; Seconi, Janet M.

    2002-01-01

    Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (dotA), oxidase (dotB), major facilitator superfamily transporter (dotC), and thioesterase (dotD). A D. pini dotA mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that dotA is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the dotA mutant confirms that the dotA gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four dot genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene, ddhA, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control Dothistroma needle blight. PMID:12039746

  1. Evolution of tryptophan biosynthetic pathway in microbial genomes: a comparative genetic study.

    PubMed

    Priya, V K; Sarkar, Susmita; Sinha, Somdatta

    2014-03-01

    Biosynthetic pathway evolution needs to consider the evolution of a group of genes that code for enzymes catalysing the multiple chemical reaction steps leading to the final end product. Tryptophan biosynthetic pathway has five chemical reaction steps that are highly conserved in diverse microbial genomes, though the genes of the pathway enzymes show considerable variations in arrangements, operon structure (gene fusion and splitting) and regulation. We use a combined bioinformatic and statistical analyses approach to address the question if the pathway genes from different microbial genomes, belonging to a wide range of groups, show similar evolutionary relationships within and between them. Our analyses involved detailed study of gene organization (fusion/splitting events), base composition, relative synonymous codon usage pattern of the genes, gene expressivity, amino acid usage, etc. to assess inter- and intra-genic variations, between and within the pathway genes, in diverse group of microorganisms. We describe these genetic and genomic variations in the tryptophan pathway genes in different microorganisms to show the similarities across organisms, and compare the same genes across different organisms to find the possible variability arising possibly due to horizontal gene transfers. Such studies form the basis for moving from single gene evolution to pathway evolutionary studies that are important steps towards understanding the systems biology of intracellular pathways. PMID:24592292

  2. Thermodynamic constraints on kinetic proofreading in biosynthetic pathways.

    PubMed Central

    Ehrenberg, M; Blomberg, C

    1980-01-01

    We develop a quantitative theory of kinetic proofreading with an arbitrary number of checking steps after the hydrolysis of a nucleoside triphosphate. In particular, we investigate the relationship between the minimum dissipation of free energy required for a given error frequency in such systems. Several conclusions can be drawn from the present treatment: first, the ultimate accuracy of error correcting selective pathways is set by the displacement from equilibrium of the nucleoside triphosphates. Second, it is advantageous to achieve a desired accuracy at a small energy dissipation with several checking steps rather than a single one. This could explain antinomies in the amino acylation reaction as well as in mRNA translation, where small structural differences lead to large differences in flow rates between right and wrong substrates. Third, all checking steps should contribute equally to the accuracy, which implies a specific and symmetrical set of rate constants for the checking events on the enzyme. PMID:7260292

  3. Complete biosynthetic pathway of anditomin: nature's sophisticated synthetic route to a complex fungal meroterpenoid.

    PubMed

    Matsuda, Yudai; Wakimoto, Toshiyuki; Mori, Takahiro; Awakawa, Takayoshi; Abe, Ikuro

    2014-10-29

    Anditomin and its precursors, andilesins, are fungal meroterpenoids isolated from Aspergillus variecolor and have unique, highly oxygenated chemical structures with a complex bridged-ring system. Previous isotope-feeding studies revealed their origins as 3,5-dimethylorsellinic acid and farnesyl pyrophosphate and suggested the possible involvement of a Diels-Alder reaction to afford the congested bicyclo[2.2.2]octane core structure of andilesins. Here we report the first identification of the biosynthetic gene cluster of anditomin and the determination of the complete biosynthetic pathway by characterizing the functions of 12 dedicated enzymes. The anditomin pathway actually does not employ a Diels-Alder reaction, but involves the nonheme iron-dependent dioxygenase AndA to synthesize the bridged-ring by an unprecedented skeletal reconstruction. Another dioxygenase, AndF, is also responsible for the structural complexification, generating the end product anditomin by an oxidative rearrangement.

  4. 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

  5. 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

  6. 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

  7. Biosynthetic pathway for acrylic acid from glycerol in recombinant Escherichia coli.

    PubMed

    Tong, Wenhua; Xu, Ying; Xian, Mo; Niu, Wei; Guo, Jiantao; Liu, Huizhou; Zhao, Guang

    2016-06-01

    Acrylic acid is an important industrial feedstock. In this study, a de novo acrylate biosynthetic pathway from inexpensive carbon source glycerol was constructed in Escherichia coli. The acrylic acid was produced from glycerol via 3-hydroxypropionaldehyde, 3-hydroxypropionyl-CoA, and acrylyl-CoA. The acrylate production was improved by screening and site-directed mutagenesis of key enzyme enoyl-CoA hydratase and chromosomal integration of some exogenous genes. Finally, our recombinant strain produced 37.7 mg/L acrylic acid under shaking flask conditions. Although the acrylate production is low, our study shows feasibility of engineering an acrylate biosynthetic pathway from inexpensive carbon source. Furthermore, the reasons for limited acrylate production and further strain optimization that should be performed in the future were also discussed. PMID:26782744

  8. Yellow flowers generated by expression of the aurone biosynthetic pathway.

    PubMed

    Ono, Eiichiro; Fukuchi-Mizutani, Masako; Nakamura, Noriko; Fukui, Yuko; Yonekura-Sakakibara, Keiko; Yamaguchi, Masaatsu; Nakayama, Toru; Tanaka, Takaharu; Kusumi, Takaaki; Tanaka, Yoshikazu

    2006-07-18

    Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4'-O-glucosyltransferase (4'CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4'CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4'CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4'-O-glucosylated in the cytoplasm, their 4'-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant. PMID:16832053

  9. 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.

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

    PubMed

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

    2015-11-01

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

  11. Yellow flowers generated by expression of the aurone biosynthetic pathway

    PubMed Central

    Ono, Eiichiro; Fukuchi-Mizutani, Masako; Nakamura, Noriko; Fukui, Yuko; Yonekura-Sakakibara, Keiko; Yamaguchi, Masaatsu; Nakayama, Toru; Tanaka, Takaharu; Kusumi, Takaaki; Tanaka, Yoshikazu

    2006-01-01

    Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4′-O-glucosyltransferase (4′CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4′CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4′CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4′-O-glucosylated in the cytoplasm, their 4′-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant. PMID:16832053

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

    PubMed

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

    2016-09-01

    Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the β-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved. PMID:27431934

  13. 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. PMID:26328935

  14. 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

  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. 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.

  17. 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

    2015-07-01

    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 (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.

  18. 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

  19. Hybrubins: Bipyrrole Tetramic Acids Obtained by Crosstalk between a Truncated Undecylprodigiosin Pathway and Heterologous Tetramic Acid Biosynthetic Genes.

    PubMed

    Zhao, Zhilong; Shi, Ting; Xu, Min; Brock, Nelson L; Zhao, Yi-Lei; Wang, Yemin; Deng, Zixin; Pang, Xiuhua; Tao, Meifeng

    2016-02-01

    Heterologous expression of bacterial artificial chromosome (BAC) clones from the genomic library of Streptomyces variabilis Snt24 in Streptomyces lividans SBT5 which carried a truncated undecylprodigiosin biosynthetic gene cluster led to the identification of hybrubins A-C. The hybrubins represent a new carbon skeleton in which a tetramic acid moiety is fused to a 2,2'-dipyrrole building block. Gene knockout experiments confirmed that hybrubins are derived from two convergent biosynthetic pathways including the remaining genomic red genes of S. lividans SBT5 as well as the BAC encoded hbn genes for the production of 5-ethylidenetetramic acid. A possible biosynthetic pathway was also proposed.

  20. Regulation of Carotenoid Biosynthesis During Fruit Development.

    PubMed

    Lado, Joanna; Zacarías, Lorenzo; Rodrigo, María Jesús

    2016-01-01

    Carotenoids are recognized as the main pigments in most fruit crops, providing colours that range from yellow and pink to deep orange and red. Moreover, the edible portion of widely consumed fruits or their derived products represent a major dietary source of carotenoids for animals and humans. Therefore, these pigments are crucial compounds contributing to fruit aesthetic and nutritional quality but may also have protecting and ecophysiological functions in coloured fruits. Among plant organs, fruits display one of the most heterogeneous carotenoids patterns in terms of diversity and abundance. In this chapter a comprehensive list of the carotenoid content and profile in the most commonly cultivated fleshy fruits is reported. The proposed fruit classification systems attending to carotenoid composition are revised and discussed. The regulation of carotenoids in fruits can be rather complex due to the dramatic changes in content and composition during ripening, which are also dependent on the fruit tissue and the developmental stage. In addition, carotenoid accumulation is a dynamic process, associated with the development of chromoplasts during ripening. As a general rule, carotenoid accumulation is highly controlled at the transcriptional level of the structural and accessory proteins of the biosynthetic and degradation pathways, but other mechanisms such as post-transcriptional modifications or the development of sink structures have been recently revealed as crucial factors in determining the levels and stability of these pigments. In this chapter common key metabolic reactions regulating carotenoid composition in fruit tissues are described in addition to others that are restricted to certain species and generate unique carotenoids patterns. The existence of fruit-specific isoforms for key steps such as the phytoene synthase, lycopene β-cyclases or catabolic carotenoid cleavage dioxygenases has allowed an independent regulation of the pathway in fruit tissues

  1. 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.

  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. Elucidation of the Biosynthetic Pathway for Okenone in Thiodictyon sp. CAD16 Leads to the Discovery of Two Novel Carotene Ketolases*

    PubMed Central

    Vogl, Kajetan; Bryant, Donald A.

    2011-01-01

    Okenone is a unique ketocarotenoid found in many purple sulfur bacteria; it is important because of its unique light absorption and photoprotection properties. Okenane, a compound formed by diagenetic reduction of okenone, is an important biomarker in geochemical analyses of sedimentary rocks. Despite its ecological and biogeochemical importance, the biochemical pathway for okenone synthesis has not yet been fully described. The genome sequence of an okenone-producing organism, Thiodictyon sp. strain CAD16, revealed four genes whose predicted proteins had strong sequence similarity to enzymes known to produce ψ-end group modifications of carotenoids in proteobacteria. These four genes encoded homologs of a 1,2-carotenoid hydratase (CrtC), an O-methyltransferase (CrtF), and two paralogs of carotenoid 3,4-desaturases (CrtD). Expression studies in lycopene- or neurosporene-producing strains of Escherichia coli confirmed the functions of crtC and crtF, but the crtD paralogs encoded enzymes with previously undescribed functions. One enzyme, CruS, was only distantly related to CrtD desaturases, was bifunctional, and performed a 3,4-desaturation and introduced a C-2 keto group into neurosporene derivatives in the presence of dioxygen. The enzyme encoded by the other crtD paralog also represents a new enzyme in carotenogenesis and was named cruO. CruO encodes the C-4/4′ ketolase uniquely required for okenone biosynthesis. The identification of CruO and the demonstration of its biochemical activity complete the elucidation of the biosynthetic pathway for okenone and other related ketocarotenoids. PMID:21921032

  4. Elucidation of the biosynthetic pathway for Okenone in Thiodictyon sp. CAD16 leads to the discovery of two novel carotene ketolases.

    PubMed

    Vogl, Kajetan; Bryant, Donald A

    2011-11-01

    Okenone is a unique ketocarotenoid found in many purple sulfur bacteria; it is important because of its unique light absorption and photoprotection properties. Okenane, a compound formed by diagenetic reduction of okenone, is an important biomarker in geochemical analyses of sedimentary rocks. Despite its ecological and biogeochemical importance, the biochemical pathway for okenone synthesis has not yet been fully described. The genome sequence of an okenone-producing organism, Thiodictyon sp. strain CAD16, revealed four genes whose predicted proteins had strong sequence similarity to enzymes known to produce ψ-end group modifications of carotenoids in proteobacteria. These four genes encoded homologs of a 1,2-carotenoid hydratase (CrtC), an O-methyltransferase (CrtF), and two paralogs of carotenoid 3,4-desaturases (CrtD). Expression studies in lycopene- or neurosporene-producing strains of Escherichia coli confirmed the functions of crtC and crtF, but the crtD paralogs encoded enzymes with previously undescribed functions. One enzyme, CruS, was only distantly related to CrtD desaturases, was bifunctional, and performed a 3,4-desaturation and introduced a C-2 keto group into neurosporene derivatives in the presence of dioxygen. The enzyme encoded by the other crtD paralog also represents a new enzyme in carotenogenesis and was named cruO. CruO encodes the C-4/4' ketolase uniquely required for okenone biosynthesis. The identification of CruO and the demonstration of its biochemical activity complete the elucidation of the biosynthetic pathway for okenone and other related ketocarotenoids. PMID:21921032

  5. Elucidation of the biosynthetic pathway for Okenone in Thiodictyon sp. CAD16 leads to the discovery of two novel carotene ketolases.

    PubMed

    Vogl, Kajetan; Bryant, Donald A

    2011-11-01

    Okenone is a unique ketocarotenoid found in many purple sulfur bacteria; it is important because of its unique light absorption and photoprotection properties. Okenane, a compound formed by diagenetic reduction of okenone, is an important biomarker in geochemical analyses of sedimentary rocks. Despite its ecological and biogeochemical importance, the biochemical pathway for okenone synthesis has not yet been fully described. The genome sequence of an okenone-producing organism, Thiodictyon sp. strain CAD16, revealed four genes whose predicted proteins had strong sequence similarity to enzymes known to produce ψ-end group modifications of carotenoids in proteobacteria. These four genes encoded homologs of a 1,2-carotenoid hydratase (CrtC), an O-methyltransferase (CrtF), and two paralogs of carotenoid 3,4-desaturases (CrtD). Expression studies in lycopene- or neurosporene-producing strains of Escherichia coli confirmed the functions of crtC and crtF, but the crtD paralogs encoded enzymes with previously undescribed functions. One enzyme, CruS, was only distantly related to CrtD desaturases, was bifunctional, and performed a 3,4-desaturation and introduced a C-2 keto group into neurosporene derivatives in the presence of dioxygen. The enzyme encoded by the other crtD paralog also represents a new enzyme in carotenogenesis and was named cruO. CruO encodes the C-4/4' ketolase uniquely required for okenone biosynthesis. The identification of CruO and the demonstration of its biochemical activity complete the elucidation of the biosynthetic pathway for okenone and other related ketocarotenoids.

  6. Divisions of labor in the thiamin biosynthetic pathway among organs of maize.

    PubMed

    Guan, Jiahn-Chou; Hasnain, Ghulam; Garrett, Timothy J; Chase, Christine D; Gregory, Jesse; Hanson, Andrew D; McCarty, Donald R

    2014-01-01

    The B vitamin thiamin is essential for central metabolism in all cellular organisms including plants. While plants synthesize thiamin de novo, organs vary widely in their capacities for thiamin synthesis. We use a transcriptomics approach to appraise the distribution of de novo synthesis and thiamin salvage pathways among organs of maize. We identify at least six developmental contexts in which metabolically active, non-photosynthetic organs exhibit low expression of one or both branches of the de novo thiamin biosynthetic pathway indicating a dependence on inter-cellular transport of thiamin and/or thiamin precursors. Neither the thiazole (THI4) nor pyrimidine (THIC) branches of the pathway are expressed in developing pollen implying a dependence on import of thiamin from surrounding floral and inflorescence organs. Consistent with that hypothesis, organs of the male inflorescence and flowers are shown to have high relative expression of the thiamin biosynthetic pathway and comparatively high thiamin contents. By contrast, divergent patterns of THIC and THI4 expression occur in the shoot apical meristem, embyro sac, embryo, endosperm, and root-tips suggesting that these sink organs acquire significant amounts of thiamin via salvage pathways. In the root and shoot meristems, expression of THIC in the absence of THI4 indicates a capacity for thiamin synthesis via salvage of thiazole, whereas the opposite pattern obtains in embryo and endosperm implying that seed storage organs are poised for pyrimidine salvage. Finally, stable isotope labeling experiments set an upper limit on the rate of de novo thiamin biosynthesis in maize leaf explants. Overall, the observed patterns of thiamin biosynthetic gene expression mirror the strategies for thiamin acquisition that have evolved in bacteria.

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

    PubMed

    Cronan, John E

    2016-06-01

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

  8. 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

  9. 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

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

    PubMed Central

    Zhao, J; Last, R L

    1996-01-01

    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. PMID:8989880

  11. 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

  12. 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.

  13. Gene fusions for the directed modification of the carotenoid biosynthesis pathway in Mucor circinelloides.

    PubMed

    Iturriaga, Enrique A; Papp, Tamás; Alvarez, María Isabel; Eslava, Arturo P

    2012-01-01

    Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by M. circinelloides is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. We used the ctrW gene of Paracoccus sp. N81106, encoding a bacterial β-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in M. circinelloides. First we fused it to the carRP gene of M. circinelloides, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by M. circinelloides of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the crtW gene of Paracoccus sp. to the crtS gene of Xanthophyllomyces dendrorhous, responsible for the synthesis of astaxanthin from β-carotene in this fungus, but which was shown to have only hydroxylase activity in M. circinelloides. The expected result in M. circinelloides transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions. PMID:22711120

  14. Biosynthesis of 3-hydroxy-5-methyl-o-methyltyrosine in the saframycin/ safracin biosynthetic pathway.

    PubMed

    Fu, Cheng-Yu; Tang, Man-Cheng; Peng, Chao; Li, Lei; He, Yan-Ling; Liu, Wen; Tang, Gong-Li

    2009-05-01

    The biosynthesis study of antibiotics saframycin (SFM) in Streptomyces lavendulae and safracin (SAC) in Pseudomonas fluorescens demonstrated that 3-hydroxy-5-methyl-Omethyltyrosine (3h5mOmTyr), a nonproteinogenic amino acid, is the precursor of the tetrahydroisoquinoline molecular core. In the biosynthetic gene cluster of SAC/SFM, sacD/ sfmD encodes a protein with high homology to each other but no sequence similarity to other known enzymes; sacF/ sfmM2 and sacG/sfmM3 encode methyltransferases for Cmethylation and O-methylation; and sacE/sfmF encodes a small protein with significant sequence similarity to the MbtH-like proteins, which are frequently found in the biosynthetic pathways of nonribosomal peptide antibiotics and siderophores. To address their function, the biosynthetic cassette of 3h5mOmTyr was heterologously expressed in S. coelicolor and P. putida, and an in-frame deletion and complementation in trans were carried out. The results revealed that (i) SfmD catalyzes the hydroxylation of aromatic rings;(ii) sacD/sacF/sacG in the SAC gene cluster and sfmD/sfmM2/sfmM3 in the SFM cluster are sufficient for the biosynthesis of 3h5mOmTyr; and (iii) the mbtH-like gene is not required for the biosynthesis of the 3h5mOmTyr precursor. PMID:19494690

  15. Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.

    PubMed Central

    Nelson, M A; Morelli, G; Carattoli, A; Romano, N; Macino, G

    1989-01-01

    The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains. Images PMID:2524647

  16. Regulation of Carotenoid Biosynthesis in Photosynthetic Organs.

    PubMed

    Llorente, Briardo

    2016-01-01

    A substantial proportion of the dazzling diversity of colors displayed by living organisms throughout the tree of life is determined by the presence of carotenoids, which most often provide distinctive yellow, orange and red hues. These metabolites play fundamental roles in nature that extend far beyond their importance as pigments. In photosynthetic lineages, carotenoids are essential to sustain life, since they have been exploited to maximize light harvesting and protect the photosynthetic machinery from photooxidative stress. Consequently, photosynthetic organisms have evolved several mechanisms that adjust the carotenoid metabolism to efficiently cope with constantly fluctuating light environments. This chapter will focus on the current knowledge concerning the regulation of the carotenoid biosynthetic pathway in leaves, which are the primary photosynthetic organs of most land plants. PMID:27485221

  17. Unravelling ionization and fragmentation pathways of carotenoids using orbitrap technology: a first step towards identification of unknowns.

    PubMed

    Bijttebier, Sebastiaan K A; D'Hondt, Els; Hermans, Nina; Apers, Sandra; Voorspoels, Stefan

    2013-06-01

    Vegetables are a major source of carotenoids and carotenoids are identified as potentially important natural antioxidants that may aid in the prevention of several human chronic degenerative diseases. Characterization of carotenoids in organic biological matrices is a crucial step in any research valorization trajectory. This study reports for the first time the use of high mass resolution and exact mass orbitrap technology for the elucidation of carotenoid fragmentation pathways. This contributes to the generation of new tools for identifying unknown carotenoids based on fragmentation patterns. Two different chromatographic methods making use of different mobile phases resulted in the generation of different ion species because of the large influence of the mobile phase solvent composition on ionization. It was shown that depending on the molecular ion species that are generated (protonated ions or radical molecular ions), different fragments are formed when applying higher energy collisional dissociation. Fragmentation and the abundance of fragments provide valuable structural information on the type of functional groups, the polyene backbone and the location of double bonds in ring structures of carotenoids. Furthermore, coherence between specific substructures in the molecules and characteristic fragmentation patterns was observed allowing the assignment of fragmentation patterns for carotenoid substructures that can theoretically be extrapolated to carotenoids with similar (sub)structures. Differentiation between isomeric carotenoids by compound specific fragments could however not be made for all the isomeric groups under study. As a wide variety of isomeric forms of carotenoids exist in nature, the combination of good chromatographic separation with high resolution mass spectrometry and other complementary qualitative structure elucidation techniques such as a photo diode array detector and/or nuclear magnetic resonance spectroscopy are indispensable for

  18. A novel interaction linking the FAS-II and phthiocerol dimycocerosate (PDIM) biosynthetic pathways.

    PubMed

    Kruh, Nicole A; Borgaro, Janine G; Ruzsicska, Béla P; Xu, Hua; Tonge, Peter J

    2008-11-14

    The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall. PMID:18703500

  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. PMID:27346378

  20. 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. PMID:22580955

  1. 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.

  2. Differential expression of carotenoid-related genes determines diversified carotenoid coloration in floral tissues of Oncidium cultivars.

    PubMed

    Chiou, Chung-Yi; Pan, Hsin-An; Chuang, Yao-Nung; Yeh, Kai-Wun

    2010-09-01

    Three cultivars of Oncidium orchid with varied coloration, such as Oncidium Gower Ramsey (yellow), Sunkist (orange), and White Jade (white), were analyzed for carotenoid metabolites and gene expression of carotenoid-biosynthetic genes. The HPLC analysis revealed that yellow Gower Ramsey accumulates violaxanthin, 9-cis-violaxanthin and neoxanthin, orange Sunkist accumulates an additional beta-carotene, and White Jade is devoid of carotenoid compounds. Molecular characterization indicated that the three Oncidium cultivars exhibited varied expression pattern and level in carotenoid-biosynthetic pathway. Among them, high expression level of beta-hydroxylase (OgHYB) and zeaxanthin epoxidase (OgZEP) was displayed in yellow Gower Ramsey, relative to the down-regulation of OgHYB and OgZEP exhibited in orange Sunkist, which results in the accumulation of beta-carotene and orange coloration in floral tissues. However, White Jade is caused by the up-regulation of OgCCD1 (Carotenoid Cleavage Dioxygenase 1), which catabolizes carotenoid metabolites. Methylation assay of OgCCD1 promoter in White Jade and Gower Ramsey revealed that a high level of DNA methylation was present in OgCCD1 promoter region of Gower Ramsey. Transient expression of OgCCD1 in yellow lip tissues of Gower Ramsey by bombardment confirmed its function of disintegrating carotenoid compounds. Our results suggest an evolutionary significance that genetic variation of carotenoid-related genes in Oncidium generates the complexity of floral pigmentation and consequently provides the profound varieties in Oncidium population.

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

    PubMed

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

    2016-09-01

    Fusarium graminearum is a devastating pathogenic fungus causing fusarium head blight (FHB) of wheat. This fungus can produce indole-3-acetic acid (IAA) and a very large amount of IAA accumulates in wheat head tissues during the first few days of infection by F. graminearum. Using liquid culture conditions, we have determined that F. graminearum can use tryptamine (TAM) and indole-3-acetonitrile (IAN) as biosynthetic intermediates to produce IAA. It is the first time that F. graminearum is shown to use the l-tryptophan-dependent TAM and IAN pathways rather than the indole-3-acetamide or indole-3-pyruvic acid pathways to produce IAA. Our experiments also showed that exogenous IAA was metabolized by F. graminearum. Exogenous IAA, TAM, and IAN inhibited mycelial growth; IAA and IAN also affected the hyphae branching pattern and delayed macroconidium germination. IAA and TAM had a small positive effect on the production of the mycotoxin 15-ADON while IAN inhibited its production. Our results showed that IAA and biosynthetic intermediates had a significant effect on F. graminearum physiology and suggested a new area of exploration for fungicidal compounds. PMID:27567719

  4. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway.

    PubMed

    Umemoto, Naoyuki; Nakayasu, Masaru; Ohyama, Kiyoshi; Yotsu-Yamashita, Mari; Mizutani, Masaharu; Seki, Hikaru; Saito, Kazuki; Muranaka, Toshiya

    2016-08-01

    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

  5. 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. PMID:25138711

  6. 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.

  7. 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.

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

    PubMed

    Karim, Ashty S; Jewett, Michael C

    2016-07-01

    Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. PMID:26996382

  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. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway.

    PubMed

    Hichri, Imène; Barrieu, François; Bogs, Jochen; Kappel, Christian; Delrot, Serge; Lauvergeat, Virginie

    2011-05-01

    Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 classes. This MYB-bHLH-WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins. Although several genes encoding transcription factors from these three families have been identified, many gaps remain in our understanding of the regulation of this biosynthetic pathway, especially about the respective roles of bHLH and WD40 proteins. A better knowledge of the regulatory mechanisms of the flavonoid pathway is likely to favour the development of new biotechnological tools for the generation of value-added plants with optimized flavonoid content.

