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Semantic Search for: "biosynthesis"

  1. Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Though the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. Here, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesismore » of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, our findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.« less
  2. Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less
  3. WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors, positively regulates glycolysis and lipid biosynthesis in Arabidopsis thaliana. Here we identify mechanistic links between KIN10, the major SUCROSE NON-FERMENTATION-1 (SNF1)-RELATED KINASE 1 (SnRK1) involved in sugar/energy homeostasis and the posttranslational regulation of WRI1. Transient expression of WRI1 with OLEOSIN1 (OLE1) in Nicotiana benthamiana stimulates triacylglycerol (TAG) accumulation, but their coexpression with KIN10 abrogates this effect by inducing proteasomal degradation of WRI1. While WRI1 lacks canonical KIN10 target sequences, we demonstrated direct KIN10-dependent phosphorylation of WRI1 using purified E. coli-expressed components. The resulting phosphorylated WRI1 was more rapidlymore » degraded than native WRI1 in cell-free degradation assays. WRI1 phosphorylation was localized to two variants of the canonical KIN10 recognition sequence, one in each of its two AP2 DNA-binding domains. Conversion of the phosphorylation sites at T70 and S166 to Ala resulted in a loss of KIN10-dependent phosphorylation, and when coexpressed with KIN10 the WRI1 double mutant accumulated to 2-3 fold higher levels than native WRI1. In conclusion, KIN10-dependent degradation of WRI1 provides a homeostatic mechanism that favors lipid biosynthesis when intracellular sugar levels are elevated and KIN10 is inhibited; conversely, glycolysis and lipid biosynthesis are curtailed as sugar levels decrease and KIN10 regains activity.« less
  4. Lignin in plant biomass represents a target for engineering strategies towards the development of a sustainable bioeconomy. In addition to the conventional lignin monomers, namely p-coumaryl, coniferyl and sinapyl alcohols, tricin has been shown to be part of the native lignin polymer in certain monocot species. Because tricin is considered to initiate the polymerization of lignin chains, elucidating its biosynthesis and mechanism of export to the cell wall constitute novel challenges for the engineering of bioenergy crops. Late steps of tricin biosynthesis require two methylation reactions involving the pathway intermediate selgin. It has recently been demonstrated in rice and maizemore » that caffeate O-methyltransferase (COMT) involved in the synthesis syringyl (S) lignin units derived from sinapyl alcohol also participates in the synthesis of tricin in planta. In this work, we validate in sorghum (Sorghum bicolor L.) that the O-methyltransferase responsible for the production of S lignin units (SbCOMT / Bmr12) is also involved in the synthesis of lignin-linked tricin. In particular, we show that biomass from the sorghum bmr12 mutant contains lower level of tricin incorporated into lignin, and that SbCOMT can methylate the tricin precursors luteolin and selgin. Our genetic and biochemical data point toward a general mechanism whereby COMT is involved in the synthesis of both tricin and S lignin units.« less
  5. Here, automatically generated bacterial metabolic models, and even some curated models, lack accuracy in predicting energy yields due to poor representation of key pathways in energy biosynthesis and the electron transport chain (ETC). Further compounding the problem, complex interlinking pathways in genome-scale metabolic models, and the need for extensive gapfilling to support complex biomass reactions, often results in predicting unrealistic yields or unrealistic physiological flux profiles. As a result, to overcome this challenge, we developed methods and tools to build high quality core metabolic models (CMM) representing accurate energy biosynthesis based on a well studied, phylogenetically diverse set of modelmore » organisms. We compare these models to explore the variability of core pathways across all microbial life, and by analyzing the ability of our core models to synthesize ATP and essential biomass precursors, we evaluate the extent to which the core metabolic pathways and functional ETCs are known for all microbes. 6,600 (80 %) of our models were found to have some type of aerobic ETC, whereas 5,100 (62 %) have an anaerobic ETC, and 1,279 (15 %) do not have any ETC. Using our manually curated ETC and energy biosynthesis pathways with no gapfilling at all, we predict accurate ATP yields for nearly 5586 (70 %) of the models under aerobic and anaerobic growth conditions. This study revealed gaps in our knowledge of the central pathways that result in 2,495 (30 %) CMMs being unable to produce ATP under any of the tested conditions. We then established a methodology for the systematic identification and correction of inconsistent annotations using core metabolic models coupled with phylogenetic analysis. In conclusion, we predict accurate energy yields based on our improved annotations in energy biosynthesis pathways and the implementation of diverse ETC reactions across the microbial tree of life. We highlighted missing annotations that were essential to energy biosynthesis in our models. We examine the diversity of these pathways across all microbial life and enable the scientific community to explore the analyses generated from this large-scale analysis of over 8000 microbial genomes.« less