  11. Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering.

    PubMed

    Farré, Gemma; Maiam Rivera, Sol; Alves, Rui; Vilaprinyo, Ester; Sorribas, Albert; Canela, Ramon; Naqvi, Shaista; Sandmann, Gerhard; Capell, Teresa; Zhu, Changfu; Christou, Paul

    2013-08-01

    Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in-depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.

  12. 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

  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. Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae▿ †

    PubMed Central

    Wang, Quan; Xu, Yanli; Perepelov, Andrei V.; Xiong, Wei; Wei, Dongmei; Shashkov, Alexander S.; Knirel, Yuriy A.; Feng, Lu; Wang, Lei

    2010-01-01

    Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound. PMID:20729354

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Regulation of the cholesterol biosynthetic pathway and its integration with fatty acid biosynthesis in the oleaginous microalga Nannochloropsis oceanica

    PubMed Central

    2014-01-01

    Background Sterols are vital structural and regulatory components in eukaryotic cells; however, their biosynthetic pathways and functional roles in microalgae remain poorly understood. Results In the oleaginous microalga Nannochloropsis oceanica, the sterol biosynthetic pathway produces phytosterols as minor products and cholesterol as the major product. The evidence together with their deduced biosynthetic pathways suggests that N. oceanica exhibits features of both higher plants and mammals. Temporal tracking of sterol profiles and sterol-biosynthetic transcripts in response to changes in light intensity and nitrogen supply reveal that sterols play roles in cell proliferation, chloroplast differentiation, and photosynthesis. Furthermore, the dynamics of fatty acid (FA) and FA-biosynthetic transcripts upon chemical inhibitor-induced sterol depletion reveal possible co-regulation of sterol production and FA synthesis, in that the squalene epoxidase inhibitor terbinafine reduces sterol content yet significantly elevates free FA production. Thus, a feedback regulation of sterol and FA homeostasis is proposed, with the 1-deoxy-D-xylulose 5-phosphate synthase (DXS, the committed enzyme in isoprenoid and sterol biosynthesis) gene potentially subject to feedback regulation by sterols. Conclusion These findings reveal features of sterol function and biosynthesis in microalgae and suggest new genetic engineering or chemical biology approaches for enhanced oil production in microalgae. PMID:24920959

  1. 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.

  2. Trehalose Polyphleates Are Produced by a Glycolipid Biosynthetic Pathway Conserved across Phylogenetically Distant Mycobacteria.

    PubMed

    Burbaud, Sophie; Laval, Françoise; Lemassu, Anne; Daffé, Mamadou; Guilhot, Christophe; Chalut, Christian

    2016-02-18

    Mycobacteria synthesize a variety of structurally related glycolipids with major biological functions. Common themes have emerged for the biosynthesis of these glycolipids, including several families of proteins. Genes encoding these proteins are usually clustered on bacterial chromosomal islets dedicated to the synthesis of one glycolipid family. Here, we investigated the function of a cluster of five genes widely distributed across non-tuberculous mycobacteria. Using defined mutant analysis and in-depth structural characterization of glycolipids from wild-type or mutant strains of Mycobacterium smegmatis and Mycobacterium abscessus, we established that they are involved in the formation of trehalose polyphleates (TPP), a family of compounds originally described in Mycobacterium phlei. Comparative genomics and lipid analysis of strains distributed along the mycobacterial phylogenetic tree revealed that TPP is synthesized by a large number of non-tuberculous mycobacteria. This work unravels a novel glycolipid biosynthetic pathway in mycobacteria and extends the spectrum of bacteria that produce TPP. PMID:27028886

  3. A Unique Tryptophan C-Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway.

    PubMed

    Parajuli, Anirudra; Kwak, Daniel H; Dalponte, Luca; Leikoski, Niina; Galica, Tomas; Umeobika, Ugochukwu; Trembleau, Laurent; Bent, Andrew; Sivonen, Kaarina; Wahlsten, Matti; Wang, Hao; Rizzi, Ermanno; De Bellis, Gianluca; Naismith, James; Jaspars, Marcel; Liu, Xinyu; Houssen, Wael; Fewer, David Peter

    2016-03-01

    Cyanobactins are a rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES-88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES-88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C-3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan-prenylated cyclic peptides. PMID:26846478

  4. 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

  5. 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.

  6. A general stereocontrolled, convergent synthesis of oligoprenols that parallels the biosynthetic pathway.

    PubMed

    Radetich, Branko; Corey, E J

    2002-03-20

    A solution is reported to the classic unsolved problem of stereoselective synthesis of all-E oligoprenols, such as E-farnesylfarnesol, by a cationic coupling analogous to the biosynthetic pathway. The simplicity and efficacy of the method, which is outlined in Scheme 1, are demonstrated by the synthesis of a series of all-E oligoprenols from C(20) to C(35) in uniformly excellent overall yield. The success of the approach is due not only to the highly E-stereoselective C-C coupling that forms the oligoprenyl chain but also to the development of efficient syntheses of allylic secondary silanes and E-oligoprenal acetals, and to a selective allylic demethoxylation reaction.

  7. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.

    PubMed

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2016-06-01

    Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum (P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria. PMID:27262062

  8. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.

    PubMed

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2016-06-01

    Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum (P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria.

  9. Cross-regulation among disparate antibiotic biosynthetic pathways of Streptomyces coelicolor.

    PubMed

    Huang, Jianqiang; Shi, Jing; Molle, Virginie; Sohlberg, Björn; Weaver, David; Bibb, Maureen J; Karoonuthaisiri, Nitsara; Lih, Chih-Jian; Kao, Camilla M; Buttner, Mark J; Cohen, Stanley N

    2005-12-01

    A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-04-01

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

  12. 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. PMID:26377817

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

    PubMed

    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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26595805

  15. 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

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

    PubMed

    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

  17. 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. PMID:26195520

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

    PubMed Central

    San Martin Galindo, Paola; Taguchi, Takaaki; Manner, Suvi; Vuorela, Pia M.; Ichinose, Koji; Metsä-Ketelä, Mikko

    2015-01-01

    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. PMID:26195520

  19. 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

  20. 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.

  1. 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

  2. Two of a Kind--The Biosynthetic Pathways of Chlorotonil and Anthracimycin.

    PubMed

    Jungmann, Katrin; Jansen, Rolf; Gerth, Klaus; Huch, Volker; Krug, Daniel; Fenical, William; Müller, Rolf

    2015-11-20

    Chlorotonil A is a novel polyketide isolated from the myxobacterium Sorangium cellulosum So ce1525 that features a unique gem-dichloro-1,3-dione moiety. It exhibits potent bioactivity, most notably against the problematic malaria pathogen Plasmodium falciparum in the nanomolar range. In addition, strong antibacterial and moderate antifungal activity were determined. The outstanding biological activity of chlorotonil A as well as its unusual chemical structure triggered our interest in elucidating its biosynthesis, a prerequisite for alteration of the scaffold by synthetic biology approaches. This endeavor was facilitated by a recent report describing the strikingly similar structure of anthracimycin from a marine streptomycete, a compound of considerable interest due to its potent antibacterial activity. In this study, we report the identification and characterization of the chlorotonil A biosynthetic gene cluster from So ce1525 and compare it with that for anthracimycin biosynthesis. Access to both gene clusters allowed us to highlight commonalities between the two pathways and revealed striking differences, some of which can plausibly explain the structural differences observed between these intriguing natural products.

  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. 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.

  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. 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. PMID:24630721

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Expression analysis of biosynthetic pathway genes vis-à-vis podophyllotoxin content in Podophyllum hexandrum Royle.

    PubMed

    Kumar, Pawan; Pal, Tarun; Sharma, Neha; Kumar, Varun; Sood, Hemant; Chauhan, Rajinder S

    2015-09-01

    Podophyllum hexandrum Royle is known for its vast medicinal properties, particularly anticancer. It contains higher amount of podophyllotoxin (4.3 %), compared to Podophyllum peltatum (0.025 %) and other plant species; as a result, it has been used worldwide in the preparation of various drugs including anticancer, antimalarial, antiviral, antioxidant, antifungal, and so on. Currently, Etoposide (VP-16-213), Vumon® (Teniposide; VM-26), Etopophos®, Pod-Ben- 25, Condofil, Verrusol, and Warticon are available in the market. Due to highly complex synthesis and low cell culture yields of podophyllotoxin (0.3 %), the supply of raw material cannot be met due to increasing industrial demands. The knowledge on podophyllotoxin biosynthetic pathway vis-à-vis expression status of genes is fragmentary. Quantitative expression analysis of 21 pathway genes has revealed 9 genes, namely SD, PD, PCH, CM, CMT, CAD, CCR, C4H, and ADH, that showed increase in transcript abundance up to 1.4 to 23.05 folds, respectively, vis-à-vis podophyllotoxin content in roots (1.37 %) and rhizomes (3.05 %) of P. hexandrum. In silico analysis of putative cis-regulatory elements in promoter regions of overexpressed genes showed the presence of common Skn-1 motif and MBS elements in CMT, CAD, CCR, C4H, and ADH genes, thereby, suggesting their common regulation. The outcome of the study has resulted in the identification of suitable candidate genes which might be contributing to podophyllotoxin biosynthesis that can act as potential targets for any genetic intervention strategies aimed at its enhanced production.

  12. An Unusual Peroxo Intermediate of the Arylamine Oxygenase of the Chloramphenicol Biosynthetic Pathway

    PubMed Central

    Makris, Thomas M.; Vu, Van V.; Meier, Katlyn K.; Komor, Anna J.; Rivard, Brent S.; Münck, Eckard; Que, Lawrence; Lipscomb, John D.

    2015-01-01

    Streptomyces venezuelae CmlI catalyzes the 6-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O2 to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t1/2 = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an 18O2-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η1:η1) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν(O–O) that is at least 60 cm−1 lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ-η1:η1-peroxo ligand; we propose that a μ-η1:η2-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway, and that the precursor is not bound to the NRPS during this step. PMID:25564306

  13. 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

    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

  14. 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

  15. 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.

  16. 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

    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.

  17. Enhancement of artemisinin content in tetraploid Artemisia annua plants by modulating the expression of genes in artemisinin biosynthetic pathway.

    PubMed

    Lin, Xiuyan; Zhou, Yin; Zhang, Jianjun; Lu, Xu; Zhang, Fangyuan; Shen, Qian; Wu, Shaoyan; Chen, Yunfei; Wang, Tao; Tang, Kexuan

    2011-01-01

    Tetraploid Artemisia annua plants were successfully inducted by using colchicine, and their ploidy was confirmed by flow cytometry. Higher stomatal length but lower frequency in tetraploids were revealed and could be considered as indicators of polyploidy. The average level of artemisinin in tetraploids was increased from 39% to 56% than that of the diploids during vegetation period, as detected by high-performance liquid chromatography-evaporative light scattering detector. Gene expressions of 10 key enzymes related to artemisinin biosynthetic pathway in different ploidy level were analyzed by semiquantitative polymerase chain reaction and significant upregulation of FPS, HMGR, and artemisinin metabolite-specific Aldh1 genes were revealed in tetraploids. Slight increased expression of ADS was also detected. Our results suggest that higher artemisinin content in tetraploid A. annua may result from the upregulated expression of some key enzyme genes related to artemisinin biosynthetic pathway. PMID:21446959

  18. A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

    PubMed

    Huang, Wenjun; Sun, Wei; Lv, Haiyan; Luo, Ming; Zeng, Shaohua; Pattanaik, Sitakanta; Yuan, Ling; Wang, Ying

    2013-01-01

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

  19. Carotenoids Play a Positive Role in the Degradation of Heterocycles by Sphingobium yanoikuyae

    PubMed Central

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

    2012-01-01

    Background 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. Findings 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. Conclusions/Significance 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. PMID:22745775

  20. 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.

  1. Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase.

    PubMed

    Saha, Saikat; Enugutti, Balaji; Rajakumari, Sona; Rajasekharan, Ram

    2006-08-01

    Triacylglycerols (TAGs) are the most important storage form of energy for eukaryotic cells. TAG biosynthetic activity was identified in the cytosolic fraction of developing peanut (Arachis hypogaea) cotyledons. This activity was NaF insensitive and acyl-coenzyme A (CoA) dependent. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG biosynthesis that acylates diacylglycerol to TAG. Soluble DGAT was identified from immature peanuts and purified by conventional column chromatographic procedures. The enzyme has a molecular mass of 41 +/- 1.0 kD. Based on the partial peptide sequence, a degenerate probe was used to obtain the full-length cDNA. The isolated gene shared less than 10% identity with the previously identified DGAT1 and 2 families, but has 13% identity with the bacterial bifunctional wax ester/DGAT. To differentiate the unrelated families, we designate the peanut gene as AhDGAT. Expression of peanut cDNA in Escherichia coli resulted in the formation of labeled TAG and wax ester from [14C]acetate. The recombinant E. coli showed high levels of DGAT activity but no wax ester synthase activity. TAGs were localized in transformed cells with Nile blue A and oil red O staining. The recombinant and native DGAT was specific for 1,2-diacylglycerol and did not utilize hexadecanol, glycerol-3-phosphate, monoacylglycerol, lysophosphatidic acid, and lysophosphatidylcholine. Oleoyl-CoA was the preferred acyl donor as compared to palmitoyl- and stearoyl-CoAs. These data suggest that the cytosol is one of the sites for TAG biosynthesis in oilseeds. The identified pathway may present opportunities of bioengineering oil-yielding plants for increased oil production.

  2. Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant.

    PubMed

    Acosta-Serrano, Alvaro; O'Rear, Jessica; Quellhorst, George; Lee, Soo Hee; Hwa, Kuo-Yuan; Krag, Sharon S; Englund, Paul T

    2004-04-01

    Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

  3. Combined biosynthetic pathway for de novo production of UDP-galactose: catalysis with multiple enzymes immobilized on agarose beads.

    PubMed

    Liu, Ziye; Zhang, Jianbo; Chen, Xi; Wang, Peng G

    2002-04-01

    Regeneration of sugar nucleotides is a critical step in the biosynthetic pathway for the formation of oligosaccharides. To alleviate the difficulties in the production of sugar nucleotides, we have developed a method to produce uridine diphosphate galactose (UDP-galactose). The combined biosynthetic pathway, which involves seven enzymes, is composed of three parts: i) the main pathway to form UDP-galactose from galactose, with the enzymes galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophosphorylase, and inorganic pyrophosphatase, ii) the uridine triphosphate supply pathway catalyzed by uridine monophosphate (UMP) kinase and nucleotide diphosphate kinase, and iii) the adenosine triphosphate (ATP) regeneration pathway catalyzed by polyphosphate kinase with polyphosphate added as an energy resource. All of the enzymes were expressed individually and immobilized through their hexahistidine tags onto nickel agarose beads ("super beads"). The reaction requires a stoichiometric amount of UMP and galactose, and catalytic amounts of ATP and glucose 1-phosphate, all inexpensive starting materials. After continuous circulation of the reaction mixture through the super-bead column for 48 h, 50 % of the UMP was converted into UDP-galactose. The results show that de novo production of UDP-galactose on the super-bead column is more efficient than in solution because of the stability of the immobilized enzymes.

  4. Polycistronic expression of a β-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to β-ionone production.

    PubMed

    Beekwilder, Jules; van Rossum, Harmen M; Koopman, Frank; Sonntag, Frank; Buchhaupt, Markus; Schrader, Jens; Hall, Robert D; Bosch, Dirk; Pronk, Jack T; van Maris, Antonius J A; Daran, Jean-Marc

    2014-12-20

    The flavour and fragrance compound β-ionone, which naturally occurs in raspberry and many other fruits and flowers, is currently produced by synthetic chemistry. This study describes a synthetic biology approach for β-ionone production from glucose by Saccharomyces cerevisiae that is partially based on polycistronic expression. Experiments with model proteins showed that the T2A sequence of the Thosea asigna virus mediated efficient production of individual proteins from a single transcript in S. cerevisiae. Subsequently, three β-carotene biosynthesis genes from the carotenoid-producing ascomycete Xanthophyllomyces dendrorhous (crtI, crtE and crtYB) were expressed in S. cerevisiae from a single polycistronic construct. In this construct, the individual crt proteins were separated by T2A sequences. Production of the individual proteins from the polycistronic construct was confirmed by Western blot analysis and by measuring the production of β-carotene. To enable β-ionone production, a carotenoid-cleavage dioxygenase from raspberry (RiCCD1) was co-expressed in the β-carotene producing strain. In glucose-grown cultures with a second phase of dodecane, β-ionone and geranylacetone accumulated in the organic phase. Thus, by introducing a polycistronic construct encoding a fungal carotenoid pathway and an expression cassette encoding a plant dioxygenase, a novel microbial production system has been established for a fruit flavour compound. PMID:24486029

  5. The diffusible factor synthase XanB2 is a bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways.

    PubMed

    Zhou, Lian; Wang, Jia-Yuan; Wu, Ji'en; Wang, Jianhe; Poplawsky, Alan; Lin, Shuangjun; Zhu, Bangshang; Chang, Changqing; Zhou, Tielin; Zhang, Lian-Hui; He, Ya-Wen

    2013-01-01

    The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end-product of the shikimate pathway, to produce 3-hydroxybenzoic acid (3-HBA) and 4-HBA. 3-HBA and 4-HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3-HBA and 4-HBA biosynthesis via a unique mechanism with the C-terminal YjgF-like domain conferring activity for 3-HBA biosynthesis and the N-terminal FGFG motif-containing domain responsible for 4-HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate-pyruvate lyase UbiC. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria. PMID:23113660

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Comparative Analysis of the Biosynthetic Gene Clusters and Pathways for Three Structurally Related Antitumor Antibiotics Bleomycin, Tallysomycin and Zorbamycin†

    PubMed Central

    Galm, Ute; Wendt-Pienkowski, Evelyn; Wang, Liyan; Huang, Sheng-Xiong; Unsin, Claudia; Tao, Meifeng; Coughlin, Jane M.; Shen, Ben

    2011-01-01

    The biosynthetic gene clusters for the glycopeptide antitumor antibiotics bleomycin (BLM), tallysomycin (TLM), and zorbamycin (ZBM) have been recently cloned and characterized from Streptomyces verticillus ATCC15003, Streptoalloteichus hindustanus E465-94 ATCC31158, and Streptomyces flavoviridis ATCC21892, respectively. The striking similarities and differences among the biosynthetic gene clusters for the three structurally related glycopeptide antitumor antibiotics prompted us to compare and contrast their respective biosynthetic pathways and to investigate various enzymatic elements. The presence of different numbers of isolated nonribosomal peptide synthetase (NRPS) domains in all three clusters does not result in major structural differences of the respective compounds. The seemingly identical domain organization of the NRPS modules responsible for heterocycle formation, on the other hand, is contrasted by the biosynthesis of two different structural entities, bithiazole and thiazolinyl-thiazole, for BLM/TLM and ZBM, respectively. Variations in sugar biosynthesis apparently dictate the glycosylation patterns distinct for each of the BLM, TLM, and ZBM glycopeptide scaffolds. These observations demonstrate nature’s ingenuity and flexibility in achieving structural differences and similarities via various mechanisms and will surely inspire combinatorial biosynthesis efforts to expand on natural product structural diversity. PMID:21210656

  12. 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

  13. Construction of a chimeric biosynthetic pathway for the de novo biosynthesis of rosmarinic acid in Escherichia coli.

    PubMed

    Bloch, Sarah E; Schmidt-Dannert, Claudia

    2014-11-01

    Hydroxycinnamic acid esters (HCEs) are widely-distributed phenylpropanoid-derived plant natural products. Rosmarinic acid (RA), 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, microbial production of HCEs could be 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, and the related HCE isorinic acid (IA), in Escherichia coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We demonstrated HCE biosynthesis using three plant rosmarinic acid synthase (RAS) orthologues, which exhibited different levels of HCE biosynthesis but produced the same ratio of IA to RA. This work serves as a proof-of-concept for a microbial production platform for HCEs by using a modular biosynthetic approach to access diverse natural and non-natural HCEs.

  14. Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol.

    PubMed

    Sheppard, Micah J; Kunjapur, Aditya M; Wenck, Spencer J; Prather, Kristala L J

    2014-09-24

    Increasingly complex metabolic pathways have been engineered by modifying natural pathways and establishing de novo pathways with enzymes from a variety of organisms. Here we apply retro-biosynthetic screening to a modular pathway design to identify a redox neutral, theoretically high yielding route to a branched C6 alcohol. Enzymes capable of converting natural E. coli metabolites into 4-methyl-pentanol (4MP) via coenzyme A (CoA)-dependent chemistry were taken from nine different organisms to form a ten-step de novo pathway. Selectivity for 4MP is enhanced through the use of key enzymes acting on acyl-CoA intermediates, a carboxylic acid reductase from Nocardia iowensis and an alcohol dehydrogenase from Leifsonia sp. strain S749. One implementation of the full pathway from glucose demonstrates selective carbon chain extension and acid reduction with 4MP constituting 81% (90±7 mg l(-1)) of the observed alcohol products. The highest observed 4MP titre is 192±23 mg l(-1). These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.

  15. 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. PMID:27285777

  16. 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

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

    PubMed

    Gómez-García, María del Rocío; 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

  18. 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

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

    PubMed

    Gómez-García, María del Rocío; 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.

  20. 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

  1. Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation.

    PubMed

    Gallagher, C E; Cervantes-Cervantes, M; Wurtzel, E T

    2003-02-01

    Carotenoids synthesized in plants but not animals are essential for human nutrition. Therefore, ongoing efforts to metabolically engineer plants for improved carotenoid content benefit from the identification of genes that affect carotenoid accumulation, possibly highlighting potential challenges when pyramiding traits represented by multiple biosynthetic pathways. We employed a heterologous bacterial system to screen for maize cDNAs encoding products that alter carotenoid accumulation either positively or negatively. Genes encoding carotenoid biosynthetic enzymes from the bacterium Erwinia uredovora were introduced into Escherichia coli cells that were subsequently transfected with a maize endosperm cDNA expression library; and these doubly transformed cells were then screened for altered carotenoid accumulation. DNA sequencing and characterization of one cDNA class conferring increased carotenoid content led to the identification of maize cDNAs encoding isopentenyl diphosphate isomerase. A cDNA that caused a reduced carotenoid content in E. coli was also identified. Based on DNA sequence analysis, DNA hybridization, and further functional testing, this latter cDNA was found to encode the small subunit of ADP-glucose pyrophosphorylase, a rate-controlling enzyme in starch biosynthesis that has been of interest for enhancing plant starch content.

  2. 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.

  3. 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

  4. Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation

    SciTech Connect

    Nawrath, C.; Poirier, Y.; Somerville, C. )

    1994-12-20

    In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plans accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-[mu]m granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand. 20 refs., 6 figs.

  5. 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

  6. 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.

  7. 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

  8. Heterologous expression, purification, and enzymatic characterization of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus.

    PubMed

    Steiger, Sabine; Mazet, Andreas; Sandmann, Gerhard

    2003-06-01

    The carotenoid 1,2-hydratase CrtC from Rubrivivax gelatinosus has been expressed in Escherichia coli in an active form and purified by affinity chromatography. The enzyme catalyzes the conversion of various acyclic carotenes including 1-hydroxy derivatives. This broad substrate specificity reflects the participation of CrtC in 1'-HO-spheroidene and in spirilloxanthin biosynthesis. Enzyme kinetic studies including the determination of substrate specificity constants indicate that among the alternative biosynthetic routes to 1'-HO-spheroidene the one via spheroidene is the dominating pathway. In contrast to CrtC from Rvi. gelatinosus, the equivalent enzyme from Rhodobacter capsulatus, a closely related bacterium which lacks the biosynthetic branch to spirilloxanthin and accumulates spheroidene instead of substantial amounts of 1'-HO-spheroidene, is extremely poor in converting 1-HO-carotenoids. The individual catalytic properties of both carotenoid 1,2-hydratases reflect the in situ carotenogenic pathways in both purple photosynthetic bacteria.

  9. 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).

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

    PubMed

    Vranish, James N; Russell, William K; Yu, Lusa E; Cox, Rachael M; Russell, David H; Barondeau, David P

    2015-01-14

    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 cluster 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. We anticipate that this cluster detection methodology will transform the study of Fe-S cluster pathways and potentially other metal cofactor biosynthetic pathways.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  12. 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

  13. 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.

  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. 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.

  16. 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

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

    PubMed

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

    2015-11-24

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

  18. 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

  19. 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

  20. 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. PMID:26893492

  1. 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.

  2. 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

  3. Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-phellandrene production in Escherichia coli cultures.

    PubMed

    Formighieri, Cinzia; Melis, Anastasios

    2014-12-01

    Escherichia coli was used as a microbial system for the heterologous synthesis of β-phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-phellandrene synthase (PHLS), alone or in combination with Picea abies geranyl-diphosphate synthase in E. coli, resulted in no β-phellandrene accumulation, in sharp contrast to observations with PHLS-transformed cyanobacteria. Lack of β-phellandrene biosynthesis in E. coli was attributed to the limited endogenous carbon partitioning through the native 2-C-methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems. PMID:25116411

  4. A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone.

    PubMed

    Martin, Collin H; Dhamankar, Himanshu; Tseng, Hsien-Chung; Sheppard, Micah J; Reisch, Christopher R; Prather, Kristala L J

    2013-01-01

    The replacement of petroleum feedstocks with biomass to produce platform chemicals requires the development of appropriate conversion technologies. 3-Hydroxy-γ-butyrolactone has been identified as one such chemical; however, there are no naturally occurring biosynthetic pathways for this molecule or its hydrolyzed form, 3,4-dihydroxybutyric acid. Here we design a novel pathway to produce various chiral 3-hydroxyacids, including 3,4-dihydroxybutyric acid, consisting of enzymes that condense two acyl-CoAs, stereospecifically reduce the resulting β-ketone and hydrolyze the CoA thioester to release the free acid. Acetyl-CoA serves as one substrate for the condensation reaction, whereas the second is produced intracellularly by a pathway enzyme that converts exogenously supplied organic acids. Feeding of butyrate, isobutyrate and glycolate results in the production of 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate and 3,4-dihydroxybutyric acid+3-hydroxy-γ-butyrolactone, respectively, molecules with potential uses in applications from materials to medicines. We also unexpectedly observe the condensation reaction resulting in the production of the 2,3-dihydroxybutyric acid isomer, a potential value-added monomer.

  5. Auxin Input Pathway Disruptions Are Mitigated by Changes in Auxin Biosynthetic Gene Expression in Arabidopsis1[W][OPEN

    PubMed Central

    Spiess, Gretchen M.; Hausman, Amanda; Yu, Peng; Cohen, Jerry D.; Rampey, Rebekah A.; Zolman, Bethany K.

    2014-01-01

    Auxin is a phytohormone involved in cell elongation and division. Levels of indole-3-acetic acid (IAA), the primary auxin, are tightly regulated through biosynthesis, degradation, sequestration, and transport. IAA is sequestered in reversible processes by adding amino acids, polyol or simple alcohols, or sugars, forming IAA conjugates, or through a two-carbon elongation forming indole-3-butyric acid. These sequestered forms of IAA alter hormone activity. To gain a better understanding of how auxin homeostasis is maintained, we have generated Arabidopsis (Arabidopsis thaliana) mutants that combine disruptions in the pathways, converting IAA conjugates and indole-3-butyric acid to free IAA. These mutants show phenotypes indicative of low auxin levels, including delayed germination, abnormal vein patterning, and decreased apical dominance. Root phenotypes include changes in root length, root branching, and root hair growth. IAA levels are reduced in the cotyledon tissue but not meristems or hypocotyls. In the combination mutants, auxin biosynthetic gene expression is increased, particularly in the YUCCA/Tryptophan Aminotransferase of Arabidopsis1 pathway, providing a feedback mechanism that allows the plant to compensate for changes in IAA input pathways and maintain cellular homeostasis. PMID:24891612

  6. 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.

  7. Diversity in the carotenoid profiles and the expression of genes related to carotenoid accumulation among citrus genotypes.

    PubMed

    Ikoma, Yoshinori; Matsumoto, Hikaru; Kato, Masaya

    2016-01-01

    Carotenoids are not only important to the plants themselves but also are beneficial to human health. Since citrus fruit is a good source of carotenoids for the human diet, it is important to study carotenoid profiles and the accumulation mechanism in citrus fruit. Thus, in the present paper, we describe the diversity in the carotenoid profiles of fruit among citrus genotypes. In regard to carotenoids, such as β-cryptoxanthin, violaxanthin, lycopene, and β-citraurin, the relationship between the carotenoid profile and the expression of carotenoid-biosynthetic genes is discussed. Finally, recent results of quantitative trait locus (QTL) analyses of carotenoid contents and expression levels of carotenoid-biosynthetic genes in citrus fruit are shown.

  8. Diversity in the carotenoid profiles and the expression of genes related to carotenoid accumulation among citrus genotypes

    PubMed Central

    Ikoma, Yoshinori; Matsumoto, Hikaru; Kato, Masaya

    2016-01-01

    Carotenoids are not only important to the plants themselves but also are beneficial to human health. Since citrus fruit is a good source of carotenoids for the human diet, it is important to study carotenoid profiles and the accumulation mechanism in citrus fruit. Thus, in the present paper, we describe the diversity in the carotenoid profiles of fruit among citrus genotypes. In regard to carotenoids, such as β-cryptoxanthin, violaxanthin, lycopene, and β-citraurin, the relationship between the carotenoid profile and the expression of carotenoid-biosynthetic genes is discussed. Finally, recent results of quantitative trait locus (QTL) analyses of carotenoid contents and expression levels of carotenoid-biosynthetic genes in citrus fruit are shown. PMID:27069398

  9. 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

  10. 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

  11. 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.

  12. 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

  13. A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation.

    PubMed

    Sevier, C S; Cuozzo, J W; Vala, A; Aslund, F; Kaiser, C A

    2001-10-01

    Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain. ERV2 encodes a luminal ER protein of relative molecular mass 22,000. Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds. Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol-disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway. PMID:11584268

  14. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. 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

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

    PubMed

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

    2016-01-01

    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.

  18. 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.

  19. Characterization of the nocardiopsin biosynthetic gene cluster reveals similarities to and differences from the rapamycin and FK-506 pathways.

    PubMed

    Bis, Dana M; Ban, Yang H; James, Elle D; Alqahtani, Norah; Viswanathan, Rajesh; Lane, Amy L

    2015-04-13

    Macrolide-pipecolate natural products, such as rapamycin (1) and FK-506 (2), are renowned modulators of FK506-binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB-M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A-D (4-7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L-lysine into the L-pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP-binding metabolites and for probing new enzymology in nature's polyketide tailoring arsenal. PMID:25755076

  20. 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

  1. 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.

  2. Evidence from mycelial studies for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi.

    PubMed Central

    Wood, S G; Gottlieb, D

    1978-01-01

    Phytophthora cinnamomi, a member of the Pythiacease, does not synthesize sterols. Small amounts of squalene, but no squalene epoxide or sterol, were isolated from the dried mycelium of this fungus after growth in sterol-free medium. The dried mycelium of Rhizoctonia solani, a sterol-synthesizing fungus grown under the same conditions, contained small amounts of squalene and squalene epoxide and large amounts of ergosterol. When the two organisms were grown in the presence of [14C]acetate, only labelled geraniol, farnesol and squalene were recovered from the P. cinnamomi mycelium, whereas labelled geraniol, farnesol, squalene, squalene epoxide and ergosterol were recovered from the R. solani mycelium. Similar results were obtained when the organisms were incubated in the presence of [2(-14)C]mevalonate; in this case, labelled lanosterol was also detected in the R. solani mycelium. Both organisms, when incubated in the presence of unlabelled squalene, squalene epoxide or lanosterol, incorporated these compounds into their mycelia; however, only the R. solani mycelium was able to convert these substrates into products further along the sterol pathway. It appears that squalene is the terminal compound in the sterol biosynthetic pathway of P. cinnamomi. PMID:637849

  3. 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

  4. 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.

  5. 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. PMID:25326165

  6. Carotenoids in Microalgae.

    PubMed

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

    2016-01-01

    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. PMID:27485224

  7. 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. PMID:27392312

  8. 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.

  9. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. 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

  12. 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.

  13. Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves

    PubMed Central

    2010-01-01

    Background Symptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (Vitis vinifera L.) cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. The reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis. Results We examined six putative constitutively expressed genes, Ubiquitin, Actin, GAPDH, EF1-a, SAND and NAD5, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using the geNorm program, a combination of two genes (Actin and NAD5) was identified as the stable set of reference genes for normalization of gene expression data obtained from grapevine leaves. By using gene-specific RT-qPCR in combination with a reliable normalization factor, we compared relative expression of the flavonoid biosynthetic pathway genes between leaves infected with Grapevine leafroll-associated virus 3 (GLRaV-3) and exhibiting GLRD symptoms and virus-free green leaves obtained from a red-fruited wine grape cultivar (cv. Merlot). The expression levels of these different genes ranged from two- to fifty-fold increase in virus-infected leaves. Among them, CHS3, F3'5'H, F3H1, LDOX, LAR1 and MybA1 showed greater than 10-fold increase suggesting that they were expressed at significantly higher levels in virus-infected symptomatic leaves. HPLC profiling of anthocyanins extracted from leaves indicated the presence of cyanidin-3-glucoside and malvidin-3-glucoside only in virus-infected symptomatic leaves. The results also showed 24% higher levels of flavonols in virus-infected symptomatic leaves than in virus-free green leaves, with quercetin followed by myricetin being the predominant compounds. Proanthocyanidins, estimated as total tannins by protein precipitation method, were 36% higher in virus-infected symptomatic

  14. The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

    PubMed Central

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

    2009-01-01

    Background 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. Methods and Findings 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 ∼600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5

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

    PubMed

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

    2014-08-29

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

  16. Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440.

    PubMed

    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

    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

  17. 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

  18. 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

  19. 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.

  20. Crystal Structure of Baeyer−Villiger Monooxygenase MtmOIV, the Key Enzyme of the Mithramycin Biosynthetic Pathway

    SciTech Connect

    Beam, Miranda P.; Bosserman, Mary A.; Noinaj, Nicholas; Wehenkel, Marie; Rohr, Jurgen; Kentucky

    2009-06-01

    Baeyer-Villiger monooxygenases (BVMOs), mostly flavoproteins, were shown to be powerful biocatalysts for synthetic organic chemistry applications and were also suggested to play key roles for the biosyntheses of various natural products. Here we present the three-dimensional structure of MtmOIV, a 56 kDa homodimeric FAD- and NADPH-dependent monooxygenase, which catalyzes the key frame-modifying step of the mithramycin biosynthetic pathway and currently the only BVMO proven to react with its natural substrate via a Baeyer-Villiger reaction. MtmOIV's structure was determined by X-ray crystallography using molecular replacement to a resolution of 2.9 A. MtmOIV cleaves a C-C bond, essential for the conversion of the biologically inactive precursor, premithramycin B, into the active drug mithramycin. The MtmOIV structure combined with substrate docking calculations and site-directed mutagenesis experiments identifies several residues that participate in cofactor and substrate binding. Future experimentation aimed at broadening the substrate specificity of the enzyme could facilitate the generation of chemically diverse mithramycin analogues through combinatorial biosynthesis.

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

    SciTech Connect

    Christianson,C.; Montavon, T.; Van Lanen, S.; Shen, B.; Bruner, S.

    2007-01-01

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

  2. 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.

  3. 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.

  4. 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

    2015-01-01

    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. PMID:25966245

  5. 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

  6. A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis.

    PubMed

    Zhao, Qing; Zhang, Yang; Wang, Gang; Hill, Lionel; Weng, Jing-Ke; Chen, Xiao-Ya; Xue, Hongwei; Martin, Cathie

    2016-04-01

    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

  7. Orthogonal Fatty Acid Biosynthetic Pathway Improves Fatty Acid Ethyl Ester Production in Saccharomyces cerevisiae.

    PubMed

    Eriksen, Dawn T; HamediRad, Mohammad; Yuan, Yongbo; Zhao, Huimin

    2015-07-17

    Fatty acid ethyl esters (FAEEs) are a form of biodiesel that can be microbially produced via a transesterification reaction of fatty acids with ethanol. The titer of microbially produced FAEEs can be greatly reduced by unbalanced metabolism and an insufficient supply of fatty acids, resulting in a commercially inviable process. Here, we report on a pathway engineering strategy in Saccharomyces cerevisiae for enhancing the titer of microbially produced FAEEs by providing the cells with an orthogonal route for fatty acid synthesis. The fatty acids generated from this heterologous pathway would supply the FAEE production, safeguarding endogenous fatty acids for cellular metabolism and growth. We investigated the heterologous expression of a Type-I fatty acid synthase (FAS) from Brevibacterium ammoniagenes coupled with WS/DGAT, the wax ester synthase/acyl-coenzyme that catalyzes the transesterification reaction with ethanol. Strains harboring the orthologous fatty acid synthesis yielded a 6.3-fold increase in FAEE titer compared to strains without the heterologous FAS. Variations in fatty acid chain length and degree of saturation can affect the quality of the biodiesel; therefore, we also investigated the diversity of the fatty acid production profile of FAS enzymes from other Actinomyces organisms. PMID:25594225

  8. Sex and season influence gonadal steroid biosynthetic pathways, end-product production and steroid conjugation in blotched blue-tongued lizards (Tiliqua nigrolutea).

    PubMed

    Edwards, Ashley; Jones, Susan M; Davies, Noel W

    2003-11-01

    We examined differences in gonadal steroid production and biosynthetic pathway activity with changing reproductive condition and between sexes in the scincid lizard, Tiliqua nigrolutea. We observed clear seasonal and sexual variation in the production of androgens and steroid conjugates, but detected no 17beta-estradiol or 5alpha-dihydrotestosterone produced by the gonads. An alternative steroid, more polar than estradiol, was detected: an investigation of this steroid is reported separately [Gen. Comp. Endocrinol. 129 (2002) 114]. There were seasonal and sex-related differences in steroid biosynthetic pathway activity. The Delta5 pathway metabolite, dehydroepiandrosterone, was detected only in males, and only from incubations using regressed testicular tissue. There was also a seasonal difference between the sexes in rates of progesterone accumulation, although the absence of corresponding elevated plasma concentrations suggests that the role of progesterone switches from a directly acting hormone to a precursor for others during the reproductive cycle in females. These results suggest that within the traditional view that vertebrate biosynthetic pathway activity and end-products are phylogenetically conserved, there is likely to be considerably species- and/or genus-specific variation.

  9. 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

  10. 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.

  11. miRNA Nomenclature: A View Incorporating Genetic Origins, Biosynthetic Pathways, and Sequence Variants.

    PubMed

    Desvignes, T; Batzel, P; Berezikov, E; Eilbeck, K; Eppig, J T; McAndrews, M S; Singer, A; Postlethwait, J H

    2015-11-01

    High-throughput sequencing of miRNAs has revealed the diversity and variability of mature and functional short noncoding RNAs, including their genomic origins, biogenesis pathways, sequence variability, and newly identified products such as miRNA-offset RNAs (moRs). Here we review known cases of alternative mature miRNA-like RNA fragments and propose a revised definition of miRNAs to encompass this diversity. We then review nomenclature guidelines for miRNAs and propose to extend nomenclature conventions to align with those for protein-coding genes established by international consortia. Finally, we suggest a system to encompass the full complexity of sequence variations (i.e., isomiRs) in the analysis of small RNA sequencing experiments.

  12. Pseudomonas aeruginosa d-Arabinofuranose Biosynthetic Pathway and Its Role in Type IV Pilus Assembly*

    PubMed Central

    Harvey, Hanjeong; Kus, Julianne V.; Tessier, Luc; Kelly, John; Burrows, Lori L.

    2011-01-01

    Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked d-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-d-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to d-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilAIV produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae. PMID:21676874

  13. Pseudomonas aeruginosa D-arabinofuranose biosynthetic pathway and its role in type IV pilus assembly.

    PubMed

    Harvey, Hanjeong; Kus, Julianne V; Tessier, Luc; Kelly, John; Burrows, Lori L

    2011-08-12

    Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked D-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-D-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to D-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilA(IV) produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae.

  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. A new caffeine biosynthetic pathway in tea leaves: utilisation of adenosine released from the S-adenosyl-L-methionine cycle.

    PubMed

    Koshiishi, C; Kato, A; Yama, S; Crozier, A; Ashihara, H

    2001-06-15

    The four-step caffeine biosynthetic pathway includes three methylation steps that utilise S-adenosyl-L-methionine (SAM) as the methyl donor. In the process SAM is converted to S-adenosyl-L-homocysteine (SAH) which in turn is hydrolysed to L-homocysteine and adenosine. Significant amounts of radioactivity from [methyl-(14)C]methionine and [methyl-(14)C]SAM were incorporated into theobromine and caffeine in young tea leaf segments, and very high SAH hydrolase activity was found in cell-free extracts from young tea leaves. Substantial amounts of radioactivity from [adenosyl-(14)C]SAH were also recovered as theobromine and caffeine in tea leaf segments, indicating that adenosine derived from SAH is utilised for the synthesis of the purine ring of caffeine. From the profiles of activity of related enzymes in tea leaf extracts, it is proposed that the major route from SAM to caffeine is a SAM-->SAH-->adenosine-->adenine-->AMP-->IMP-->XMP-->xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine pathway. In addition, direct adenosine kinase-catalysed formation of AMP from adenosine may participate as an alternative minor route. The activity of two of the three N-methyltransferase activities involved in caffeine biosynthesis and part of the activities of SAH hydrolase, adenosine nucleosidase, adenine phosphoribosyltransferase and adenosine kinase were located in tea chloroplasts. In contrast, no detectable activity of SAM synthetase was associated with the purified chloroplast fraction. This is a first demonstration that the purine skeleton of caffeine is synthesised from adenosine released from the SAM cycle.

  16. 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.

  17. Structure and function of the RedJ protein, a thioesterase from the prodiginine biosynthetic pathway in Streptomyces coelicolor.

    PubMed

    Whicher, Jonathan R; Florova, Galina; Sydor, Paulina K; Singh, Renu; Alhamadsheh, Mamoun; Challis, Gregory L; Reynolds, Kevin A; Smith, Janet L

    2011-06-24

    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.

  18. The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence.

    PubMed

    de Gontijo, Fabiano Assis; Pascon, Renata C; Fernandes, Larissa; Machado, Joel; Alspaugh, J Andrew; Vallim, Marcelo A

    2014-09-01

    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

  19. Hereditary Tyrosinemia and the Heme Biosynthetic Pathway. PROFOUND INHIBITION OF δ-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY BY SUCCINYLACETONE

    PubMed Central

    Sassa, Shigeru; Kappas, Attallah

    1983-01-01

    Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of δ-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 × 10-7 M to 3 × 10-7 M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic

  20. 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

  1. 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.

  2. 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

  3. 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

  4. An unexpectedly branched biosynthetic pathway for bacteriochlorophyll b capable of absorbing near-infrared light

    PubMed Central

    Tsukatani, Yusuke; Yamamoto, Haruki; Harada, Jiro; Yoshitomi, Taichi; Nomata, Jiro; Kasahara, Masahiro; Mizoguchi, Tadashi; Fujita, Yuichi; Tamiaki, Hitoshi

    2013-01-01

    Chlorophyllous pigments are essential for photosynthesis. Bacteriochlorophyll (BChl) b has the characteristic C8-ethylidene group and therefore is the sole naturally occurring pigment having an absorption maximum at near-infrared light wavelength. Here we report that chlorophyllide a oxidoreductase (COR), a nitrogenase-like enzyme, showed distinct substrate recognition and catalytic reaction between BChl a- and b-producing proteobacteria. COR from BChl b-producing Blastochloris viridis synthesized the C8-ethylidene group from 8-vinyl-chlorophyllide a. In contrast, despite the highly conserved primary structures, COR from BChl a-producing Rhodobacter capsulatus catalyzes the C8-vinyl reduction as well as the previously known reaction of the C7 = C8 double bond reduction on 8-vinyl-chlorophyllide a. The present data indicate that the plasticity of the nitrogenase-like enzyme caused the branched pathways of BChls a and b biosynthesis, ultimately leading to ecologically different niches of BChl a- and b-based photosynthesis differentiated by more than 150 nm wavelength. PMID:23386973

  5. 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

  6. 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

  7. Small rho GTPases and cholesterol biosynthetic pathway intermediates in African swine fever virus infection.

    PubMed

    Quetglas, Jose I; Hernáez, Bruno; Galindo, Inmaculada; Muñoz-Moreno, Raquel; Cuesta-Geijo, Miguel A; Alonso, Covadonga

    2012-02-01

    The integrity of the cholesterol biosynthesis pathway is required for efficient African swine fever virus (ASFV) infection. Incorporation of prenyl groups into Rho GTPases plays a key role in several stages of ASFV infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. We found that Rho GTPase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions. Furthermore, abundant defective virions reached the plasma membrane, and filopodia formation in exocytosis was abrogated. Rac1 was activated at early ASFV infection stages, coincident with microtubule acetylation, a process that stabilizes microtubules for virus transport. Rac1 inhibition did not affect the viral entry step itself but impaired subsequent virus production. We found that specific Rac1 inhibition impaired viral induced microtubule acetylation and viral intracellular transport. These findings highlight that viral infection is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell; this modulation results critical for virus morphogenesis and in turn, triggers cytoskeleton remodeling, such as microtubule stabilization for viral transport during early infection.

  8. Small Rho GTPases and Cholesterol Biosynthetic Pathway Intermediates in African Swine Fever Virus Infection

    PubMed Central

    Quetglas, Jose I.; Hernáez, Bruno; Galindo, Inmaculada; Muñoz-Moreno, Raquel; Cuesta-Geijo, Miguel A.

    2012-01-01

    The integrity of the cholesterol biosynthesis pathway is required for efficient African swine fever virus (ASFV) infection. Incorporation of prenyl groups into Rho GTPases plays a key role in several stages of ASFV infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. We found that Rho GTPase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions. Furthermore, abundant defective virions reached the plasma membrane, and filopodia formation in exocytosis was abrogated. Rac1 was activated at early ASFV infection stages, coincident with microtubule acetylation, a process that stabilizes microtubules for virus transport. Rac1 inhibition did not affect the viral entry step itself but impaired subsequent virus production. We found that specific Rac1 inhibition impaired viral induced microtubule acetylation and viral intracellular transport. These findings highlight that viral infection is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell; this modulation results critical for virus morphogenesis and in turn, triggers cytoskeleton remodeling, such as microtubule stabilization for viral transport during early infection. PMID:22114329

  9. 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.

  10. 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

  11. 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. PMID:22144352

  12. 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.

  13. A Common Biosynthetic Pathway Governs the Dimerization and Secretion of Inhibin and Related Transforming Growth Factor β (TGFβ) Ligands*

    PubMed Central

    Walton, Kelly L.; Makanji, Yogeshwar; Wilce, Matthew C.; Chan, Karen L.; Robertson, David M.; Harrison, Craig A.

    2009-01-01

    The assembly and secretion of transforming growth factor β superfamily ligands is dependent upon non-covalent interactions between their pro- and mature domains. Despite the importance of this interaction, little is known regarding the underlying regulatory mechanisms. In this study, the binding interface between the pro- and mature domains of the inhibin α-subunit was characterized using in vitro mutagenesis. Three hydrophobic residues near the N terminus of the prodomain (Leu30, Phe37, Leu41) were identified that, when mutated to alanine, disrupted heterodimer assembly and secretion. It is postulated that these residues mediate dimerization by interacting non-covalently with hydrophobic residues (Phe271, Ile280, Pro283, Leu338, and Val340) on the outer convex surface of the mature α-subunit. Homology modeling indicated that these mature residues are located at the interface between two β-sheets of the α-subunit and that their side chains form a hydrophobic packing core. Mutation of these residues likely disturbs the conformation of this region, thereby disrupting non-covalent interactions with the prodomain. A similar hydrophobic interface was identified spanning the pro- and mature domains of the inhibin βA-subunit. Mutation of key residues, including Ile62, Leu66, Phe329, and Pro341, across this interface was disruptive for the production of both inhibin A and activin A. In addition, mutation of Ile62 and Leu66 in the βA-propeptide reduced its ability to bind, or inhibit the activity of, activin A. Conservation of the identified hydrophobic motifs in the pro- and mature domains of other transforming growth factor β superfamily ligands suggests that we have identified a common biosynthetic pathway governing dimer assembly. PMID:19193648

  14. 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.

  15. 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

  16. 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

  17. Biosynthetic pathway toward carbohydrate-like moieties of alnumycins contains unusual steps for C-C bond formation and cleavage

    PubMed Central

    Oja, Terhi; Klika, Karel D.; Appassamy, Laura; Sinkkonen, Jari; Mäntsälä, Pekka; Niemi, Jarmo; Metsä-Ketelä, Mikko

    2012-01-01

    Carbohydrate moieties are important components of natural products, which are often imperative for the solubility and biological activity of the compounds. The aromatic polyketide alnumycin A contains an extraordinary sugar-like 4′-hydroxy-5′-hydroxymethyl-2′,7′-dioxane moiety attached via a carbon-carbon bond to the aglycone. Here we have extensively investigated the biosynthesis of the dioxane unit through 13C labeling studies, gene inactivation experiments and enzymatic synthesis. We show that AlnA and AlnB, members of the pseudouridine glycosidase and haloacid dehalogenase enzyme families, respectively, catalyze C-ribosylation conceivably through Michael-type addition of d-ribose-5-phosphate and dephosphorylation. The ribose moiety may be attached both in furanose (alnumycin C) and pyranose (alnumycin D) forms. The C1′-C2′ bond of alnumycin C is subsequently cleaved and the ribose unit is rearranged into an unprecedented dioxolane (cis-bicyclo[3.3.0]-2′,4′,6′-trioxaoctan-3′β-ol) structure present in alnumycin B. The reaction is catalyzed by Aln6, which belongs to a previously uncharacterized enzyme family. The conversion was accompanied with consumption of O2 and formation of H2O2, which allowed us to propose that the reaction may proceed via hydroxylation of C1′ followed by retro-aldol cleavage and acetal formation. Interestingly, no cofactors could be detected and the reaction was also conducted in the presence of metal chelating agents. The last step is the conversion of alnumycin B into the final end-product alnumycin A catalyzed by Aln4, an NADPH-dependent aldo-keto reductase. This characterization of the dioxane biosynthetic pathway sets the basis for the utilization of C-C bound ribose, dioxolane and dioxane moieties in the generation of improved biologically active compounds. PMID:22474343

  18. 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

  19. Carotenoid Distribution in Nature.

    PubMed

    Alcaíno, Jennifer; Baeza, Marcelo; Cifuentes, Víctor

    2016-01-01

    Carotenoids are naturally occurring red, orange and yellow pigments that are synthesized by plants and some microorganisms and fulfill many important physiological functions. This chapter describes the distribution of carotenoid in microorganisms, including bacteria, archaea, microalgae, filamentous fungi and yeasts. We will also focus on their functional aspects and applications, such as their nutritional value, their benefits for human and animal health and their potential protection against free radicals. The central metabolic pathway leading to the synthesis of carotenoids is described as the three following principal steps: (i) the synthesis of isopentenyl pyrophosphate and the formation of dimethylallyl pyrophosphate, (ii) the synthesis of geranylgeranyl pyrophosphate and (iii) the synthesis of carotenoids per se, highlighting the differences that have been found in several carotenogenic organisms and providing an evolutionary perspective. Finally, as an example, the synthesis of the xanthophyll astaxanthin is discussed. PMID:27485217

  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.

  1. 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. PMID:25438140

  2. 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

  3. RNA interference-based gene silencing of phytoene synthase impairs growth, carotenoids, and plastid phenotype in Oncidium hybrid orchid.

    PubMed

    Liu, Jian-Xin; Chiou, Chung-Yi; Shen, Chin-Hui; Chen, Peng-Jen; Liu, Yao-Chung; Jian, Chin-Der; Shen, Xiao-Lan; Shen, Fu-Quan; Yeh, Kai-Wun

    2014-01-01

    Phytoene synthase (PSY) is the first rate-limiting regulatory enzyme in the carotenoid biosynthesis pathway. In order to modify the floral color pattern by reducing carotenoid contents, a phytoene synthase-RNAi construct was delivered into protocorm-like body (PLB) of Oncidium hybrid orchid. The transgenic orchids show down-regulated level of PSY and geranyl synthase gene. They displayed semi-dwarf phenotype and brilliant green leaves. The microscopic anatomy revealed development-arrested plastids with rare grana. The total carotenoid content was decreased and the efficiency of the photosynthetic electron transport was declined. The chlorophyll level and the expression of chlorophyll biosynthetic genes, such as OgGLUTR and OgCS were dramatically reduced. HPLC analysis showed that the endogenous level of gibberellic acid and abscisic acid in the dwarf transformants are 4-fold lower than in wild type plants. In addition, chilling tolerance of the transgenic Oncidium plants was reduced. The data showed that down-regulation of PSY resulted in alterations of gene expression in enzymes involved in many metabolic pathways, such as carotenoid, gibberellic acid, abscisic acid and chlorophyll biosynthetic pathway as well as causes predominant defects in plant growth and development. PMID:25221736

  4. Biosynthetic Studies of 13-Desmethylspirolide C Produced by Alexandrium ostenfeldii (= A. peruvianum): Rationalization of the Biosynthetic Pathway Following Incorporation of (13)C-Labeled Methionine and Application of the Odd-Even Rule of Methylation.

    PubMed

    Anttila, Matthew; Strangman, Wendy; York, Robert; Tomas, Carmelo; Wright, Jeffrey L C

    2016-03-25

    Understanding the biosynthesis of dinoflagellate polyketides presents many unique challenges. Because of the remaining hurdles to dinoflagellate genome sequencing, precursor labeling studies remain the only viable way to investigate dinoflagellate biosynthesis. However, prior studies have shown that polyketide chain assembly does not follow any of the established processes. Additionally, acetate, the common precursor for polyketides, is frequently scrambled, thus compromising interpretation. These factors are further compounded by low production yields of the compounds of interest. A recent report on the biosynthesis of spirolides, a group belonging to the growing class of toxic spiroimines, provided some insight into the polyketide assembly process based on acetate labeling studies, but many details were left uncertain. By feeding (13)C methyl-labeled methionine to cultures of Alexandrium ostenfeldii, the producing organism of 13-desmethylspirolide C, and application of the odd-even methylation rule, the complete biosynthetic pathway has been established.

  5. 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

  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. 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

  8. 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.

  9. Genetic manipulation of carotenoid biosynthesis in the green sulfur bacterium Chlorobium tepidum.

    PubMed

    Frigaard, Niels-Ulrik; Maresca, Julia A; Yunker, Colleen E; Jones, A Daniel; Bryant, Donald A

    2004-08-01

    The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur

  10. Carotenoids' production from halophilic bacteria.

    PubMed

    de Lourdes Moreno, María; Sánchez-Porro, Cristina; García, María Teresa; Mellado, Encarnación

    2012-01-01

    Carotenoids have received considerable attention due to their interesting industrial applications and, more importantly, their potential beneficial effects on human health. Halophiles comprise a heterogeneous group of microorganisms that need salts for optimal growth. The pigments produced by these halophilic organisms comprise phytoene, β-carotene, lycopene, derivatives of bacterioruberin, and salinixanthin. Here, we describe the procedure to obtain salinixanthin from the extremely halophilic bacterium Salinibacter ruber. Additionally, we describe the expression of the β-carotene biosynthetic genes crtE, crtY, crtI, and crtB from Pantoea agglomerans in the moderately halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure β-carotene. Thus, the use of these halophilic microorganisms as a source of carotenoids constitutes an important commercial alternative in the production of carotenoids from biological sources. PMID:22623305

  11. Increase in nutritionally important sweet corn kernel carotenoids following Mesotrione and atrazine applications.

    PubMed

    Kopsell, Dean A; Armel, Gregory R; Mueller, Thomas C; Sams, Carl E; Deyton, Dennis E; McElroy, J Scott; Kopsell, David E

    2009-07-22

    The herbicide mesotrione inhibits a critical enzyme, phytoene desaturase, in plant carotenoid biosynthesis. Mesotrione is currently labeled for selective weed control in sweet corn ( Zea mays var. rugosa). Mesotrione applied alone, or in mixtures with the photosystem II inhibitor atrazine, acted to increase concentrations of kernel antheraxanthin, lutein, and zeaxanthin carotenoids in several sweet corn genotypes. Kernel lutein and zeaxanthin levels significantly increased 15.6% after mesotrione + atrazine early postemergence applications, as compared to the control treatment. It appears that mesotrione applications resulted in greater pools of kernel carotenoids once the sweet corn genotypes expressing moderate injury overcame the initial herbicidal photo-oxidative stress. This is the first report of herbicides directly up-regulating the carotenoid biosynthetic pathway in corn kernels, which is associated with the nutritional quality of sweet corn. Enhanced accumulation of lutein and zeaxanthin is important because dietary carotenoids function in suppressing aging eye diseases such as macular degeneration, now affecting 1.75 million older Americans. PMID:19537793

  12. Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum.

    PubMed

    Kadono, Takashi; Kira, Nozomu; Suzuki, Kengo; Iwata, Osamu; Ohama, Takeshi; Okada, Shigeru; Nishimura, Tomohiro; Akakabe, Mai; Tsuda, Masashi; Adachi, Masao

    2015-08-01

    Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

  13. Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum

    PubMed Central

    Kadono, Takashi; Kira, Nozomu; Suzuki, Kengo; Iwata, Osamu; Ohama, Takeshi; Okada, Shigeru; Nishimura, Tomohiro; Akakabe, Mai; Tsuda, Masashi; Adachi, Masao

    2015-01-01

    Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase. PMID:26308005

  14. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum.

    PubMed

    Huang, Wenjun; Khaldun, A B M; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus

  15. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum

    PubMed Central

    Huang, Wenjun; Khaldun, A. B. M.; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus

  16. Inhibitory effect of eugenol on aflatoxin B1 production in Aspergillus parasiticus by downregulating the expression of major genes in the toxin biosynthetic pathway.

    PubMed

    Jahanshiri, Zahra; Shams-Ghahfarokhi, Masoomeh; Allameh, Abdolamir; Razzaghi-Abyaneh, Mehdi

    2015-07-01

    Aflatoxin contamination of grains and agro-products is a serious food safety issue and a significant economic concern worldwide. In the present study, the effects of eugenol on Aspergillus parasiticus growth and aflatoxin production were studied in relation to the expression of some essential genes involved in aflatoxin biosynthetic pathway. The fungus was cultured in presence of serial two-fold concentrations of eugenol (15.62-500 μg mL(-1)) for 3 days at 28 °C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography. The expression of aflatoxin biosynthetic genes including ver-1, nor-1, pksA, omtA and aflR were evaluated by real-time PCR. Eugenol strongly inhibited A. parasiticus growth in the range of 19.16-95.83 % in a dose-dependent manner. Aflatoxin B1 production was also inhibited by the compound in the range of 15.07-98.0 %. The expressions of ver-1, nor-1, pksA, omtA and aflR genes were significantly suppressed by eugenol at concentrations of 62.5 and 125 μg mL(-1). These results indicate that eugenol may be considered as a good candidate to control toxigenic fungal growth and the subsequent contamination of food, feed and agricultural commodities by carcinogenic aflatoxins.

  17. Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway.

    PubMed

    Takos, Adam M; Knudsen, Camilla; Lai, Daniela; Kannangara, Rubini; Mikkelsen, Lisbeth; Motawia, Mohammed S; Olsen, Carl E; Sato, Shusei; Tabata, Satoshi; Jørgensen, Kirsten; Møller, Birger L; Rook, Fred

    2011-10-01

    Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.

  18. Functional and Structural Analysis of the Siderophore Synthetase AsbB through Reconstitution of the Petrobactin Biosynthetic Pathway from Bacillus anthracis*

    PubMed Central

    Nusca, Tyler D.; Kim, Youngchang; Maltseva, Natalia; Lee, Jung Yeop; Eschenfeldt, William; Stols, Lucy; Schofield, Michael M.; Scaglione, Jamie B.; Dixon, Shandee D.; Oves-Costales, Daniel; Challis, Gregory L.; Hanna, Philip C.; Pfleger, Brian F.; Joachimiak, Andrzej; Sherman, David H.

    2012-01-01

    Petrobactin, a mixed catechol-carboxylate siderophore, is required for full virulence of Bacillus anthracis, the causative agent of anthrax. The asbABCDEF operon encodes the biosynthetic machinery for this secondary metabolite. Here, we show that the function of five gene products encoded by the asb operon is necessary and sufficient for conversion of endogenous precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. In solving the crystal structure of the B. anthracis siderophore biosynthesis protein B (AsbB), we disclose a three-dimensional model of a nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. Structural characteristics provide new insight into how this bifunctional condensing enzyme can bind and adenylate multiple citrate-containing substrates followed by incorporation of both natural and unnatural polyamine nucleophiles. This activity enables formation of multiple end-stage products leading to final assembly of petrobactin. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides show that the alternative products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. These studies identify potential therapeutic targets to halt deadly infections caused by B. anthracis and other pathogenic bacteria and suggest new avenues for the chemoenzymatic synthesis of novel compounds. PMID:22408253

  19. PtoMYB92 is a Transcriptional Activator of the Lignin Biosynthetic Pathway During Secondary Cell Wall Formation in Populus tomentosa.

    PubMed

    Li, Chaofeng; Wang, Xianqiang; Ran, Lingyu; Tian, Qiaoyan; Fan, Di; Luo, Keming

    2015-12-01

    Wood is the most abundant biomass in perennial woody plants and is mainly made up of secondary cell wall. R2R3-MYB transcription factors are important regulators of secondary wall biosynthesis in plants. In this study, we describe the identification and characterization of a poplar MYB transcription factor PtoMYB92, a homolog of Arabidopsis MYB42 and MYB85, which is involved in the regulation of secondary cell wall biosynthesis. PtoMYB92 is specifically expressed in xylem tissue in poplar. Subcellular localization and transcriptional activation analysis suggest that PtoMYB92 is a nuclear-localized transcriptional activator. Overexpression of PtoMYB92 in poplar resulted in an increase in secondary cell wall thickness in stems and ectopic deposition of lignin in leaves. Quantitative real-time PCR showed that PtoMYB92 specifically activated the expression of lignin biosynthetic genes. Furthermore, transient expression assays using a β-glucuronidase (GUS) reporter gene revealed that PtoMYB92 is an activator in the lignin biosynthetic pathway during secondary cell wall formation. Taken together, our results suggest that PtoMYB92 is involved in the regulation of secondary cell wall formation in poplar by controlling the biosynthesis of monolignols.

  20. Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay.

    PubMed

    Blaszczyk-Thurin, M; Sarnesto, A; Thurin, J; Hindsgaul, O; Koprowski, H

    1988-02-29

    The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases. PMID:3348768

  1. Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses.

    PubMed

    Kampa, Annette; Gagunashvili, Andrey N; Gulder, Tobias A M; Morinaka, Brandon I; Daolio, Cristina; Godejohann, Markus; Miao, Vivian P W; Piel, Jörn; Andrésson, Ólafur S

    2013-08-13

    Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.

  2. Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses

    PubMed Central

    Kampa, Annette; Gagunashvili, Andrey N.; Gulder, Tobias A. M.; Morinaka, Brandon I.; Daolio, Cristina; Godejohann, Markus; Miao, Vivian P. W.; Piel, Jörn; Andrésson, Ólafur S.

    2013-01-01

    Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites. PMID:23898213

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

    PubMed

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

    2014-06-27

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  6. Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the LLD-ACV:bisACV ratio on the flux control.

    PubMed

    Theilgaard, H A; Nielsen, J

    1999-01-01

    An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up. It includes the formation and reduction of the dimer bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) from the first pathway intermediate LLD-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS). The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both LLD-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione. The inhibition constant of LLD-ACV, KACV is determined to be 0.54 mm. With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of LLD-ACV:bisACV. It is concluded that the flux control totally resides at the IPNS. This is a result of the regulation of the ACVS by both the LLD-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition. The measurement of an intracellular ratio of LLD-ACV:bisACV to be in the range of 1-2 moles per moles emphasises the importance of a fast conversion of LLD-ACV to IPN, and accumulation of LLD-ACV above the K(m)-value of the IPNS should therefore be avoided.

  7. Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains1

    PubMed Central

    Gandía-Herrero, Fernando; Escribano, Josefa; García-Carmona, Francisco

    2005-01-01

    Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis and in the enzymatic browning of fruits and vegetables. The role of tyrosinase in the secondary metabolism of plants still remains unclear, but its implication in betalain biosynthesis has been proposed. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this article, the betaxanthins, tyrosine-betaxanthin (portulacaxanthin II) and dopaxanthin, are reported to be physiological substrates for tyrosinase. The direct activity of tyrosinase on selected betaxanthins is characterized in depth, and conversion of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are described. Identity of the reaction products was studied by high-performance liquid chromatography and electrospray ionization-mass spectrometry. Masses determined for the reaction products were the same in all cases, 389 m/z ([M + H]+) and equal to that determined for betanidin. Data indicate that dopaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. Kinetic parameters for tyrosinase acting on dopaxanthin were evaluated, showing a high affinity for this substrate (Km = 84.3 μm). The biosynthetic scheme of betalains is reviewed and a branch is proposed based on the description of physiological substrates for tyrosinase. Lampranthus productus, Glottiphylum oligocarpum, and Glottiphylum pigmaeum are described as sources of stereopure (2S/S)-dopaxanthin. PMID:15805475

  8. Structure and Excitation Transfer Pathways in the Chlorophyll-Carotenoid Aggregate of the Photosynthetic Unit of Purple Bacteria

    NASA Astrophysics Data System (ADS)

    Schulten, Klaus

    1998-03-01

    The absorption of light by light harvesting complexes and transfer of electronic excitation to the photosynthetic reaction center (RC) has been investigated on the basis of an atomic level model of the so-called photosynthetic unit of the photosynthetic bacterium Rb. sphaeroides. The photosynthetic unit combines in the intracytoplasmic membrane a nanometric (20-100 nm) assembly of three protein complexes: (i) the photosynthetic reaction center, (ii) a ring-shaped light harvesting complex LH-I, and (iii) multiple copies of a similar complex, LH-II. The unit has been modeled using the known structure of LH-II of Rs. molischianum. The lecture describes in detail the organization of chromophores involved in primary light absorption and excitation transfer: a hierarchy of ring-shaped chlorophyl aggregates with attached carotenoids. A quantum-mechanical description of the entire light harvesting process is developed employing electron structure calculations of individual and aggregated chlorophylls and carotenoids and associated effective Hamiltonian descriptions. The transfer times calculated, ranging between 100 fs and 100 ps for various processes, are found in close agreement with measured transfer rates. The results suggest that excitons are the key carriers of the excitation transfered. The photoprotection of chlorophylls by chlorophylls through triplet excitation transfer is also described.

  9. Expression analysis of flavonoid biosynthesis genes during Arabidopsis thaliana silique and seed development with a primary focus on the proanthocyanidin biosynthetic pathway

    PubMed Central

    2010-01-01

    Background The coordinated activity of different flavonoid biosynthesis genes in Arabidopsis thaliana results in tissue-specific accumulation of flavonols, anthocyanins and proanthocyanidins (PAs). These compounds possess diverse functions in plants including light-attenuation and oxidative stress protection. Flavonoids accumulate in a stimulus- and/or development-dependent manner in specific parts of the plant. PAs accumulate in the seed coat (testa). Findings We describe the biological material and the preparation of total RNA for the AtGenExpress developmental silique and seed series. AtGenExpress ATH1 GeneChip expression data from the different stages were reanalyzed and verified using quantitative real time PCR (qPCR). We observed organ-specific transcript accumulation of specific flavonoid biosynthetic genes consistent with previously published data and our PA compound accumulation data. In addition, we investigated the regulation of PA accumulation in developing A. thaliana seeds by correlating gene expression patterns of specific flavonoid biosynthesis genes with different seed embryonic developmental stages and organs and present two useful marker genes for isolated valve and replum organs, as well as one seed-specific marker. Conclusions Potential caveats of array-based expression data are discussed based on comparisons with qPCR data. Results from ATH1 microarray and qPCR experiments revealed a shift in gene activity from general flavonoid biosynthesis at early stages of seed development to PA synthesis at late (mature) stages of embryogenesis. The examined PA accumulation-associated genes, including biosynthetic and regulatory genes, were found to be exclusively expressed in immature seeds. Accumulation of PAs initiates at the early heart stage of silique and seed development. Our findings provide new insights for further studies targeting the PA pathway in seeds. PMID:20929528

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

    PubMed Central

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

    2016-01-01

    The plant-specific WUSCHEL-related homeobox (WOX) nuclear proteins have important roles in the transcriptional regulation of many developmental processes. Among the rice (Oryza sativa) WOX proteins, a loss of OsWOX3A function in narrow leaf2 (nal2) nal3 double mutants (termed nal2/3) causes pleiotropic effects, such as narrow and curly leaves, opened spikelets, narrow grains, more tillers, and fewer lateral roots, but almost normal plant height. To examine OsWOX3A function in more detail, transgenic rice overexpressing OsWOX3A (OsWOX3A-OX) were generated; unexpectedly, all of them consistently exhibited severe dwarfism with very short and wide leaves, a phenotype that resembles that of gibberellic acid (GA)-deficient or GA-insensitive mutants. Exogenous GA3 treatment fully rescued the developmental defects of OsWOX3A-OX plants, suggesting that constitutive overexpression of OsWOX3A downregulates GA biosynthesis. Quantitative analysis of GA intermediates revealed significantly reduced levels of GA20 and bioactive GA1 in OsWOX3A-OX, possibly due to downregulation of the expression of KAO, which encodes ent-kaurenoic acid oxidase, a GA biosynthetic enzyme. Yeast one-hybrid and electrophoretic mobility shift assays revealed that OsWOX3A directly interacts with the KAO promoter. OsWOX3A expression is drastically and temporarily upregulated by GA3 and downregulated by paclobutrazol, a blocker of GA biosynthesis. These data indicate that OsWOX3A is a GA-responsive gene and functions in the negative feedback regulation of the GA biosynthetic pathway for GA homeostasis to maintain the threshold levels of endogenous GA intermediates throughout development. PMID:26767749

  11. Novel Bread Wheat Lines Enriched in Carotenoids Carrying Hordeum chilense Chromosome Arms in the ph1b Background

    PubMed Central

    Rey, María-Dolores; Calderón, María-Carmen; Rodrigo, María Jesús; Zacarías, Lorenzo; Alós, Enriqueta; Prieto, Pilar

    2015-01-01

    The use of crop wild relative species to improve major crops performance is well established. Hordeum chilense has a high potential as a genetic donor to increase the carotenoid content of wheat. Crosses between the 7Hch H. chilense substitution lines in wheat and the wheat pairing homoeologous1b (ph1b) mutant allowed the development of wheat-H. chilense translocation lines for both 7Hchα and 7Hchβ chromosome arms in the wheat background. These translocation lines were characterized by in situ hybridization and using molecular markers. In addition, reverse phase chromatography (HPLC) analysis was carried out to evaluate the carotenoid content and both 7Hchα∙7AL and 7AS∙7Hchβ disomic translocation lines. The carotenoid content in 7Hchα∙7AL and 7AS∙7Hchβ disomic translocation lines was higher than the wheat-7Hch addition line and double amount of carotenoids than the wheat itself. A proteomic analysis confirmed that the presence of chromosome 7Hch introgressions in wheat scarcely altered the proteomic profile of the wheat flour. The Psy1 (Phytoene Synthase1) gene, which is the first committed step in the carotenoid biosynthetic pathway, was also cytogenetically mapped on the 7Hchα chromosome arm. These new wheat-H. chilense translocation lines can be used as a powerful tool in wheat breeding programs to enrich the diet in bioactive compounds. PMID:26241856

  12. De Novo Transcriptome Analysis of an Aerial Microalga Trentepohlia jolithus: Pathway Description and Gene Discovery for Carbon Fixation and Carotenoid Biosynthesis

    PubMed Central

    Li, Qianqian; Liu, Jianguo; Zhang, Litao; Liu, Qian

    2014-01-01

    Background Algae in the order Trentepohliales have a broad geographic distribution and are generally characterized by the presence of abundant β-carotene. The many monographs published to date have mainly focused on their morphology, taxonomy, phylogeny, distribution and reproduction; molecular studies of this order are still rare. High-throughput RNA sequencing (RNA-Seq) technology provides a powerful and efficient method for transcript analysis and gene discovery in Trentepohlia jolithus. Methods/Principal Findings Illumina HiSeq 2000 sequencing generated 55,007,830 Illumina PE raw reads, which were assembled into 41,328 assembled unigenes. Based on NR annotation, 53.28% of the unigenes (22,018) could be assigned to gene ontology classes with 54 subcategories and 161,451 functional terms. A total of 26,217 (63.44%) assembled unigenes were mapped to 128 KEGG pathways. Furthermore, a set of 5,798 SSRs in 5,206 unigenes and 131,478 putative SNPs were identified. Moreover, the fact that all of the C4 photosynthesis genes exist in T. jolithus suggests a complex carbon acquisition and fixation system. Similarities and differences between T. jolithus and other algae in carotenoid biosynthesis are also described in depth. Conclusions/Significance This is the first broad transcriptome survey for T. jolithus, increasing the amount of molecular data available for the class Ulvophyceae. As well as providing resources for functional genomics studies, the functional genes and putative pathways identified here will contribute to a better understanding of carbon fixation and fatty acid and carotenoid biosynthesis in T. jolithus. PMID:25254555

  13. A new member of the 4-methylideneimidazole-5-one-containing aminomutase family from the enediyne kedarcidin biosynthetic pathway.

    PubMed

    Huang, Sheng-Xiong; Lohman, Jeremy R; Huang, Tingting; Shen, Ben

    2013-05-14

    4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of L-α-amino acids to β-amino acids with either an (R) or an (S) configuration. L-phenylalanine and L-tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an (R)-2-aza-3-chloro-β-tyrosine moiety reminiscent of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for (R)-2-aza-3-chloro-β-tyrosine biosynthesis starting from 2-aza-L-tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza-L-tyrosine to (R)-2-aza-β-tyrosine, exhibiting no detectable activity toward 2-aza-L-phenylalanine or L-tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of L-tyrosine to (S)-β-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza-L-tyrosine as an alternative substrate but generating (S)-2-aza-β-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong; Hassan, Maizom

    2016-01-01

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

  16. Plastids and Carotenoid Accumulation.

    PubMed

    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    2016-01-01

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants. PMID:27485226

  17. Plastids and Carotenoid Accumulation.

    PubMed

    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    2016-01-01

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants.

  18. Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase (AaPAL1) from Artemisia annua.

    PubMed

    Zhang, Ying; Fu, Xueqing; Hao, Xiaolong; Zhang, Lida; Wang, Luyao; Qian, Hongmei; Zhao, Jingya

    2016-07-01

    Phenylalanine ammonia-lyase (PAL) is the key enzyme in the biosynthetic pathway of salicylic acid (SA). In this study, a full-length cDNA of PAL gene (named as AaPAL1) was cloned from Artemisia annua. The gene contains an open reading frame of 2,151 bps encoding 716 amino acids. Comparative and bioinformatics analysis revealed that the polypeptide protein of AaPAL1 was highly homologous to PALs from other plant species. Southern blot analysis revealed that it belonged to a gene family with three members. Quantitative RT-PCR analysis of various tissues of A. annua showed that AaPAL1 transcript levels were highest in the young leaves. A 1160-bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including W-box, TGACG-motif, and TC-rich repeats. Quantitative RT-PCR indicated that AaPAL1 was upregulated by salinity, drought, wounding, and SA stresses, which were corroborated positively with the identified cis-elements within the promoter region. AaPAL1 was successfully expressed in Escherichia. coli and the enzyme activity of the purified AaPAL1 was approximately 287.2 U/mg. These results substantiated the involvement of AaPAL1 in the phenylalanine pathway. PMID:26040426

  19. The characterization of transgenic tomato overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit growth, higher yield, and alteration of the gibberellin biosynthetic pathway.

    PubMed

    García-Hurtado, Noemí; Carrera, Esther; Ruiz-Rivero, Omar; López-Gresa, Maria Pilar; Hedden, Peter; Gong, Fan; García-Martínez, José Luis

    2012-10-01

    Fruit-set and growth in tomato depend on the action of gibberellins (GAs). To evaluate the role of the GA biosynthetic enzyme GA 20-oxidase (GA20ox) in that process, the citrus gene CcGA20ox1 was overexpressed in tomato (Solanum lycopersicum L.) cv Micro-Tom. The transformed plants were taller, had non-serrated leaves, and some flowers displayed a protruding stigma due to a longer style, thus preventing self-pollination, similar to GA(3)-treated plants. Flowering was delayed compared with wild-type (WT) plants. Both yield and number of fruits per plant, some of them seedless, were higher in the transgenic plants. The Brix index value of fruit juice was also higher due to elevated citric acid content, but not glucose or fructose content. When emasculated, 14-30% of ovaries from transgenic flowers developed parthenocarpically, whereas no parthenocarpy was found in emasculated WT flowers. The presence of early-13-hydroxylation and non-13-hydroxylation GA pathways was demonstrated in the shoot and fruit of Micro-Tom, as well as in two tall tomato cultivars (Ailsa Craig and UC-82). The transgenic plants had altered GA profiles containing higher concentrations of GA(4), from the non-13-hydroxylation pathway, which is generally a minor active GA in tomato. The effect of GA(4) application in enhancing stem growth and parthenocarpic fruit development was proportional to dose, with the same activity as GA(1). The results support the contention that GA20ox overexpression diverts GA metabolism from the early-13-hydroxylation pathway to the non-13-hydroxylation pathway. This led to enhanced GA(4) synthesis and higher yield, although the increase in GA(4) content in the ovary was not sufficient to induce full parthenocarpy. PMID:22945942

  20. Ecdysteroid biosynthesis in varroa mites: identification of halloween genes from the biosynthetic pathway and their regulation during reproduction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Carotenoid-based coloration in cichlid fishes

    PubMed Central

    Sefc, Kristina M.; Brown, Alexandria C.; Clotfelter, Ethan D.

    2014-01-01

    Animal colors play important roles in communication, ecological interactions and speciation. Carotenoid pigments are responsible for many yellow, orange and red hues in animals. Whereas extensive knowledge on the proximate mechanisms underlying carotenoid coloration in birds has led to testable hypotheses on avian color evolution and signaling, much less is known about the expression of carotenoid coloration in fishes. Here, we promote cichlid fishes (Perciformes: Cichlidae) as a system in which to study the physiological and evolutionary significance of carotenoids. Cichlids include some of the best examples of adaptive radiation and color pattern diversification in vertebrates. In this paper, we examine fitness correlates of carotenoid pigmentation in cichlids and review hypotheses regarding the signal content of carotenoid-based ornaments. Carotenoid-based coloration is influenced by diet and body condition and is positively related to mating success and social dominance. Gaps in our knowledge are discussed in the last part of this review, particularly in the understanding of carotenoid metabolism pathways and the genetics of carotenoid coloration. We suggest that carotenoid metabolism and transport are important proximate mechanisms responsible for individual and population-differences in cichlid coloration that may ultimately contribute to diversification and speciation. PMID:24667558

  2. A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

    PubMed Central

    2011-01-01

    Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits against experimental data

  3. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    PubMed

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-01

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts. PMID:19206228

  4. Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

    PubMed

    Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

    2009-03-01

    The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

  5. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase.

    PubMed

    Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P; Geng, Jiafeng; Liu, Aimin; Modi, Anuja; Dawson, John H; Sono, Masanori; Shumskaya, Maria; Ampomah-Dwamena, Charles; Love, James D; Wurtzel, Eleanore T

    2015-08-01

    Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15' carbon-carbon double bond in 9,15,9'-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

  6. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase

    PubMed Central

    Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P.; Geng, Jiafeng; Liu, Aimin; Modi, Anuja; Dawson, John H.; Sono, Masanori; Shumskaya, Maria; Ampomah-Dwamena, Charles; Love, James D.; Wurtzel, Eleanore T.

    2015-01-01

    Plants synthesize carotenoids essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway leading to all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not certain whether isomerization was achieved by Z-ISO alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-to-trans isomerization of the 15–15′ C=C bond in 9,15,9′-cis-ζ-carotene to produce the substrate required by the following biosynthetic pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand-switching between the heme iron and alternate Z-ISO amino acid residues. Heme b-dependent isomerization of a large, hydrophobic compound in a membrane is unprecedented. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially utilizes a novel chemical mechanism. PMID:26075523

  7. Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data

    PubMed Central

    Kawasaki, Regiane; Carepo, Marta S. P.; Oliveira, Rui; Marques, Rodolfo; Ramos, Rommel T. J.; Schneider, Maria P. C.

    2016-01-01

    Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments. PMID:27595107

  8. A cautionary tale of structure-guided inhibitor development against an essential enzyme in the aspartate-biosynthetic pathway.

    PubMed

    Pavlovsky, Alexander G; Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

    2014-12-01

    The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate β-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme-inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.

  9. Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data

    PubMed Central

    Kawasaki, Regiane; Carepo, Marta S. P.; Oliveira, Rui; Marques, Rodolfo; Ramos, Rommel T. J.; Schneider, Maria P. C.

    2016-01-01

    Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.

  10. Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway.

    PubMed

    de Sena Filho, Jose Guedes; Quin, Maureen B; Spakowicz, Daniel J; Shaw, Jeffrey J; Kucera, Kaury; Dunican, Brian; Strobel, Scott A; Schmidt-Dannert, Claudia

    2016-08-01

    Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products.

  11. Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae)

    PubMed Central

    Martins, Talline R.; Berg, Jeremy J.; Blinka, Steven; Rausher, Mark D.; Baum, David A.

    2012-01-01

    Summary Petal spots are widespread in Angiosperms and are often implicated in pollinator attraction. Clarkia gracilis petals each have a single red-purple spot that contrasts against a pink background. The position and presence of spots in C. gracilis are determined by the epistatic interaction of alleles at two, as-yet-unidentified loci.We used HPLC to identify the different pigments produced in the petals, and qualitative and quantitative RT-PCR to assay for spatio-temporal patterns of expression of different anthocyanin pathway genes.We found that spots contain different pigments from the remainder of the petal, being composed of cyanidin/peonidin-based, instead of malvidin-based anthocyanins. Expression assays of anthocyanin pathway genes show that Dfr2 has a spot-specific expression pattern and acts as a switch for spot production. Co-segregation analyses implicate the gene products of the P and I loci as trans-regulators of this switch. Spot pigments appear earlier in development due to early expression of Dfr2 and F3′h1. Pigments in the background appear later, due to later expression of Dfr1 and F3′5′h1.The evolution of this spot production mechanism appears to have been facilitated by duplication of the Dfr gene and to have required substantial reworking of the anthocyanin pathway regulatory network. PMID:23231386

  12. Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data.

    PubMed

    Kawasaki, Regiane; Baraúna, Rafael A; Silva, Artur; Carepo, Marta S P; Oliveira, Rui; Marques, Rodolfo; Ramos, Rommel T J; Schneider, Maria P C

    2016-01-01

    Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments. PMID:27595107

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

    PubMed

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

    2015-05-01

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

  14. Discovery and Characterization of BlsE, a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

    PubMed Central

    Feng, Jun; Wu, Jun; Dai, Nan; Lin, Shuangjun; Xu, H. Howard; Deng, Zixin; He, Xinyi

    2013-01-01

    BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]2+ cluster. Mutagenesis studies demonstrated that Cys31, Cys35, Cys38 in the C×××C×MC motif and Gly73, Gly74, Glu75, Pro76 in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met37 had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis. PMID:23874663

  15. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives. PMID:27439219

  16. Evolution of high-level ethambutol-resistant tuberculosis through interacting mutations in decaprenylphosphoryl-β-D-arabinose biosynthetic and utilization pathway genes.

    PubMed

    Safi, Hassan; Lingaraju, Subramanya; Amin, Anita; Kim, Soyeon; Jones, Marcus; Holmes, Michael; McNeil, Michael; Peterson, Scott N; Chatterjee, Delphi; Fleischmann, Robert; Alland, David

    2013-10-01

    To study the evolution of drug resistance, we genetically and biochemically characterized Mycobacterium tuberculosis strains selected in vitro for ethambutol resistance. Mutations in decaprenylphosphoryl-β-D-arabinose (DPA) biosynthetic and utilization pathway genes Rv3806c, Rv3792, embB and embC accumulated to produce a wide range of ethambutol minimal inhibitory concentrations (MICs) that depended on mutation type and number. Rv3806c mutations increased DPA synthesis, causing MICs to double from 2 to 4 μg/ml in a wild-type background and to increase from 16 to 32 μg/ml in an embB codon 306 mutant background. Synonymous mutations in Rv3792 increased the expression of downstream embC, an ethambutol target, resulting in MICs of 8 μg/ml. Multistep selection was required for high-level resistance. Mutations in embC or very high embC expression were observed at the highest resistance level. In clinical isolates, Rv3806c mutations were associated with high-level resistance and had multiplicative effects with embB mutations on MICs. Ethambutol resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance.

  17. TRC8, A KIDNEY CANCER-ASSOCIATED UBIQUITIN LIGASE, IS STEROL-REGULATED AND INTERACTS WITH LIPID AND PROTEIN BIOSYNTHETIC PATHWAYS

    PubMed Central

    Lee, Jason P.; Brauweiler, Anne; Rudolph, Michael; Hooper, Joan E.; Drabkin, Harry A.; Gemmill, Robert M.

    2009-01-01

    TRC8/RNF139 encodes an ER-resident E3-ubiquitin ligase that inhibits growth in a RING- and ubiquitylation-dependent manner. TRC8 also contains a predicted sterol-sensing domain. Here we report that TRC8 protein levels are sterol-responsive, and that it binds and stimulates ubiquitylation of the ER-anchor protein, INSIG. Induction of TRC8 destabilized the precursor forms of the transcription factors, SREBP-1 and SREBP-2. Loss of SREBP precursors was proteasome-dependent, required a functional RING domain, occurred without generating processed nuclear forms and suppressed SREBP target genes. TRC8 knockdown had opposite effects in sterol-deprived cells. In Drosophila, growth inhibition by DTrc8 was genetically suppressed by loss of specific MPN domain-containing proteins found in the COP9 signalosome and eIF3. DTrc8 genetically and physicially interacted with two eIF3 subunits, eIF3f and eIF3h. Co-immunoprecipitation experiments confirmed these interactions in mammalian cells and TRC8 over-expression suppressed polysome profiles. Moreover, high molecular weight ubiquitylated proteins were observed in eIF3 immunoprecipitations from TRC8 over-expressing cells. Thus, TRC8 function may provide a regulatory link between the lipid and protein biosynthetic pathways. PMID:20068067

  18. Inhibition of green tea and the catechins against 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway.

    PubMed

    Hui, Xian; Liu, Hui; Tian, Fang-Lin; Li, Fei-Fei; Li, Heng; Gao, Wen-Yun

    2016-09-01

    1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the MEP terpenoid biosynthetic pathway and also a validated antimicrobial target. Green tea which is rich in polyphenolic components such as the catechins, possesses a plenty of pharmacological activities, in particular an antibacterial effect. To uncover the antibacterial mechanism of green tea and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of green tea and its main antimicrobial catechins were investigated in this study. The results show that the raw extract of green tea and its ethyl acetate fraction are able to suppress DXR activity explicitly. Further determination of the DXR inhibitory capacity of eight catechin compounds demonstrates that the most active compound is gallocatechin gallate that is able to inhibit around 50% activity of DXR at 25μM. Based on these data, the primary structure-activity relationship of the catechins against DXR is discussed. This study would be very helpful to elucidate the antimicrobial mechanism of green tea and the catechins and also would be very useful to direct the rational utilization of them as food additives.

  19. The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus.

    PubMed

    Kong, Qing; Chi, Chen; Yu, Jiujiang; Shan, Shihua; Li, Qiyu; Li, Qianting; Guan, Bin; Nierman, William C; Bennett, Joan W

    2014-06-01

    Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.

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

    PubMed

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

    2015-09-01

    miRNAs are emerging as potential regulators of the gene expression. Their proven promising role in regulating biosynthetic pathways related gene networks may hold the key to understand the genetic regulation of these pathways which may assist in selection and manipulation to get high performing plant genotypes with better secondary metabolites yields and increased biomass. miRNAs associated with genes of steviol glycosides biosynthetic pathway, however, have not been identified so far. In this study miRNAs targeting genes of steviol glycosides biosynthetic pathway were identified for the first time whose precursors were potentially generated from ESTs and nucleotide sequences of Stevia rebaudiana. Thereafter, stem-loop coupled real time PCR based expressions of these miRNAs in different tissues of Stevia rebaudiana were investigated and their relationship pattern was analysed with the expression levels of their target mRNAs as well as steviol glycoside contents. All the miRNAs investigated showed differential expressions in all the three tissues studied, viz. leaves, flowers and stems. Out of the eleven miRNAs validated, the expression levels of nine miRNAs (miR319a, miR319b, miR319c, miR319d, miR319e, miR319f, miR319h, miRstv_7, miRstv_9) were found to be inversely related, while expression levels of the two, i.e. miR319g and miRstv_11 on the contrary, showed direct relation with the expression levels of their target mRNAs and steviol glycoside contents in the leaves, flowers and stems. This study provides a platform for better understanding of the steviol glycosides biosynthetic pathway and these miRNAs can further be employed to manipulate the biosynthesis of these metabolites to enhance their contents and yield in S. rebaudiana. PMID:26042546

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

    PubMed

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

    2015-09-01

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

  2. The biosynthetic pathway of curcuminoid in turmeric (Curcuma longa) as revealed by 13C-labeled precursors.

    PubMed

    Kita, Tomoko; Imai, Shinsuke; Sawada, Hiroshi; Kumagai, Hidehiko; Seto, Haruo

    2008-07-01

    In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid > ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

  3. Indole-3-acetic acid biosynthetic pathway and aromatic amino acid aminotransferase activities in Pantoea dispersa strain GPK.

    PubMed

    Kulkarni, G B; Nayak, A S; Sajjan, S S; Oblesha, A; Karegoudar, T B

    2013-05-01

    This investigation deals with the production of IAA by a bacterial isolate Pantoea dispersa strain GPK (PDG) identified by 16S rRNA gene sequence analysis. HPLC and Mass spectral analysis of metabolites from bacterial spent medium revealed that, IAA production by PDG is Trp-dependent and follows indole-3-pyruvic acid (IPyA) pathway. Substrate specificity study of aromatic amino acid aminotransferase (AAT) showed high activities, only when tryptophan (Trp) and α-ketoglutarate (α-kg) were used as substrates. AAT is highly specific for Trp and α-kg as amino group donor and acceptor, respectively. The effect of exogenous IAA on bacterial growth was established. Low concentration of exogenous IAA induced the growth, whereas high concentration decreased the growth of bacterium. PDG treatment significantly increased the root length, shoot length and dry mass of the chickpea and pigeon pea plants. PMID:23448265

  4. Sequence Diversity in Coding Regions of Candidate Genes in the Glycoalkaloid Biosynthetic Pathway of Wild Potato Species

    PubMed Central

    Manrique-Carpintero, Norma C.; Tokuhisa, James G.; Ginzberg, Idit; Holliday, Jason A.; Veilleux, Richard E.

    2013-01-01

    Natural variation in five candidate genes of the steroidal glycoalkaloid (SGA) metabolic pathway and whole-genome single nucleotide polymorphism (SNP) genotyping were studied in six wild [Solanum chacoense (chc 80-1), S. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, S. stoloniferum] and cultivated S. tuberosum Group Phureja (phu DH) potato species with contrasting levels of SGAs. Amplicons were sequenced for five candidate genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2) and 2.3-squalene epoxidase (SQE) of primary metabolism, and solanidine galactosyltransferase (SGT1), and glucosyltransferase (SGT2) of secondary metabolism. SNPs (n = 337) producing 354 variations were detected within 3.7 kb of sequenced DNA. More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism. Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima’s D test suggested purifying selection and genetic hitchhiking in the gene fragments. In addition, patterns of dN/dS ratios across the SGA pathway suggested constraint by natural selection. Comparison of nucleotide diversity estimates and dN/dS ratios showed stronger selective constraints for genes of primary rather than secondary metabolism. SNPs (n = 24) with an exclusive genotype for either phu DH (low SGA) or chc 80-1 (high SGA) were identified for HMG2, SQE, SGT1 and SGT2. The SolCAP 8303 Illumina Potato SNP chip genotyping revealed eight informative SNPs on six pseudochromosomes, with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation. These results can be used to evaluate SGA accumulation in segregating or association mapping populations. PMID:23853090

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed Central

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

    2016-01-01

    We aimed to identify novel molecular mechanisms for muscle growth during administration of anabolic agents. Growing pigs (Duroc/(Landrace/Large-White)) were administered Ractopamine (a beta-adrenergic agonist; BA; 20 ppm in feed) or Reporcin (recombinant growth hormone; GH; 10 mg/48 hours injected) and compared to a control cohort (feed only; no injections) over a 27-day time course (1, 3, 7, 13 or 27-days). Longissimus Dorsi muscle gene expression was analyzed using Agilent porcine transcriptome microarrays and clusters of genes displaying similar expression profiles were identified using a modified maSigPro clustering algorithm. Anabolic agents increased carcass (p = 0.002) and muscle weights (Vastus Lateralis: p < 0.001; Semitendinosus: p = 0.075). Skeletal muscle mRNA expression of serine/one-carbon/glycine biosynthesis pathway genes (Phgdh, Psat1 and Psph) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase-M (Pck2/PEPCK-M), increased during treatment with BA, and to a lesser extent GH (p < 0.001, treatment x time interaction). Treatment with BA, but not GH, caused a 2-fold increase in phosphoglycerate dehydrogenase (PHGDH) protein expression at days 3 (p < 0.05) and 7 (p < 0.01), and a 2-fold increase in PEPCK-M protein expression at day 7 (p < 0.01). BA treated pigs exhibit a profound increase in expression of PHGDH and PEPCK-M in skeletal muscle, implicating a role for biosynthetic metabolic pathways in muscle growth. PMID:27350173

  7. CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells.

    PubMed

    Courdavault, Vincent; Thiersault, Martine; Courtois, Martine; Gantet, Pascal; Oudin, Audrey; Doireau, Pierre; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie

    2005-04-01

    CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common alpha subunit and a specific beta subunit. We have established RNA interference cell lines targeting the beta subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants.

  8. Analysis of biochemical compounds and differentially expressed genes of the anthocyanin biosynthetic pathway in variegated peach flowers.

    PubMed

    Hassani, D; Liu, H L; Chen, Y N; Wan, Z B; Zhuge, Q; Li, S X

    2015-10-28

    Variegated plants are highly valuable in the floricultural market, yet the genetic mechanism underlying this attractive phenomenon has not been completely elucidated. In this study, we identified and measured different compounds in pink and white flower petals of peach (Prunus persica) by high-performance liquid chromatography and liquid chromatography/mass spectrometry analyses. No cyanidin-based or pelargonidin-based compounds were detected in white petals, but high levels of these compounds were found in pink petals. Additionally, we sequenced and analyzed the expression of six key structural genes in the anthocyanin biosynthesis pathway (CHI, CHS, DFR, F3'H, ANS, and UFGT) in both white and pink petals. Quantitative real-time polymerase chain reaction revealed all six genes to be expressed at greatly reduced levels in white flower petals, relative to pink. No allelic variations were found in the transcribed sequences. However, alignment of transcribed and genomic sequences of the ANS gene detected alternative splicing, resulting in transcripts of 1.071 and 942 bp. Only the longer transcript was observed in white flower petals. Since ANS is the key intermediate enzyme catalyzing the colorless leucopelargonidin and leucocyanidin to substrates required for completion of anthocyanin biosynthesis, the ANS gene is implicated in flower color variegation and should be explored in future studies. This article, together with a previous transcriptome study, elucidates the mechanism underlying peach flower color variegation in terms of the key structural genes involved in anthocyanin biosynthesis.

  9. Analysis of biochemical compounds and differentially expressed genes of the anthocyanin biosynthetic pathway in variegated peach flowers.

    PubMed

    Hassani, D; Liu, H L; Chen, Y N; Wan, Z B; Zhuge, Q; Li, S X

    2015-01-01

    Variegated plants are highly valuable in the floricultural market, yet the genetic mechanism underlying this attractive phenomenon has not been completely elucidated. In this study, we identified and measured different compounds in pink and white flower petals of peach (Prunus persica) by high-performance liquid chromatography and liquid chromatography/mass spectrometry analyses. No cyanidin-based or pelargonidin-based compounds were detected in white petals, but high levels of these compounds were found in pink petals. Additionally, we sequenced and analyzed the expression of six key structural genes in the anthocyanin biosynthesis pathway (CHI, CHS, DFR, F3'H, ANS, and UFGT) in both white and pink petals. Quantitative real-time polymerase chain reaction revealed all six genes to be expressed at greatly reduced levels in white flower petals, relative to pink. No allelic variations were found in the transcribed sequences. However, alignment of transcribed and genomic sequences of the ANS gene detected alternative splicing, resulting in transcripts of 1.071 and 942 bp. Only the longer transcript was observed in white flower petals. Since ANS is the key intermediate enzyme catalyzing the colorless leucopelargonidin and leucocyanidin to substrates required for completion of anthocyanin biosynthesis, the ANS gene is implicated in flower color variegation and should be explored in future studies. This article, together with a previous transcriptome study, elucidates the mechanism underlying peach flower color variegation in terms of the key structural genes involved in anthocyanin biosynthesis. PMID:26535657

  10. Molecular Cloning and Characterization of DXS and DXR Genes in the Terpenoid Biosynthetic Pathway of Tripterygium wilfordii

    PubMed Central

    Tong, Yuru; Su, Ping; Zhao, Yujun; Zhang, Meng; Wang, Xiujuan; Liu, Yujia; Zhang, Xianan; Gao, Wei; Huang, Luqi

    2015-01-01

    1-Deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) genes are the key enzyme genes of terpenoid biosynthesis but still unknown in Tripterygium wilfordii Hook. f. Here, three full-length cDNA encoding DXS1, DXS2 and DXR were cloned from suspension cells of T. wilfordii with ORF sizes of 2154 bp (TwDXS1, GenBank accession no.KM879187), 2148 bp (TwDXS2, GenBank accession no.KM879186), 1410 bp (TwDXR, GenBank accession no.KM879185). And, the TwDXS1, TwDXS2 and TwDXR were characterized by color complementation in lycopene accumulating strains of Escherichia coli, which indicated that they encoded functional proteins and promoted lycopene pathway flux. TwDXS1 and TwDXS2 are constitutively expressed in the roots, stems and leaves and the expression level showed an order of roots > stems > leaves. After the suspension cells were induced by methyl jasmonate, the mRNA expression level of TwDXS1, TwDXS2, and TwDXR increased, and triptophenolide was rapidly accumulated to 149.52 µg·g−1, a 5.88-fold increase compared with the control. So the TwDXS1, TwDXS2, and TwDXR could be important genes involved in terpenoid biosynthesis in Tripterygium wilfordii Hook. f. PMID:26512659

  11. Metformin inhibits hepatocellular glucose, lipid and cholesterol biosynthetic pathways by transcriptionally suppressing steroid receptor coactivator 2 (SRC-2)

    PubMed Central

    Madsen, Andre; Bozickovic, Olivera; Bjune, Jan-Inge; Mellgren, Gunnar; Sagen, Jørn V.

    2015-01-01

    The ability of the anti-diabetic drug metformin to inhibit anabolic processes including gluconeogenesis and lipogenesis is partly attributable to activation of the AMP-activated protein kinase (AMPK) pathway. The p160 steroid receptor coactivator 2 (SRC-2) is a key regulator of cellular metabolism and drives expression of the gluconeogenic enzyme glucose-6-phosphatase (G6Pc). Here, we uncovered a role for SRC-2 in the metabolic reprogramming imposed by metformin. In FaO cells, metformin dose-dependently reduced mRNA expression of SRC-2. Microarray analysis of metformin-treated cells revealed an overrepresentation of downregulated genes involved in biosynthesis of lipids and cholesterol. Several metformin-regulated genes including fatty acid synthase (FASN) were validated as transcriptional targets of SRC-2 with promoters characterized by sterol regulatory element (SRE) binding protein (SREBP) recognition sequences. Transactivation assays of the FASN promoter confirmed that SRC-2 is a coactivator of SREBP-1. By suppressing SRC-2 at the transcriptional level, metformin impeded recruitment of SRC-2 and RNA polymerase II to the G6Pc promoter and to SREs of mutual SRC-2/SREBP-1 target gene promoters. Hepatocellular fat accretion was reduced by metformin or knock-down of both SRC-2 and SREBP-1. Accordingly we propose that metformin inhibits glucose and lipid biosynthesis partly by downregulating SRC-2 gene expression. PMID:26548416

  12. Metformin inhibits hepatocellular glucose, lipid and cholesterol biosynthetic pathways by transcriptionally suppressing steroid receptor coactivator 2 (SRC-2).

    PubMed

    Madsen, Andre; Bozickovic, Olivera; Bjune, Jan-Inge; Mellgren, Gunnar; Sagen, Jørn V

    2015-01-01

    The ability of the anti-diabetic drug metformin to inhibit anabolic processes including gluconeogenesis and lipogenesis is partly attributable to activation of the AMP-activated protein kinase (AMPK) pathway. The p160 steroid receptor coactivator 2 (SRC-2) is a key regulator of cellular metabolism and drives expression of the gluconeogenic enzyme glucose-6-phosphatase (G6Pc). Here, we uncovered a role for SRC-2 in the metabolic reprogramming imposed by metformin. In FaO cells, metformin dose-dependently reduced mRNA expression of SRC-2. Microarray analysis of metformin-treated cells revealed an overrepresentation of downregulated genes involved in biosynthesis of lipids and cholesterol. Several metformin-regulated genes including fatty acid synthase (FASN) were validated as transcriptional targets of SRC-2 with promoters characterized by sterol regulatory element (SRE) binding protein (SREBP) recognition sequences. Transactivation assays of the FASN promoter confirmed that SRC-2 is a coactivator of SREBP-1. By suppressing SRC-2 at the transcriptional level, metformin impeded recruitment of SRC-2 and RNA polymerase II to the G6Pc promoter and to SREs of mutual SRC-2/SREBP-1 target gene promoters. Hepatocellular fat accretion was reduced by metformin or knock-down of both SRC-2 and SREBP-1. Accordingly we propose that metformin inhibits glucose and lipid biosynthesis partly by downregulating SRC-2 gene expression. PMID:26548416

  13. Crystal Structure of Vancosaminyltransferase GtfD from the Vancomycin Biosynthetic Pathway: Interactions with Acceptor and Nucleotide Ligands

    SciTech Connect

    Mulichak, A.M.; Lu, W.; Losey, H.C.; Walsh, C.T.; Garavito, R.M.

    2010-03-08

    The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that are effective against vancomycin resistant bacterial strains. We have determined the X-ray crystal structure of GtfD as a complex with TDP and the natural glycopeptide substrate at 2.0 {angstrom} resolution. GtfD, a member of the bidomain GT-B glycosyltransferase superfamily, binds TDP in the interdomain cleft, while the aglycone acceptor binds in a deep crevice in the N-terminal domain. However, the two domains are more interdependent in terms of substrate binding and overall structure than was evident in the structures of closely related glycosyltransferases GtfA and GtfB. Structural and kinetic analyses support the identification of Asp13 as a catalytic general base, with a possible secondary role for Thr10. Several residues have also been identified as being involved in donor sugar binding and recognition.

  14. Modulation of carotenoid accumulation in transgenic potato by inducing chromoplast formation with enhanced sink strength

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An increasing interest in carotenoids as nutritional sources of provitamin A and health-promoting compounds has prompted a significant effort in metabolic engineering of carotenoid content and composition in food crops. The strategy commonly used in plants is to increase the biosynthetic capacity by...

  15. Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil.

    PubMed

    Battini, Fabio; Bernardi, Rodolfo; Turrini, Alessandra; Agnolucci, Monica; Giovannetti, Manuela

    2016-10-01

    In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3'-hydroxylase isoform 1 (CS3'H iso1) genes, 5.7-fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3'H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health.

  16. Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil.

    PubMed

    Battini, Fabio; Bernardi, Rodolfo; Turrini, Alessandra; Agnolucci, Monica; Giovannetti, Manuela

    2016-10-01

    In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3'-hydroxylase isoform 1 (CS3'H iso1) genes, 5.7-fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3'H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health. PMID:27179537

  17. Omega-3 phospholipids from fish suppress hepatic steatosis by integrated inhibition of biosynthetic pathways in dietary obese mice.

    PubMed

    Rossmeisl, Martin; Medrikova, Dasa; van Schothorst, Evert M; Pavlisova, Jana; Kuda, Ondrej; Hensler, Michal; Bardova, Kristina; Flachs, Pavel; Stankova, Barbora; Vecka, Marek; Tvrzicka, Eva; Zak, Ales; Keijer, Jaap; Kopecky, Jan

    2014-02-01

    Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Antiinflammatory, hypolipidemic, and insulin-sensitizing effects ofDHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHlF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated downregulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex downregulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment. PMID:24295779

  18. Violet/blue chrysanthemums--metabolic engineering of the anthocyanin biosynthetic pathway results in novel petal colors.

    PubMed

    Brugliera, Filippa; Tao, Guo-Qing; Tems, Ursula; Kalc, Gianna; Mouradova, Ekaterina; Price, Kym; Stevenson, Kim; Nakamura, Noriko; Stacey, Iolanda; Katsumoto, Yukihisa; Tanaka, Yoshikazu; Mason, John G

    2013-10-01

    Chrysanthemums (Chrysanthemum×morifolium Ramat.) are an important cut-flower and potted plant crop in the horticultural industry world wide. Chrysanthemums express the flavonoid 3'-hydroxylase (F3'H) gene and thus accumulate anthocyanins derived from cyanidin in their inflorescences which appear pink/red. Delphinidin-based anthocyanins are lacking due to the deficiency of a flavonoid 3', 5'-hydroxylase (F3'5'H), and so violet/blue chrysanthemum flower colors are not found. In this study, together with optimization of transgene expression and selection of the host cultivars and gene source, F3'5'H genes have been successfully utilized to produce transgenic bluish chrysanthemums that accumulate delphinidin-based anthocyanins. HPLC analysis and feeding experiments with a delphinidin precursor identified 16 cultivars of chrysanthemums out of 75 that were predicted to turn bluish upon delphinidin accumulation. A selection of eight cultivars were successfully transformed with F3'5'H genes under the control of different promoters. A pansy F3'5'H gene under the control of a chalcone synthase promoter fragment from rose resulted in the effective diversion of the anthocyanin pathway to produce delphinidin in transgenic chrysanthemum flower petals. The resultant petal color was bluish, with 40% of total anthocyanidins attributed to delphinidin. Increased delphinidin levels (up to 80%) were further achieved by hairpin RNA interference-mediated silencing of the endogenous F3'H gene. The resulting petal colors were novel bluish hues, not possible by hybridization breeding. This is the first report of the production of anthocyanins derived from delphinidin in chrysanthemum petals leading to novel flower color.

  19. Diversity, biological roles and biosynthetic pathways for sugar-glycerate containing compatible solutes in bacteria and archaea.

    PubMed

    Empadinhas, Nuno; da Costa, Milton S

    2011-08-01

    A decade ago the compatible solutes mannosylglycerate (MG) and glucosylglycerate (GG) were considered to be rare in nature. Apart from two species of thermophilic bacteria, Thermus thermophilus and Rhodothermus marinus, and a restricted group of hyperthermophilic archaea, the Thermococcales, MG had only been identified in a few red algae. Glucosylglycerate was considered to be even rarer and had only been detected as an insignificant solute in two halophilic microorganisms, a cyanobacterium, as a component of a polysaccharide and of a glycolipid in two actinobacteria. Unlike the hyper/thermophilic MG-accumulating microorganisms, branching close to the root of the Tree of Life, those harbouring GG shared a mesophilic lifestyle. Exceptionally, the thermophilic bacterium Persephonella marina was reported to accumulate GG. However, and especially owing to the identification of the key-genes for MG and GG synthesis and to the escalating numbers of genomes available, a plethora of new organisms with the resources to synthesize these solutes has been recognized. The accumulation of GG as an 'emergency' compatible solute under combined salt stress and nitrogen-deficient conditions now seems to be a disseminated survival strategy from enterobacteria to marine cyanobacteria. In contrast, the thermophilic and extremely radiation-resistant bacterium Rubrobacter xylanophilus is the only actinobacterium known to accumulate MG, and under all growth conditions tested. This review addresses the environmental factors underlying the accumulation of MG, GG and derivatives in bacteria and archaea and their roles during stress adaptation or as precursors for more elaborated macromolecules. The diversity of pathways for MG and GG synthesis as well as those for some of their derivatives is also discussed. The importance of glycerate-derived organic solutes in the microbial world is only now being recognized. Their stress-dependent accumulation and the molecular aspects of their

  20. Structural and Functional Analysis of Campylobacter jejuni PseG: a Udp-sugarhydrolase from the Pseudaminic Acid Biosynthetic Pathway

    SciTech Connect

    E Rangarajan; A Proteau; Q Cui; S Logan; Z Potetinova; D Whitfield; E Purisima; M Cygler; A Matte; et al.

    2011-12-31

    Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-{beta}-l-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-l-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 {angstrom} resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His{sup 17}-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His{sup 17} functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C-O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.

  1. Chemistry of carotenoid neutral radicals.

    PubMed

    Ligia Focsan, A; Magyar, Adam; Kispert, Lowell D

    2015-04-15

    Proton loss from the carotenoid radical cations (Car(+)) to form neutral radicals (#Car) was investigated by numerous electrochemical, EPR, ENDOR and DFT studies described herein. The radical cation and neutral radicals were formed in solution electrochemically and stabilized on solid silica-alumina and MCM-41 matrices. Carotenoid neutral radicals were recently identified in Arabidopsis thaliana plant and photosystem II samples. Deprotonation at the terminal ends of a zeaxanthin radical cation could provide a secondary photoprotection pathway which involves quenching excited state chlorophyll by the long-lived zeaxanthin neutral radicals formed. PMID:25687648

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. Stimulation of carotenoid metabolism in arbuscular mycorrhizal roots.

    PubMed

    Fester, Thomas; Schmidt, Diana; Lohse, Swanhild; Walter, Michael H; Giuliano, Giovanni; Bramley, Peter M; Fraser, Paul D; Hause, Bettina; Strack, Dieter

    2002-11-01

    Development of arbuscular mycorrhizal roots is correlated with accumulation of various isoprenoids, i.e. acyclic C(14) polyene 'mycorradicin' and C(13) cyclohexenone derivatives. We present data indicating a strong stimulation of carotenoid metabolism in such roots. Carotenoid profiling revealed mycorrhiza-specific accumulation of zeta-carotene in Zea mays and Medicago truncatula. Precursor accumulation after inhibition of phytoene desaturase (Pds) activity by norflurazon indicated an increased phytoene biosynthetic capacity in mycorrhizal roots of all species analyzed. Nicotiana tabacum plants transformed with a PDS promoter- GUS construct showed a cell-specific induction of PDS promoter activity in root cells containing arbuscules. Mycorradicin biosynthesis and, partially, mycorrhization were impaired in maize mutants deficient in carotenoid biosynthesis. These data indicate that (1) mycorradicin is probably synthesized via a C(40) precursor carotenoid, (2) carotenoid biosynthesis is induced in mycorrhizal roots, (3) induction occurs, at least partially, at the transcriptional level, and (4) that this may play a functional role during mycorrhization.

  5. Formal modeling and analysis of the hexosamine biosynthetic pathway: role of O-linked N-acetylglucosamine transferase in oncogenesis and cancer progression

    PubMed Central

    Saeed, Muhammad Tariq; Holowatyj, Andreana N.; Sheikh, Iftikhar A.; Zafar Paracha, Rehan; Shafi, Aamir; Siddiqa, Amnah; Bibi, Zurah; Khan, Mukaram

    2016-01-01

    The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP) drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation) and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT), an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer. PMID:27703839

  6. In vivo and in vitro evidence that chronic activation of the hexosamine biosynthetic pathway interferes with leptin-dependent STAT3 phosphorylation

    PubMed Central

    Zimmerman, Arthur D.

    2015-01-01

    We previously reported that a 2-day peripheral infusion of glucosamine caused leptin resistance in rats, suggesting a role for the hexosamine biosynthetic pathway (HBP) in the development of leptin resistance. Here we tested leptin responsiveness in mice in which HBP activity was stimulated by offering 30% sucrose solution in addition to chow and water or by infusing glucosamine. Mice were leptin resistant after 33 days of access to sucrose. Resistance was associated with increased activity of the HBP and with phosphorylation of transcription factor signal transducer and activator of transcription-3 Tyr705 [pSTAT3(Y705)] but inhibition of suppressor of cytokine signaling 3 in the liver and hypothalamus. Intravenous infusion of glucosamine for 3 h stimulated pSTAT3(Y705) but prevented leptin-induced phosphorylation of STAT3(S727). In an in vitro system, glucose, glucosamine, and leptin each dose dependently increased O-linked β-N-acetylglucosamine (O-GlcNAc) protein and pSTAT3(Y705) in HepG2 cells. To test the effect of glucose on leptin responsiveness cells were incubated in 5.5 mM (LG) or 20 mM (HG) glucose for 18 h and were treated with 0 or 50 ng/ml leptin for 15 min. HG alone and LG + leptin produced similar increases in O-GlcNAc protein, glutamine fructose-6-phosphate amidotransferase (GFAT), and pSTAT3(Y705) compared with LG media. Leptin did not stimulate these proteins in HG cells, suggesting leptin resistance. Leptin-induced pSTAT3(S727) was prevented by HG media. Inhibition of GFAT with azaserine prevented LG + leptin and HG stimulation of pSTAT3. These data demonstrate development of leptin resistance in sucrose-drinking mice and provide new evidence of leptin-induced stimulation of the HBP. PMID:25568075

  7. Primitive Extracellular Lipid Components on the Surface of the Charophytic Alga Klebsormidium flaccidum and Their Possible Biosynthetic Pathways as Deduced from the Genome Sequence

    PubMed Central

    Kondo, Satoshi; Hori, Koichi; Sasaki-Sekimoto, Yuko; Kobayashi, Atsuko; Kato, Tsubasa; Yuno-Ohta, Naoko; Nobusawa, Takashi; Ohtaka, Kinuka; Shimojima, Mie; Ohta, Hiroyuki

    2016-01-01

    Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles. PMID:27446179

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

    PubMed

    Nakada, Yuji; Itoh, Yoshifumi

    2003-03-01

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

  9. Primitive Extracellular Lipid Components on the Surface of the Charophytic Alga Klebsormidium flaccidum and Their Possible Biosynthetic Pathways as Deduced from the Genome Sequence.

    PubMed

    Kondo, Satoshi; Hori, Koichi; Sasaki-Sekimoto, Yuko; Kobayashi, Atsuko; Kato, Tsubasa; Yuno-Ohta, Naoko; Nobusawa, Takashi; Ohtaka, Kinuka; Shimojima, Mie; Ohta, Hiroyuki

    2016-01-01

    Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles. PMID:27446179

  10. Induction of potato steroidal glycoalkaloid biosynthetic pathway by overexpression of cDNA encoding primary metabolism HMG-CoA reductase and squalene synthase.

    PubMed

    Ginzberg, Idit; Thippeswamy, Muddarangappa; Fogelman, Edna; Demirel, Ufuk; Mweetwa, Alice M; Tokuhisa, James; Veilleux, Richard E

    2012-06-01

    Potato steroidal glycoalkaloids (SGAs) are toxic secondary metabolites whose total content in tubers must be regulated. SGAs are biosynthesized by the sterol branch of the mevalonic acid/isoprenoid pathway. In a previous study, we showed a correlation between SGA levels and the abundance of transcript coding for HMG-CoA reductase 1 (HMG1) and squalene synthase 1 (SQS1) in potato tissues and potato genotypes varying in SGA content. Here, Solanum tuberosum cv. Desirée (low SGA producer) was transformed with a gene construct containing the coding region of either HMG1 or SQS1 of Solanum chacoense Bitt. clone 8380-1, a high SGA producer. SGA levels in transgenic HMG-plants were either greater than (in eight of 14 plants) or no different from untransformed controls, whereas only four of 12 SQS-transgenics had greater SGA levels than control, as determined by HPLC. Quantitative real-time PCR was used to estimate relative steady-state transcript levels of isoprenoid-, steroid-, and SGA-related genes in leaves of the transgenic plants compared to nontransgenic controls. HMG-transgenic plants exhibited increased transcript accumulation of SQS1, sterol C24-methyltransferase type1 (SMT1), and solanidine glycosyltransferase 2 (SGT2), whereas SQS-transgenic plants, had consistently lower transcript levels of HMG1 and variable SMT1 and SGT2 transcript abundance among different transgenics. HMG-transgenic plants exhibited changes in transcript accumulation for some sterol biosynthetic genes as well. Taken together, the data suggest coordinated regulation of isoprenoid metabolism and SGA secondary metabolism.

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

    PubMed

    Yahyaa, Mosaab; Bar, Einat; Dubey, Neeraj Kumar; Meir, Ayala; Davidovich-Rikanati, Rachel; Hirschberg, Joseph; Aly, Radi; Tholl, Dorothea; Simon, Philipp W; Tadmor, Yaakov; Lewinsohn, Efraim; Ibdah, Mwafaq

    2013-12-18

    Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and β-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and β-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.

  12. The MORPH Algorithm: Ranking Candidate Genes for Membership in Arabidopsis and Tomato Pathways[C][W

    PubMed Central

    Tzfadia, Oren; Amar, David; Bradbury, Louis M.T.; Wurtzel, Eleanore T.; Shamir, Ron

    2012-01-01

    Closing gaps in our current knowledge about biological pathways is a fundamental challenge. The development of novel computational methods along with high-throughput experimental data carries the promise to help in the challenge. We present an algorithm called MORPH (for module-guided ranking of candidate pathway genes) for revealing unknown genes in biological pathways. The method receives as input a set of known genes from the target pathway, a collection of expression profiles, and interaction and metabolic networks. Using machine learning techniques, MORPH selects the best combination of data and analysis method and outputs a ranking of candidate genes predicted to belong to the target pathway. We tested MORPH on 230 known pathways in Arabidopsis thaliana and 93 known pathways in tomato (Solanum lycopersicum) and obtained high-quality cross-validation results. In the photosynthesis light reactions, homogalacturonan biosynthesis, and chlorophyll biosynthetic pathways of Arabidopsis, genes ranked highly by MORPH were recently verified to be associated with these pathways. MORPH candidates ranked for the carotenoid pathway from Arabidopsis and tomato are derived from pathways that compete for common precursors or from pathways that are coregulated with or regulate the carotenoid biosynthetic pathway. PMID:23204403

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

    PubMed Central

    Pluskal, Tomáš; Ueno, Masaru; Yanagida, Mitsuhiro

    2014-01-01

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

  14. Carotenoids in algae: distributions, biosyntheses and functions.

    PubMed

    Takaichi, Shinichi

    2011-01-01

    For photosynthesis, phototrophic organisms necessarily synthesize not only chlorophylls but also carotenoids. Many kinds of carotenoids are found in algae and, recently, taxonomic studies of algae have been developed. In this review, the relationship between the distribution of carotenoids and the phylogeny of oxygenic phototrophs in sea and fresh water, including cyanobacteria, red algae, brown algae and green algae, is summarized. These phototrophs contain division- or class-specific carotenoids, such as fucoxanthin, peridinin and siphonaxanthin. The distribution of α-carotene and its derivatives, such as lutein, loroxanthin and siphonaxanthin, are limited to divisions of Rhodophyta (macrophytic type), Cryptophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta. In addition, carotenogenesis pathways are discussed based on the chemical structures of carotenoids and known characteristics of carotenogenesis enzymes in other organisms; genes and enzymes for carotenogenesis in algae are not yet known. Most carotenoids bind to membrane-bound pigment-protein complexes, such as reaction center, light-harvesting and cytochrome b(6)f complexes. Water-soluble peridinin-chlorophyll a-protein (PCP) and orange carotenoid protein (OCP) are also established. Some functions of carotenoids in photosynthesis are also briefly summarized.

  15. Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis.

    PubMed

    Cordero, Baldo F; Couso, Inmaculada; León, Rosa; Rodríguez, Herminia; Vargas, M Angeles

    2011-07-01

    The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering. PMID:21519934

  16. Biosynthetic infochemical communication.

    PubMed

    Olsson, S B; Challiss, R A J; Cole, M; Gardeniers, J G E; Gardner, J W; Guerrero, A; Hansson, B S; Pearce, T C

    2015-07-09

    There is an ever-increasing demand for data to be embedded in our environment at ever-decreasing temporal and spatial scales. Whilst current communication and storage technologies generally exploit the electromagnetic properties of media, chemistry offers us a new alternative for nanoscale signaling using molecules as messengers with high information content. Biological systems effectively overcome the challenges of chemical communication using highly specific biosynthetic pathways for signal generation together with specialized protein receptors and nervous systems. Here we consider a new approach for information transmission based upon nature's quintessential example of infochemical communication, the moth pheromone system. To approach the sensitivity, specificity and versatility of infochemical communication seen in nature, we describe an array of biologically-inspired technologies for the production, transmission, detection, and processing of molecular signals. We show how it is possible to implement each step of the moth pheromone pathway for biosynthesis, transmission, receptor protein binding/transduction, and antennal lobe processing of monomolecular and multimolecular signals. For each implemented step, we discuss the value, current limitations, and challenges for the future development and integration of infochemical communication technologies. Together, these building blocks provide a starting point for future technologies that can utilize programmable emission and detection of multimolecular information for a new and robust means of communicating chemical information.

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

    DOE PAGES

    Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; Rottmann, William H.; Kuhn, Sean A.; Foster, Cliff E.; Ziebell, Angela; Turner, Geoffrey B.; Decker, Stephen R.; Hinchee, Maud A. W.; et al

    2015-08-27

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

  18. CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds[W

    PubMed Central

    Gonzalez-Jorge, Sabrina; Ha, Sun-Hwa; Magallanes-Lundback, Maria; Gilliland, Laura Ullrich; Zhou, Ailing; Lipka, Alexander E.; Nguyen, Yen-Nhu; Angelovici, Ruthie; Lin, Haining; Cepela, Jason; Little, Holly; Buell, C. Robin; Gore, Michael A.; DellaPenna, Dean

    2013-01-01

    Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops. PMID:24368792

  19. Aflatoxin biosynthetic pathway and pathway genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus is the most economically important because it produces the toxic and carcinogenic aflatoxins. A. flavus fungus is capable of surviving on many organic nutrient sources and is one of the most abundant soil-borne molds on earth. Aflatoxins were first identified in 1960. Over the la...

  20. Untargeted metabolite profiling reveals that nitric oxide bioynthesis is an endogenous modulator of carotenoid biosynthesis in Deinococcus radiodurans and is required for extreme ionizing radiation resistance.

    PubMed

    Hansler, Alex; Chen, Qiuying; Ma, Yuliang; Gross, Steven S

    2016-01-01

    Deinococcus radiodurans (Drad) is the most radioresistant organism known. Although mechanisms that underlie the extreme radioresistance of Drad are incompletely defined, resistance to UV irradiation-induced killing was found to be greatly attenuated in an NO synthase (NOS) knockout strain of Drad (Δnos). We now show that endogenous NO production is also critical for protection of Drad against γ-irradiation (3000 Gy), a result of accelerated growth recovery, not protection against killing. NO-donor treatment rescued radiosensitization in Δnos Drad but did not influence radiosensitivity in wild type Drad. To discover molecular mechanisms by which endogenous NO confers radioresistance, metabolite profiling studies were performed. Untargeted LC-MS-based metabolite profiling in Drad quantified relative abundances of 1425 molecules and levels of 294 of these were altered by >5-fold (p < 0.01). Unexpectedly, these studies identified a dramatic perturbation in carotenoid biosynthetic intermediates in Δnos Drad, including a reciprocal switch in the pathway end-products from deoxydeinoxanthin to deinoxanthin. NO supplementation rescued these nos deletion-associated changes in carotenoid biosynthesis, and fully-restored radioresistance to wildtype levels. Because carotenoids were shown to be important contributors to radioprotection in Drad, our findings suggest that endogenously-produced NO serves to maintain a spectrum of carotenoids critical for Drad's ability to withstand radiation insult.

  1. Carotenoids and Neurobiological Health.

    PubMed

    Guest, J; Grant, R

    2016-01-01

    The consumption of carotenoid phytonutrients, largely as part of plant tissue, has been associated with a number of health benefits. Epidemiological and other studies support a link between higher dietary intake and tissue concentrations of carotenoids and lower risk of chronic diseases such as heart disease, diabetes, and some cancers. Evidence also suggests that increased levels of carotenoids can help maintain healthy cognitive function, especially into older age. Carotenoids mediate their beneficial effects via several mechanisms including cell growth regulation and modulation of gene expression and immune activity. However their primary protective mechanism is thought to be due to their potent antioxidant properties that effectively scavenge free radicals and reduce the risk of oxidative damage. This chapter discusses the impact of carotenoids on neurological health by first reviewing their chemical characteristics, dietary sources, and general mechanisms of action before examining in some detail the available evidence for a protective role for various carotenoids in neurodegenerative disease. PMID:27651255

  2. Synthetic biology and metabolic engineering for marine carotenoids: new opportunities and future prospects.

    PubMed

    Wang, Chonglong; Kim, Jung-Hun; Kim, Seon-Won

    2014-09-17

    Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations.

  3. Synthetic Biology and Metabolic Engineering for Marine Carotenoids: New Opportunities and Future Prospects

    PubMed Central

    Wang, Chonglong; Kim, Jung-Hun; Kim, Seon-Won

    2014-01-01

    Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations. PMID:25233369

  4. Biotechnological production of value-added carotenoids from microalgae

    PubMed Central

    Wichuk, Kristine; Brynjólfsson, Sigurður; Fu, Weiqi

    2014-01-01

    We recently evaluated the relationship between abiotic environmental stresses and lutein biosynthesis in the green microalga Dunaliella salina and suggested a rational design of stress-driven adaptive evolution experiments for carotenoids production in microalgae. Here, we summarize our recent findings regarding the biotechnological production of carotenoids from microalgae and outline emerging technology in this field. Carotenoid metabolic pathways are characterized in several representative algal species as they pave the way for biotechnology development. The adaptive evolution strategy is highlighted in connection with enhanced growth rate and carotenoid metabolism. In addition, available genetic modification tools are described, with emphasis on model species. A brief discussion on the role of lights as limiting factors in carotenoid production in microalgae is also included. Overall, our analysis suggests that light-driven metabolism and the photosynthetic efficiency of microalgae in photobioreactors are the main bottlenecks in enhancing biotechnological potential of carotenoid production from microalgae. PMID:24691165

  5. The plant pathogen Streptomyces scabies 87-22 has a functional pyochelin biosynthetic pathway that is regulated by TetR- and AfsR-family proteins.

    PubMed

    Seipke, Ryan F; Song, Lijiang; Bicz, Joanna; Laskaris, Paris; Yaxley, Alice M; Challis, Gregory L; Loria, Rosemary

    2011-09-01

    Siderophores are high-affinity iron-chelating compounds produced by bacteria for iron uptake that can act as important virulence determinants for both plant and animal pathogens. Genome sequencing of the plant pathogen Streptomyces scabies 87-22 revealed the presence of a putative pyochelin biosynthetic gene cluster (PBGC). Liquid chromatography (LC)-MS analyses of culture supernatants of S. scabies mutants, in which expression of the cluster is upregulated and which lack a key biosynthetic gene from the cluster, indicated that pyochelin is a product of the PBGC. LC-MS comparisons with authentic standards on a homochiral stationary phase confirmed that pyochelin and not enantio-pyochelin (ent-pyochelin) is produced by S. scabies. Transcription of the S. scabies PBGC occurs via ~19 kb and ~3 kb operons and transcription of the ~19 kb operon is regulated by TetR- and AfsR-family proteins encoded by the cluster. This is the first report, to our knowledge, of pyochelin production by a Gram-positive bacterium; interestingly regulation of pyochelin production is distinct from characterized PBGCs in Gram-negative bacteria. Though pyochelin-mediated iron acquisition by Pseudomonas aeruginosa is important for virulence, in planta bioassays failed to demonstrate that pyochelin production by S. scabies is required for development of disease symptoms on excised potato tuber tissue or radish seedlings. PMID:21757492

  6. Chemical biology: Biosynthetic interceptors

    NASA Astrophysics Data System (ADS)

    Pryk, Niclas; Schulz, Frank

    2015-02-01

    Mutated enzymes are useful tools for the investigation of the biosynthetic routes to natural products. Now, they are used in a new approach to carry functionalized substrates through the synthesis and produce simplified or modified unnatural compounds with useful properties.

  7. Encapsulation of Carotenoids

    NASA Astrophysics Data System (ADS)

    Ribeiro, Henelyta S.; Schuchmann, Heike P.; Engel, Robert; Walz, Elke; Briviba, Karlis

    Carotenoids are natural pigments, which are synthesized by microorganisms and plants. More than 600 naturally occurring carotenoids have been found in the nature. The main sources of carotenoids are fruits, vegetables, leaves, peppers, and certain types of fishes, sea foods, and birds. Carotenoids may protect cells against photosensitization and work as light-absorbing pigments during photosynthesis. Some carotenoids may inhibit the destructive effect of reactive oxygen species. Due to the antioxidative properties of carotenoids, many investigations regarding their protective effects against cardiovascular diseases and certain types of cancers, as well as other degenerative illnesses, have been carried out in the last years (Briviba et al. 2004; Krinsky et al. 2004; Kirsh et al. 2006). A diet rich in carotenoids may also contribute to photoprotection against UV radiation (Stahl et al. 2006). In vitro studies have shown that carotenoids such as β-cryptoxanthin and lycopene stimulate bone formation and mineralization. The results may be related to prevention of osteoporosis (Kim et al. 2003; Yamaguchi and Uchiyama 2003; 2004; Yamaguchi et al. 2005).

  8. Carotenoid metabolism in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carotenoids are mostly C40 terpenoids, a class of hydrocarbons that participate in various biological processes in plants, such as photosynthesis, photomorphogenesis, photoprotection, and development. Carotenoids also serve as precursors for two plant hormones and a diverse set of apocarotenoids. Th...

  9. Biosynthesis of Carotenoids in Plants: Enzymes and Color.

    PubMed

    Rosas-Saavedra, Carolina; Stange, Claudia

    2016-01-01

    Carotenoids are the most important biocolor isoprenoids responsible for yellow, orange and red colors found in nature. In plants, they are synthesized in plastids of photosynthetic and sink organs and are essential molecules for photosynthesis, photo-oxidative damage protection and phytohormone synthesis. Carotenoids also play important roles in human health and nutrition acting as vitamin A precursors and antioxidants. Biochemical and biophysical approaches in different plants models have provided significant advances in understanding the structural and functional roles of carotenoids in plants as well as the key points of regulation in their biosynthesis. To date, different plant models have been used to characterize the key genes and their regulation, which has increased the knowledge of the carotenoid metabolic pathway in plants. In this chapter a description of each step in the carotenoid synthesis pathway is presented and discussed.

  10. Biosynthesis of Carotenoids in Plants: Enzymes and Color.

    PubMed

    Rosas-Saavedra, Carolina; Stange, Claudia

    2016-01-01

    Carotenoids are the most important biocolor isoprenoids responsible for yellow, orange and red colors found in nature. In plants, they are synthesized in plastids of photosynthetic and sink organs and are essential molecules for photosynthesis, photo-oxidative damage protection and phytohormone synthesis. Carotenoids also play important roles in human health and nutrition acting as vitamin A precursors and antioxidants. Biochemical and biophysical approaches in different plants models have provided significant advances in understanding the structural and functional roles of carotenoids in plants as well as the key points of regulation in their biosynthesis. To date, different plant models have been used to characterize the key genes and their regulation, which has increased the knowledge of the carotenoid metabolic pathway in plants. In this chapter a description of each step in the carotenoid synthesis pathway is presented and discussed. PMID:27485218

  11. Functions of Intact Carotenoids

    NASA Astrophysics Data System (ADS)

    Britton, George

    The traditional view that carotenoids are a class of plant pigments does not do justice to their versatility. This versatility will become clear from the overview of the biological roles of carotenoids, in animals and microorganisms as well as in plants, that is given in this Chapter. It has become customary and convenient to differentiate biological effects of carotenoids into functions, actions and associations [1]. `Functions' have been defined as effects or properties that are essential for the normal well-being of the organism. Biological responses that follow the administration of carotenoids in the diet or as supplements are considered as `actions'. When an effect is seen but a causal relationship to the carotenoid has not been demonstrated, this is described as an `association'. The line between these is often not clear, however.

  12. Analysis of carotenoid accumulation and expression of carotenoid biosynthesis genes in different organs of Chinese cabbage (Brassica rapa subsp. pekinensis).

    PubMed

    Tuan, Pham Anh; Kim, Jae Kwang; Lee, Jeongyeo; Park, Woo Tae; Kwon, Do Yeon; Kim, Yeon Bok; Kim, Haeng Hoon; Kim, Hye Ran; Park, Sang Un

    2012-01-01

    The relationship between carotenoid accumulation and expression of carotenoid biosynthesis genes was investigated in the flowers, stems, young leaves, old leaves, and roots of Chinese cabbage (Brassica rapa subsp. pekinensis). Quantitative real-time PCR analysis showed that the mRNA levels of BrPSY, BrPDS, BrZDS, BrLCYB, BrLCYE, BrCHXB, and BrZEP leading to the production of carotenoids were highest in the flowers or the leaves and lowest in the roots of Chinese cabbage. In contrast, the mRNA expression of BrNCED, a gene involved in abscisic acid (ABA) biosynthesis, was highest in the roots. High-performance liquid chromatography revealed that carotenoids, namely, lutein and β-carotene, were distributed predominantly in the flowers and leaves, with very little in the underground organ, the roots. Specifically, old leaves contained 120.3 μg/g lutein and 103.93 μg/g β-carotene, which is the most potent dietary precursor of vitamin A. Moreover, we found a relatively large amount of cis isomers of β-carotene, namely, 9-cis β-carotene and 13-cis β-carotene, in Chinese cabbage. These results provide insight into carotenoid biosynthetic mechanisms in Chinese cabbage and may be helpful in the metabolic engineering of carotenoid biosynthesis in plants.

  13. Biosynthetic engineering of nonribosomal peptide synthetases.

    PubMed

    Kries, Hajo

    2016-09-01

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

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

    PubMed Central

    Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

    2013-01-01

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

  15. Carotenoids and Photosynthesis.

    PubMed

    Hashimoto, Hideki; Uragami, Chiasa; Cogdell, Richard J

    2016-01-01

    Carotenoids are ubiquitous and essential pigments in photosynthesis. They absorb in the blue-green region of the solar spectrum and transfer the absorbed energy to (bacterio-)chlorophylls, and so expand the wavelength range of light that is able to drive photosynthesis. This is an example of singlet-singlet energy transfer, and so carotenoids serve to enhance the overall efficiency of photosynthetic light reactions. Carotenoids also act to protect photosynthetic organisms from the harmful effects of excess exposure to light. Triplet-triplet energy transfer from chlorophylls to carotenoids plays a key role in this photoprotective reaction. In the light-harvesting pigment-protein complexes from purple photosynthetic bacteria and chlorophytes, carotenoids have an additional role of structural stabilization of those complexes. In this article we review what is currently known about how carotenoids discharge these functions. The molecular architecture of photosynthetic systems will be outlined first to provide a basis from which to describe carotenoid photochemistry, which underlies most of their important functions in photosynthesis. PMID:27485220

  16. Carotenoids and Photosynthesis.

    PubMed

    Hashimoto, Hideki; Uragami, Chiasa; Cogdell, Richard J

    2016-01-01

    Carotenoids are ubiquitous and essential pigments in photosynthesis. They absorb in the blue-green region of the solar spectrum and transfer the absorbed energy to (bacterio-)chlorophylls, and so expand the wavelength range of light that is able to drive photosynthesis. This is an example of singlet-singlet energy transfer, and so carotenoids serve to enhance the overall efficiency of photosynthetic light reactions. Carotenoids also act to protect photosynthetic organisms from the harmful effects of excess exposure to light. Triplet-triplet energy transfer from chlorophylls to carotenoids plays a key role in this photoprotective reaction. In the light-harvesting pigment-protein complexes from purple photosynthetic bacteria and chlorophytes, carotenoids have an additional role of structural stabilization of those complexes. In this article we review what is currently known about how carotenoids discharge these functions. The molecular architecture of photosynthetic systems will be outlined first to provide a basis from which to describe carotenoid photochemistry, which underlies most of their important functions in photosynthesis.

  17. Manipulation of Carotenoid Content in Plants to Improve Human Health.

    PubMed

    Alós, Enriqueta; Rodrigo, Maria Jesús; Zacarias, Lorenzo

    2016-01-01

    Carotenoids are essential components for human nutrition and health, mainly due to their antioxidant and pro-vitamin A activity. Foods with enhanced carotenoid content and composition are essential to ensure carotenoid feasibility in malnourished population of many countries around the world, which is critical to alleviate vitamin A deficiency and other health-related disorders. The pathway of carotenoid biosynthesis is currently well understood, key steps of the pathways in different plant species have been characterized and the corresponding genes identified, as well as other regulatory elements. This enables the manipulation and improvement of carotenoid content and composition in order to control the nutritional value of a number of agronomical important staple crops. Biotechnological and genetic engineering-based strategies to manipulate carotenoid metabolism have been successfully implemented in many crops, with Golden rice as the most relevant example of β-carotene improvement in one of the more widely consumed foods. Conventional breeding strategies have been also adopted in the bio-fortification of carotenoid in staple foods that are highly consumed in developing countries, including maize, cassava and sweet potatoes, to alleviate nutrition-related problems. The objective of the chapter is to summarize major breakthroughs and advances in the enhancement of carotenoid content and composition in agronomical and nutritional important crops, with special emphasis to their potential impact and benefits in human nutrition and health. PMID:27485228

  18. Manipulation of Carotenoid Content in Plants to Improve Human Health.

    PubMed

    Alós, Enriqueta; Rodrigo, Maria Jesús; Zacarias, Lorenzo

    2016-01-01

    Carotenoids are essential components for human nutrition and health, mainly due to their antioxidant and pro-vitamin A activity. Foods with enhanced carotenoid content and composition are essential to ensure carotenoid feasibility in malnourished population of many countries around the world, which is critical to alleviate vitamin A deficiency and other health-related disorders. The pathway of carotenoid biosynthesis is currently well understood, key steps of the pathways in different plant species have been characterized and the corresponding genes identified, as well as other regulatory elements. This enables the manipulation and improvement of carotenoid content and composition in order to control the nutritional value of a number of agronomical important staple crops. Biotechnological and genetic engineering-based strategies to manipulate carotenoid metabolism have been successfully implemented in many crops, with Golden rice as the most relevant example of β-carotene improvement in one of the more widely consumed foods. Conventional breeding strategies have been also adopted in the bio-fortification of carotenoid in staple foods that are highly consumed in developing countries, including maize, cassava and sweet potatoes, to alleviate nutrition-related problems. The objective of the chapter is to summarize major breakthroughs and advances in the enhancement of carotenoid content and composition in agronomical and nutritional important crops, with special emphasis to their potential impact and benefits in human nutrition and health.

  19. Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin.

    PubMed

    Liu, Yanli; Liu, Shasha; Yang, Tingting; Guo, Xiaoxia; Jiang, Yali; Zahid, Kashif Rafiq; Liu, Ke; Liu, Jinlin; Yang, Jihong; Zhao, Haobin; Yang, Yi; Li, Aiying; Qi, Chao

    2015-12-01

    Medermycin, as a prominent member of benzoisochromanequinones, possesses strong antitumor activity and is biosynthesized under the control of a 29-ORF-containing biosynthetic gene cluster. Most of ORFs in this gene cluster have not been characterized, including a small protein encoding gene med-ORF10, proposed to play a regulatory role in biosynthesis of medermycin in an unknown mode. In this study, we reported the expression, protein preparation, crystallization and preliminary X-ray diffraction analyses of Med-ORF10 of the wild type Streptomyces strain. Firstly, we cloned and overexpressed med-ORF10 in Escherichia coli and purified the protein with 98% purity and 3 mg/L yield. Then, we crystallized the protein at concentration of 20 mg/mL in condition 22% PEG 3350, 0.2 M magnesium formate and collected the data at 1.78 Å resolution. Finally, we detected the expression of Med-ORF10 in Streptomyces by western blotting. In conclusion, this study confirmed the expression of Med-ORF10 protein in the wild-type strain of Streptomyces AM-7161 and collected the X-ray diffraction data of Med-ORF10 crystal at 1.78 Å resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.

  20. Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains.

    PubMed

    Qin, Xiaoqiong; Zhang, Wenjun; Dubcovsky, Jorge; Tian, Li

    2012-12-01

    Carotenoid β-hydroxylases attach hydroxyl groups to the β-ionone rings (β-rings) of carotenoid substrates, resulting in modified structures and functions of carotenoid molecules. We cloned and characterized two genes (each with three homeologs), HYD1 and HYD2, which encode β-hydroxylases in wheat. The results from bioinformatic and nested degenerate PCR analyses collectively suggest that HYD1 and HYD2 may represent the entire complement of non-heme di-iron β-hydroxylases in wheat. The homeologs of wheat HYDs exhibited major β-ring and minor ε-ring hydroxylation activities in carotenoid-accumulating E. coli strains. Distinct expression patterns were observed for different HYD genes and homeologs in vegetative tissues and developing grains of tetraploid and hexaploid wheat, suggesting their functional divergence and differential regulatory control in tissue-, grain development-, and ploidy-specific manners. An intriguing observation was that the expression of HYD1, particularly HYD-B1, reached highest levels at the last stage of tetraploid and hexaploid grain development, suggesting that carotenoids (at least xanthophylls) were still actively synthesized in mature grains. This result challenges the common perception that carotenoids are simply being turned over during wheat grain development after their initial biosynthesis at the early grain development stages. Overall, this improved understanding of carotenoid biosynthetic gene expression and carotenoid metabolism in wheat grains will contribute to the improvement of the nutritional value of wheat grains for human consumption.

  1. Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains.

    PubMed

    Qin, Xiaoqiong; Zhang, Wenjun; Dubcovsky, Jorge; Tian, Li

    2012-12-01

    Carotenoid β-hydroxylases attach hydroxyl groups to the β-ionone rings (β-rings) of carotenoid substrates, resulting in modified structures and functions of carotenoid molecules. We cloned and characterized two genes (each with three homeologs), HYD1 and HYD2, which encode β-hydroxylases in wheat. The results from bioinformatic and nested degenerate PCR analyses collectively suggest that HYD1 and HYD2 may represent the entire complement of non-heme di-iron β-hydroxylases in wheat. The homeologs of wheat HYDs exhibited major β-ring and minor ε-ring hydroxylation activities in carotenoid-accumulating E. coli strains. Distinct expression patterns were observed for different HYD genes and homeologs in vegetative tissues and developing grains of tetraploid and hexaploid wheat, suggesting their functional divergence and differential regulatory control in tissue-, grain development-, and ploidy-specific manners. An intriguing observation was that the expression of HYD1, particularly HYD-B1, reached highest levels at the last stage of tetraploid and hexaploid grain development, suggesting that carotenoids (at least xanthophylls) were still actively synthesized in mature grains. This result challenges the common perception that carotenoids are simply being turned over during wheat grain development after their initial biosynthesis at the early grain development stages. Overall, this improved understanding of carotenoid biosynthetic gene expression and carotenoid metabolism in wheat grains will contribute to the improvement of the nutritional value of wheat grains for human consumption. PMID:23015203

  2. Molecular characterisation and the light-dark regulation of carotenoid biosynthesis in sprouts of tartary buckwheat (Fagopyrum tataricum Gaertn.).

    PubMed

    Tuan, Pham Anh; Thwe, Aye Aye; Kim, Jae Kwang; Kim, Yeon Bok; Lee, Sanghyun; Park, Sang Un

    2013-12-15

    Seven partial-length cDNAs and 1 full-length cDNA that were involved in carotenoid biosynthesis and 2 partial-length cDNAs that encoded carotenoid cleavage dioxygenases were first isolated and characterised in 2 tartary buckwheat cultivars (Fagopyrum tataricum Gaertn.), Hokkai T8 and Hokkai T10. They were constitutively expressed at high levels in the leaves and flowers, where carotenoids are mostly distributed. During the seed development of tartary buckwheat, an inverse correlation between transcription level of carotenoid cleavage dioxygenase and carotenoid content was observed. The light-grown sprouts exhibited higher levels of expression of carotenoid biosynthetic genes in T10 and carotenoid content in both T8 and T10 compared to the dark-grown sprouts. The predominant carotenoids in tartary buckwheat were lutein and β-carotene, and very abundant amounts of these carotenoids were found in light-grown sprouts. This study might broaden our understanding of the molecular mechanisms involved in carotenoid biosynthesis and indicates targets for increasing the production of carotenoids in tartary buckwheat.

  3. Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1), which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes, modulates carotenoid metabolism in transgenic tomato.

    PubMed

    Endo, Tomoko; Fujii, Hiroshi; Sugiyama, Aiko; Nakano, Michiharu; Nakajima, Naoko; Ikoma, Yoshinori; Omura, Mitsuo; Shimada, Takehiko

    2016-02-01

    To explore the transcription factors associated with carotenoid metabolism in citrus fruit, one transcription factor (CubHLH1) was selected through microarray screening in Satsuma mandarin (Citrus unshiu Marc.) fruit, which was treated with exogenous ethylene or gibberellin (GA), accelerating or retarding carotenoid accumulation in peel, respectively. The amino acid sequence of CubHLH1 has homology to Arabidopsis activation-tagged bri1 suppressor 1 (ATBS1) interacting factor (AIF), which is functionally characterized as a negative regulator of the brassinolide (BR) signalling pathway. Yeast two-hybrid analysis revealed that protein for CubHLH1 could interact with Arabidopsis and tomato ATBS1. Overexpression of CubHLH1 caused a dwarf phenotype in transgenic tomato (Solanum lycopersicum L.), suggesting that CubHLH1 has a similar function to Arabidopsis AIF. In the transgenic tomato fruit at ripening stage, the lycopene content was reduced along with the changes in carotenoid biosynthetic gene expression. The abscisic acid (ABA) content of all the transgenic tomato fruit was higher than that of the wild type. These results implied that CubHLH1 is considered to have a similar function to Arabidopsis AIFs and might be directly involved in carotenoid metabolism in mature citrus fruit.

  4. Carotenoids and chronic diseases.

    PubMed

    Agarwal, S; Rao, A V

    2000-01-01

    Chronic diseases such as cancer and cardiovascular diseases are the major causes of deaths in North America. Dietary intake of fruits and vegetables has been suggested to have protective effects against such chronic diseases. Carotenoids are important plant pigments which are thought to contribute towards the beneficial effects of fruit and vegetable consumption. This review focuses on the role of carotenoids and particularly lycopene in chronic diseases.

  5. Structure of the Bifunctional Acyltransferase/Decarboxylase LnmK from the Leinamycin Biosynthetic Pathway Revealing Novel Activity for a Double-Hot-Dog Fold

    SciTech Connect

    Lohman, Jeremy R.; Bingman, Craig A.; George N. Phillips Jr.; Shen, Ben

    2013-01-15

    The β-branched C3 unit in leinamycin biosynthesis is installed by a set of four proteins, LnmFKLM. In vitro biochemical investigation confirmed that LnmK is a bifunctional acyltransferase/decarboxylase (AT/DC) that catalyzes first self-acylation using methylmalonyl-CoA as a substrate and subsequently transacylation of the methylmalonyl group to the phosphopantetheinyl group of the LnmL acyl carrier protein [Liu, T., Huang, Y., and Shen, B. (2009) J. Am. Chem. Soc. 131, 6900–6901]. LnmK shows no sequence homology to proteins of known function, representing a new family of AT/DC enzymes. Here we report the X-ray structure of LnmK. LnmK is homodimer with each of the monomers adopting a double-hot-dog fold. Cocrystallization of LnmK with methylmalonyl-CoA revealed an active site tunnel terminated by residues from the dimer interface. But, to canonical AT and ketosynthase enzymes that employ Ser or Cys as an active site residue, none of these residues are found in the vicinity of the LnmK active site. Instead, three tyrosines were identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residue for the AT activity of LnmK. Moreover, LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date. The LnmK structure sets the stage for probing of the DC activity of LnmK through site-directed mutagenesis. These findings highlight natural product biosynthetic machinery as a rich source of novel enzyme activities, mechanisms, and structures.

  6. Characterization of a SAM-dependent fluorinase from a latent biosynthetic pathway for fluoroacetate and 4-fluorothreonine formation in Nocardia brasiliensis

    PubMed Central

    Qu, Xudong

    2014-01-01

    Fluorination has been widely used in chemical synthesis, but is rare in nature. The only known biological fluorination scope is represented by the fl pathway from Streptomyces cattleya that produces fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Here we report the identification of a novel pathway for FAc and 4-FT biosynthesis from the actinomycetoma-causing pathogen Nocardia brasiliensis ATCC 700358. The new pathway shares overall conservation with the fl pathway in S. cattleya. Biochemical characterization of the conserved domains revealed a novel fluorinase NobA that can biosynthesize 5’-fluoro-5’-deoxyadenosine (5’-FDA) from inorganic fluoride and S-adenosyl-l-methionine (SAM). The NobA shows similar halide specificity and characteristics to the fluorination enzyme FlA of the fl pathway. Kinetic parameters for fluoride ( K m 4153 μM, k cat 0.073 min -1) and SAM ( K m 416 μM, k cat 0.139 min -1) have been determined, revealing that NobA is slightly (2.3 fold) slower than FlA. Upon sequence comparison, we finally identified a distinct loop region in the fluorinases that probably accounts for the disparity of fluorination activity. PMID:24795808

  7. Radical SAM, A Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods

    SciTech Connect

    Sofia, Heidi J.; Chen, Guang; Hetzler, Elizabeth G.; Reyes Spindola, Jorge F.; Miller, Nancy E.

    2001-03-01

    A large protein superfamily with over 500 members has been discovered and analyzed using powerful new bioinformatics and information visualization methods. Evidence exists that these proteins generate a 5?-deoxyadenosyl radical by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. Radical SAM superfamily proteins function in DNA precursor, vitamin, cofactor, antibiotic, and herbicide biosynthesis in a collection of basic and familiar pathways. One of the members is interferon-inducible and is considered a candidate drug target for osteoporosis. The identification of this superfamily suggests that radical-based catalysis is important in a number of previously well-studied but unresolved biochemical pathways.

  8. A molecular genetic analysis of carotenoid biosynthesis and the effects of carotenoid mutations on other photosynthetic genes in Rhodobacter capsulatus

    SciTech Connect

    Armstrong, G.A.

    1989-04-01

    The nine known R. capsulatus carotenoid genes are contained within the 46 kilobase (kb) photosynthesis gene cluster. An 11 kb subcluster containing eight of these genes has been cloned and its nucleotide sequence determined. A new gene, crtK, has been located in the middle of the subcluster. The carotenoid gene cluster contains sequences homologous to Escherichia coli ..omega../sup 70/ promoters, rho-independent transcription terminators, and prokaryotic transcriptional factor binding sites. The phenotypes and genotypes of ten transposon Tn5.7 insertion mutations within the carotenoid gene cluster have been analyzed, by characterization of the carotenoids accumulated and high resolution mapping of the Tn5.7 insertions. The enzymatic blockages in previously uncharacterized early carotenoid mutants have been determined using a new in vitro synthesis system, suggesting specific roles for the CrtB and CrtE gene products. The expression of six of the eight carotenoid genes in the cluster is induced upon the shift from dark chemoheterotrophic to anaerobic photosynthetic growth. The magnitude of the induction is equivalent to that of genes encoding structural photosynthesis polypeptides, although the carotenoid genes are induced earlier after the growth shift. Different means of regulating photosynthesis genes in R. capsulatus are discussed, and a rationale for the temporal pattern of expression of the carotenoid genes during photosynthetic adaptation is presented. Comparison of the deduced amino acid sequences of the two dehydrogenases of the R. capsulatus carotenoid biosynthesis pathway reveals two regions of strong similarity. The effect of carotenoid mutations on the photosynthetic phenotype has been studied by examining growth rates, pigments, pigment-protein complexes and gene expression for a complete set of carotenoid mutants. 161 refs.

  9. New Biosynthetic Step in the Melanin Pathway of Wangiella (Exophiala) dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced as a pentaketide directly by an iter...

  10. Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway*

    PubMed Central

    Moore, Simon J.; Biedendieck, Rebekka; Lawrence, Andrew D.; Deery, Evelyne; Howard, Mark J.; Rigby, Stephen E. J.; Warren, Martin J.

    2013-01-01

    The anaerobic pathway for the biosynthesis of cobalamin (vitamin B12) has remained poorly characterized because of the sensitivity of the pathway intermediates to oxygen and the low activity of enzymes. One of the major bottlenecks in the anaerobic pathway is the ring contraction step, which has not been observed previously with a purified enzyme system. The Gram-positive aerobic bacterium Bacillus megaterium has a complete anaerobic pathway that contains an unusual ring contraction enzyme, CbiH60, that harbors a C-terminal extension with sequence similarity to the nitrite/sulfite reductase family. To improve solubility, the enzyme was homologously produced in the host B. megaterium DSM319. CbiH60 was characterized by electron paramagnetic resonance and shown to contain a [4Fe-4S] center. Assays with purified recombinant CbiH60 demonstrate that the enzyme converts both cobalt-precorrin-3 and cobalt factor III into the ring-contracted product cobalt-precorrin-4 in high yields, with the latter transformation dependent upon DTT and an intact Fe-S center. Furthermore, the ring contraction process was shown not to involve a change in the oxidation state of the central cobalt ion of the macrocycle. PMID:23155054

  11. Carotenoid Formation by Staphylococcus aureus

    PubMed Central

    Hammond, Ray K.; White, David C.

    1970-01-01

    The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; ζ-carotene; δ-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-14C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of 14C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of 14C for 2.5 bacterial doublings. The incorporation and turnover of 14C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids. PMID:5423369

  12. [Carotenoids as natural antioxidants].

    PubMed

    Igielska-Kalwat, Joanna; Gościańska, Joanna; Nowak, Izabela

    2015-04-07

    Human organisms have many defence mechanisms able to neutralise the harmful effects of the reactive species of oxygen. Antioxidants play an important role in reducing the oxidative damage to the human organism. Carotenoids are among the strongest antioxidants. They have 11 coupled double bonds, so they can be classified as polyisoprenoids, show low polarity and can occur in acyclic, monocyclic or bicyclic forms. The carotenoids of the strongest antioxidant properties are lycopene, lutein, astaxanthin and β-carotene. Carotenoids with strong antioxidant properties have found wide application in medical, pharmaceutical and cosmetic industries. These compounds are highly active against both reactive oxygen species and free radicals. Comparing β-carotene, astaxanthin and lycopene with other antioxidants (e.g. vitamin C and E), it can be concluded that these compounds have higher antioxidant activity, e.g. against singlet oxygen. Astaxanthin is a stronger antioxidant compared to β-carotene, vitamin E and vitamin C, respectively 54, 14 and 65 times. Carotenoids have a salutary effect on our body, making it more resistant and strong to fight chronic diseases. The purpose of this article is to review the literature concerning free radicals and their adverse effects on the human body and carotenoids, as strong, natural antioxidants.

  13. Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803

    PubMed Central

    Selstam, Eva; Norling, Birgitta

    2015-01-01

    The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that “light” plasma membranes have a high carotenoid/protein ratio, when compared to “heavier” plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone. PMID:26083372

  14. ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

    PubMed

    Xing, Shufan; van Deenen, Nicole; Magliano, Pasqualina; Frahm, Lea; Forestier, Edith; Nawrath, Christiane; Schaller, Hubert; Gronover, Christian S; Prüfer, Dirk; Poirier, Yves

    2014-07-01

    Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids. PMID:24844815

  15. ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

    PubMed

    Xing, Shufan; van Deenen, Nicole; Magliano, Pasqualina; Frahm, Lea; Forestier, Edith; Nawrath, Christiane; Schaller, Hubert; Gronover, Christian S; Prüfer, Dirk; Poirier, Yves

    2014-07-01

    Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

  16. Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants

    SciTech Connect

    Talon, M. Zeevaart, J.A.D. ); Koornneef, M. )

    1990-10-01

    Twenty gibberellins (GAs) have been identified in extracts from shoots of the Landsberg erecta line of Arabidopsis thaliana by full-scan gas chromatography-mass spectrometry and Kovats retention indices. Eight of them are members of the early-13-hydroxylation pathway (GA{sub 53}, GA{sub 44}, GA{sub 19}, GA{sub 17}, GA{sub 20}, GA{sub 1}, GA{sub 29}, and GA{sub 8}), six are members of the early-3-hydroxylation pathway (GA{sub 37}, GA{sub 27}, GA{sub 36}, GA{sub 13}, GA{sub 4}, and GA{sub 34}), and the remaining six are members of the non-3,13-hydroxylation pathway (GA{sub 12}, GA{sub 15}, GA{sub 24}, GA{sub 25}, GA{sub 9}, and GFA{sub 51}). Seven of these GAs were quantified in the Landsberg erecta line of Arabidopsis and in the semidwarf ga4 and ga5 mutants by gas chromatography-selected ion monitoring (SIM) using internal standards. The relative levels of the remaining 13 GAs were compared by the use of ion intensities only. The growth-response data, as well as the accumulation of GA{sub 9} in the ga4 mutant, indicate that GA{sub 9} is not active in Arabidopsis, but it must be 3{beta}-hydroxytlated to GA{sub 4} to become bioactive. It is concluded that the reduced levels of the 3{beta}-hydroxy-GAs, GA{sub 1} and GA{sub 4}, are the cause of the semidwarf growth habit of both mutants.

  17. The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

    PubMed Central

    Gao, Shouhong; Saechao, Saengking; Di, Peng; Chen, Junfeng; Chen, Wansheng

    2011-01-01

    Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors. PMID:22242141

  18. Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae

    PubMed Central

    2013-01-01

    Background Xanthophylls, oxygenated derivatives of carotenes, play critical roles in photosynthetic apparatus of cyanobacteria, algae, and higher plants. Although the xanthophylls biosynthetic pathway of algae is largely unknown, it is of particular interest because they have a very complicated evolutionary history. Carotenoid hydroxylase (CHY) is an important protein that plays essential roles in xanthophylls biosynthesis. With the availability of 18 sequenced algal genomes, we performed a comprehensive comparative analysis of chy genes and explored their distribution, structure, evolution, origins, and expression. Results Overall 60 putative chy genes were identified and classified into two major subfamilies (bch and cyp97) according to their domain structures. Genes in the bch subfamily were found in 10 green algae and 1 red alga, but absent in other algae. In the phylogenetic tree, bch genes of green algae and higher plants share a common ancestor and are of non-cyanobacterial origin, whereas that of red algae is of cyanobacteria. The homologs of cyp97a/c genes were widespread only in green algae, while cyp97b paralogs were seen in most of algae. Phylogenetic analysis on cyp97 genes supported the hypothesis that cyp97b is an ancient gene originated before the formation of extant algal groups. The cyp97a gene is more closely related to cyp97c in evolution than to cyp97b. The two cyp97 genes were isolated from the green alga Haematococcus pluvialis, and transcriptional expression profiles of chy genes were observed under high light stress of different wavelength. Conclusions Green algae received a β-xanthophylls biosynthetic pathway from host organisms. Although red algae inherited the pathway from cyanobacteria during primary endosymbiosis, it remains unclear in Chromalveolates. The α-xanthophylls biosynthetic pathway is a common feature in green algae and higher plants. The origination of cyp97a/c is most likely due to gene duplication before divergence of

  19. Fungal peroxisomes as biosynthetic organelles.

    PubMed

    Stehlik, Thorsten; Sandrock, Björn; Ast, Julia; Freitag, Johannes

    2014-12-01

    Peroxisomes are nearly ubiquitous single-membrane organelles harboring multiple metabolic pathways beside their prominent role in the β-oxidation of fatty acids. Here we review the diverse metabolic functions of peroxisomes in fungi. A variety of fungal metabolites are at least partially synthesized inside peroxisomes. These include the essential co-factor biotin but also different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters for example β-oxidation intermediates. In several ascomycetes a subtype of peroxisomes has been identified that is metabolically inactive but is required to plug the septal pores of wounded hyphae. Thus, peroxisomes are versatile organelles that can adapt their function to the life style of an organism. This remarkable variability suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive. Moreover, in fungi peroxisomes are non-essential under laboratory conditions making them attractive organelles for biotechnological approaches and the design of novel metabolic pathways in customized peroxisomes.

  20. Carotenoid fluorescence in Dunaliella salina

    PubMed Central

    van Es, Marjon A.; Janssen, Marcel; Brandenburg, Willem A.; Wijffels, René H.

    2010-01-01

    Dunaliella salina is a halotolerant green alga that is well known for its carotenoid producing capacity. The produced carotenoids are mainly stored in lipid globules. For various research purposes, such as production and extraction kinetics, we would like to determine and/or localise the carotenoid globules in vivo. In this study, we show that the carotenoid-rich globules emit clear green fluorescence, which can be used in, for example, fluorescence microscopy (e.g. CLSM) to obtain pictures of the cells and their carotenoid content. PMID:20835349

  1. Carotenoid fluorescence in Dunaliella salina.

    PubMed

    Kleinegris, Dorinde M M; van Es, Marjon A; Janssen, Marcel; Brandenburg, Willem A; Wijffels, René H

    2010-10-01

    Dunaliella salina is a halotolerant green alga that is well known for its carotenoid producing capacity. The produced carotenoids are mainly stored in lipid globules. For various research purposes, such as production and extraction kinetics, we would like to determine and/or localise the carotenoid globules in vivo. In this study, we show that the carotenoid-rich globules emit clear green fluorescence, which can be used in, for example, fluorescence microscopy (e.g. CLSM) to obtain pictures of the cells and their carotenoid content.

  2. Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway.

    PubMed

    Wallace, Simon; Chater, Caspar C; Kamisugi, Yasuko; Cuming, Andrew C; Wellman, Charles H; Beerling, David J; Fleming, Andrew J

    2015-01-01

    The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

  3. Plastids and carotenoid accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plastids are ubiquitously in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids except proplastids can synth...

  4. The floral transcriptome of ylang ylang (Cananga odorata var. fruticosa) uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase

    PubMed Central

    Jin, Jingjing; Kim, Mi Jung; Dhandapani, Savitha; Tjhang, Jessica Gambino; Yin, Jun-Lin; Wong, Limsoon; Sarojam, Rajani; Chua, Nam-Hai; Jang, In-Cheol

    2015-01-01

    The pleasant fragrance of ylang ylang varieties (Cananga odorata) is mainly due to volatile organic compounds (VOCs) produced by the flowers. Floral scents are a key factor in plant–insect interactions and are vital for successful pollination. C. odorata var. fruticosa, or dwarf ylang ylang, is a variety of ylang ylang that is popularly grown in Southeast Asia as a small shrub with aromatic flowers. Here, we describe the combined use of bioinformatics and chemical analysis to discover genes for the VOC biosynthesis pathways and related genes. The scented flowers of C. odorata var. fruticosa were analysed by gas chromatography/mass spectrometry and a total of 49 VOCs were identified at four different stages of flower development. The bulk of these VOCs were terpenes, mainly sesquiterpenes. To identify the various terpene synthases (TPSs) involved in the production of these essential oils, we performed RNA sequencing on mature flowers. From the RNA sequencing data, four full-length TPSs were functionally characterized. In vitro assays showed that two of these TPSs were mono-TPSs. CoTPS1 synthesized four products corresponding to β-thujene, sabinene, β-pinene, and α-terpinene from geranyl pyrophosphate and CoTPS4 produced geraniol from geranyl pyrophosphate. The other two TPSs were identified as sesqui-TPSs. CoTPS3 catalysed the conversion of farnesyl pyrophosphate to α-bergamotene, whereas CoTPS2 was found to be a multifunctional and novel TPS that could catalyse the synthesis of three sesquiterpenes, β-ylangene, β-copaene, and β-cubebene. Additionally, the activities of the two sesqui-TPSs were confirmed in planta by transient expression of these TPS genes in Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. PMID:25956881

  5. The floral transcriptome of ylang ylang (Cananga odorata var. fruticosa) uncovers biosynthetic pathways for volatile organic compounds and a multifunctional and novel sesquiterpene synthase.

    PubMed

    Jin, Jingjing; Kim, Mi Jung; Dhandapani, Savitha; Tjhang, Jessica Gambino; Yin, Jun-Lin; Wong, Limsoon; Sarojam, Rajani; Chua, Nam-Hai; Jang, In-Cheol

    2015-07-01

    The pleasant fragrance of ylang ylang varieties (Cananga odorata) is mainly due to volatile organic compounds (VOCs) produced by the flowers. Floral scents are a key factor in plant-insect interactions and are vital for successful pollination. C. odorata var. fruticosa, or dwarf ylang ylang, is a variety of ylang ylang that is popularly grown in Southeast Asia as a small shrub with aromatic flowers. Here, we describe the combined use of bioinformatics and chemical analysis to discover genes for the VOC biosynthesis pathways and related genes. The scented flowers of C. odorata var. fruticosa were analysed by gas chromatography/mass spectrometry and a total of 49 VOCs were identified at four different stages of flower development. The bulk of these VOCs were terpenes, mainly sesquiterpenes. To identify the various terpene synthases (TPSs) involved in the production of these essential oils, we performed RNA sequencing on mature flowers. From the RNA sequencing data, four full-length TPSs were functionally characterized. In vitro assays showed that two of these TPSs were mono-TPSs. CoTPS1 synthesized four products corresponding to β-thujene, sabinene, β-pinene, and α-terpinene from geranyl pyrophosphate and CoTPS4 produced geraniol from geranyl pyrophosphate. The other two TPSs were identified as sesqui-TPSs. CoTPS3 catalysed the conversion of farnesyl pyrophosphate to α-bergamotene, whereas CoTPS2 was found to be a multifunctional and novel TPS that could catalyse the synthesis of three sesquiterpenes, β-ylangene, β-copaene, and β-cubebene. Additionally, the activities of the two sesqui-TPSs were confirmed in planta by transient expression of these TPS genes in Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration. PMID:25956881

  6. Effects of overexpressing individual lignin biosynthetic enzymes on feeding and growth of corn earworms and fall armyworms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lignin is an important insect resistance component of plants. Enhancing or disrupting the lignin biosynthetic pathway for different bioenergy uses may alter pest resistance. The lignin biosynthetic pathway is complex, and a number of pathway compounds are also involved in the biosynthesis of simpler...

  7. Carotenoid-induced non-photochemical quenching in the cyanobacterial chlorophyll synthase-HliC/D complex.

    PubMed

    Niedzwiedzki, Dariusz M; Tronina, Tomasz; Liu, Haijun; Staleva, Hristina; Komenda, Josef; Sobotka, Roman; Blankenship, Robert E; Polívka, Tomáš

    2016-09-01

    Chl synthase (ChlG) is an important enzyme of the Chl biosynthetic pathway catalyzing attachment of phytol/geranylgeraniol tail to the chlorophyllide molecule. Here we have investigated the Flag-tagged ChlG (f.ChlG) in a complex with two different high-light inducible proteins (Hlips) HliD and HliC. The f.ChlG-Hlips complex binds a Chl a and three different carotenoids, β-carotene, zeaxanthin and myxoxanthophyll. Application of ultrafast time-resolved absorption spectroscopy performed at room and cryogenic temperatures revealed excited-state dynamics of complex-bound pigments. After excitation of Chl a in the complex, excited Chl a is efficiently quenched by a nearby carotenoid molecule via energy transfer from the Chl a Qy state to the carotenoid S1 state. The kinetic analysis of the spectroscopic data revealed that quenching occurs with a time constant of ~2ps and its efficiency is temperature independent. Even though due to its long conjugation myxoxanthophyll appears to be energetically best suited for role of Chl a quencher, based on comparative analysis and spectroscopic data we propose that β-carotene bound to Hlips acts as the quencher rather than myxoxanthophyll and zeaxanthin, which are bound at the f.ChlG and Hlips interface. The S1 state lifetime of the quencher has been determined to be 13ps at room temperature and 21ps at 77K. These results demonstrate that Hlips act as a conserved functional module that prevents photodamage of protein complexes during photosystem assembly or Chl biosynthesis. PMID:27133505

  8. HPLC-MS/MS Analyses Show That the Near-Starchless aps1 and pgm Leaves Accumulate Wild Type Levels of ADPglucose: Further Evidence for the Occurrence of Important ADPglucose Biosynthetic Pathway(s) Alternative to the pPGI-pPGM-AGP Pathway

    PubMed Central

    Muñoz, Francisco José; Li, Jun; Almagro, Goizeder; Montero, Manuel; Pujol, Pablo; Galarza, Regina; Kaneko, Kentaro; Oikawa, Kazusato; Wada, Kaede; Mitsui, Toshiaki; Pozueta-Romero, Javier

    2014-01-01

    In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low. PMID:25133777

  9. Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

    PubMed Central

    2012-01-01

    Background Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. Results Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ɛ-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained. Conclusion C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions

  10. Arabidopsis cpSRP54 regulates carotenoid accumulation in Arabidopsis and Brassica napus

    PubMed Central

    Gruber, Margaret Y.; Hannoufa, Abdelali

    2012-01-01

    An Arabidopsis thaliana mutant, cbd (carotenoid biosynthesis deficient), was recovered from a mutant population based on its yellow cotyledons, yellow-first true leaves, and stunted growth. Seven-day-old seedlings and mature seeds of this mutant had lower chlorophyll and total carotenoids than the wild type (WT). Genetic and molecular characterization revealed that cbd was a recessive mutant caused by a T-DNA insertion in the gene cpSRP54 encoding the 54kDa subunit of the chloroplast signal recognition particle. Transcript levels of most of the main carotenoid biosynthetic genes in cbd were unchanged relative to WT, but expression increased in carotenoid and abscisic acid catabolic genes. The chloroplasts of cbd also had developmental defects that contributed to decreased carotenoid and chlorophyll contents. Transcription of AtGLK1 (Golden 2-like 1), AtGLK2, and GUN4 appeared to be disrupted in the cbd mutant suggesting that the plastid-to-nucleus retrograde signal may be affected, regulating the changes in chloroplast functional and developmental states and carotenoid content flux. Transformation of A. thaliana and Brassica napus with a gDNA encoding the Arabidopsis cpSRP54 showed the utility of this gene in enhancing levels of seed carotenoids without affecting growth or seed yield. PMID:22791829

  11. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus. PMID:22428138

  12. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.

  13. Changes in carotenoid profiles and in the expression pattern of the genes in carotenoid metabolisms during fruit development and ripening in four watermelon cultivars.

    PubMed

    Lv, Pin; Li, Na; Liu, Hui; Gu, Huihui; Zhao, Wen-En

    2015-05-01

    Changes in carotenoid profiles during fruit ripening were investigated in four watermelon cultivars: red-fleshed "CN66", pink-fleshed "CN62", yellow-fleshed "ZXG381" and white-fleshed "ZXG507". The expression pattern of twelve genes (GGPS, PSY, PSY-A, PDS, ZDS, CRTISO, LCYB, CHYB, ZEP, NCED1, NCED2 and NCED3) was analysed. In "CN66" and "CN62", lycopene appeared at 12 DAP and became a main carotenoid increased at the later stages. The transcript levels of carotenogenic genes in "CN66" sharply increased during 18-30 DAP, and concomitantly, fruit accumulated the massive amounts of carotenoids. In "ZXG381", violaxanthin and lutein contents were positively correlated, respectively, with CHYB and ZEP transcript levels during fruit ripening. The trace amounts of carotenoids in "ZXG507" were accompanied with the low transcript levels of most biosynthetic genes. The results suggest that differential transcriptional regulation of carotenoid metabolic genes is very important in determining the amount and type of specific carotenoids accumulated during fruit development and ripening.

  14. Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and b-cryptoxanthin by ferret carotene-9, 10-monooxygenase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xanthophyll carotenoids, such as lutein, zeaxanthin and b-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,150-monooxygenase (CMO1) h...

  15. Tryptophan biosynthetic enzymes of Staphylococcus aureus.

    PubMed

    Proctor, A R; Kloos, W E

    1973-04-01

    Tryptophan biosynthetic enzymes were assayed in various tryptophan mutants of Staphylococcus aureus strain 655 and the wild-type parent. All mutants, except trpB mutants, lacked only the activity corresponding to the particular biosynthetic block, as suggested previously by analysis of accumulated intermediates and auxonography. Tryptophan synthetase A was not detected in extracts of either trpA or trpB mutants but appeared normal in other mutants. Mutants in certain other classes exhibited partial loss of another particular tryptophan enzyme activity. Tryptophan synthetase B activity was not detected in cell extract preparations but was detected in whole cells. The original map order proposed for the S. aureus tryptophan gene cluster was clarified by the definition of trpD (phosphoribosyl transferase(-)) and trpF (phosphoribosyl anthranilate isomerase(-)) mutants. These mutants were previously unresolved and designated as trp(DF) mutants (anthranilate accumulators). Phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase enzymes were separable by molecular sieve chromatography, suggesting that these functions are coded by separate loci. Molecular sieve chromatography failed to reveal aggregates involving anthranilate synthetase, phosphoribosyl transferase, phosphoribosyl anthranilate isomerase, and indole-3-glycerol phosphate synthetase, and this procedure provided an estimate of the molecular weights of these enzymes. Tryptophan was shown to repress synthesis of all six tryptophan biosynthetic enzymes, and derepression of all six activities was incident upon tryptophan starvation. Tryptophan inhibited the activity of anthranilate synthetase, the first enzyme of the pathway. PMID:4698207

  16. Biosynthetic engineering of nonribosomal peptide synthetases.

    PubMed

    Kries, Hajo

    2016-09-01

    From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27465074

  17. Biotechnological production of value-added carotenoids from microalgae: Emerging technology and prospects.

    PubMed

    Wichuk, Kristine; Brynjólfsson, Sigurður; Fu, Weiqi

    2014-01-01

    We recently evaluated the relationship between abiotic environmental stresses and lutein biosynthesis in the green microalga Dunaliella salina and suggested a rational design of stress-driven adaptive evolution experiments for carotenoids production in microalgae. Here, we summarize our recent findings regarding the biotechnological production of carotenoids from microalgae and outline emerging technology in this field. Carotenoid metabolic pathways are characterized in several representative algal species as they pave the way for biotechnology development. The adaptive evolution strategy is highlighted in connection with enhanced growth rate and carotenoid metabolism. In addition, available genetic modification tools are described, with emphasis on model species. A brief discussion on the role of lights as limiting factors in carotenoid production in microalgae is also included. Overall, our analysis suggests that light-driven metabolism and the photosynthetic efficiency of microalgae in photobioreactors are the main bottlenecks in enhancing biotechnological potential of carotenoid production from microalgae.

  18. Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves1

    PubMed Central

    Lätari, Kira; Wüst, Florian; Hübner, Michaela; Schaub, Patrick; Beisel, Kim Gabriele; Matsubara, Shizue; Beyer, Peter; Welsch, Ralf

    2015-01-01

    Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly β-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C13 apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of β-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation. PMID:26134165

  19. Evolution of carotenoid pigmentation in caciques and meadowlarks (Icteridae): repeated gains of red plumage coloration by carotenoid C4-oxygenation.

    PubMed

    Friedman, Nicholas R; McGraw, Kevin J; Omland, Kevin E

    2014-03-01

    Many animals use carotenoid pigments to produce yellow, orange, and red coloration. In birds, at least 10 carotenoid compounds have been documented in red feathers; most of these are produced through metabolic modification of dietary precursor compounds. However, it is poorly understood how lineages have evolved the biochemical mechanisms for producing red coloration. We used high-performance liquid chromatography to identify the carotenoid compounds present in feathers from 15 species across two clades of blackbirds (the meadowlarks and allies, and the caciques and oropendolas; Icteridae), and mapped their presence or absence on a phylogeny. We found that the red plumage found in meadowlarks includes different carotenoid compounds than the red plumage found in caciques, indicating that these gains of red color are convergent. In contrast, we found that red coloration in two closely related lineages of caciques evolved twice by what appear to be similar biochemical mechanisms. The C4-oxygenation of dietary carotenoids was responsible for each observed transition from yellow to red plumage coloration, and has been commonly reported by other researchers. This suggests that the C4-oxygenation pathway may be a readily evolvable means to gain red coloration using carotenoids. PMID:24164419

  20. Carotenoids and lung cancer prevention

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the molecular actions of carotenoids is critical for human studies involving carotenoids for prevention of lung cancer and cancers at other tissue sites. While the original hypothesis prompting the beta-carotene intervention trials was that beta-carotene exerts beneficial effects thro...