  6. Flavonoids represent a large family of specialized metabolites involved in plant growth, development, and adaptation. Chalcone synthase (CHS) catalyzes the first step of flavonoid biosynthesis by directing carbon flux from general phenylpropanoid metabolism to flavonoid pathway. Despite extensive characterization of its function and transcriptional regulation, the molecular basis governing its posttranslational modification is enigmatic. Here, we report the discovery of a proteolytic regulator of CHS, namely, KFB CHS, a Kelch domain-containing F-box protein in Arabidopsis thaliana. KFB CHS physically interacts with CHS and specifically mediates its ubiquitination and degradation. KFB CHS exhibits developmental expression patterns in Arabidopsis leaves, stems, andmore » siliques and strongly responds to the dark-to-light (or the light-to-dark) switch, the blue, red, and far-red light signals, and UV-B irradiation. Alteration of KFB CHS expression negatively correlates to the cellular concentration of CHS and the production of flavonoids. Our study suggests that KFB CHS serves as a crucial negative regulator, via mediating CHS degradation, coordinately controlling flavonoid biosynthesis in response to the developmental cues and environmental stimuli.« less
  7. Suberin, a lipophilic polymer deposited in the outer integument of the Arabidopsis ( Arabidopsis thaliana) seed coat, represents an essential sealing component controlling water and solute movement and protecting seed from pathogenic infection. Although many genes responsible for suberin synthesis are identified, the regulatory components controlling its biosynthesis have not been definitively determined. Here, we show that the Arabidopsis MYB107 transcription factor acts as a positive regulator controlling suberin biosynthetic gene expression in the seed coat. MYB107 coexpresses with suberin biosynthetic genes in a temporal manner during seed development. Disrupting MYB107 particularly suppresses the expression of genes involved in suberinmore » but not cutin biosynthesis, lowers seed coat suberin accumulation, alters suberin lamellar structure, and consequently renders higher seed coat permeability and susceptibility to abiotic stresses. Furthermore, MYB107 directly binds to the promoters of suberin biosynthetic genes, verifying its primary role in regulating their expression. Identifying MYB107 as a positive regulator for seed coat suberin synthesis offers a basis for discovering the potential transcriptional network behind one of the most abundant lipid-based polymers in nature.« less
  8. S-Adenosyl-l-methionine (SAM) dependent xanthosine methyltransferase (XMT) is the key enzyme that catalyzes the first methyl transfer in the caffeine biosynthesis pathway to produce the intermediate 7-methylxanthosine (7mXR). Although XMT has been a subject of extensive discussions, the catalytic mechanism and nature of the substrate involved in the catalysis are still unclear. Here in this paper, quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy (potential of mean force or PMF) simulations are undertaken to determine the catalytic mechanism of the XMT-catalyzed reaction. Both xanthosine and its monoanionic form with N3 deprotonated are used as the substrates for the methylation.more » It is found that while the methyl group can be transferred to the monoanionic form of xanthosine with a reasonable free energy barrier (about 17 kcal/mol), that is not the case for the neutral xanthosine. The results suggest that the substrate for the first methylation step in the caffeine biosynthesis pathway is likely to be the monoanionic form of xanthosine rather than the neutral form as widely adopted. This conclusion is supported by the p K a value on N3 of xanthosine both measured in aqueous phase and calculated in the enzymatic environment. As a result, the structural and dynamics information from both the X-ray structure and MD simulations is also consistent with the monoanionic xanthosine scenario. Finally, we discuss the implications of this conclusion for caffeine biosynthesis.« less
  9. We report pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wildmore » type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.« less
  10. In Arabidopsis, anthocyanin biosynthesis is controlled by a MYB-bHLH-WD40 (MBW) transcriptional activator complex. The MBW complex activates the transcription of late biosynthesis genes in the flavonoid pathway, leading to the production of anthocyanins. A similar MBW complex regulates epidermal cell fate by activating the transcription of GLABRA2 (GL2), a homeodomain transcription factor required for trichome formation in shoots and non-hair cell formation in roots. Here we provide experimental evidence to show that GL2 also plays a role in regulating anthocyanin biosynthesis in Arabidopsis. From an activation-tagged mutagenized population of Arabidopsis plants, we isolated a dominant, gain-of-function mutant with reduced anthocyanins.more » Molecular cloning revealed that this phenotype is caused by an elevated expression of GL2, thus the mutant was named gl2-1D. Consistent with the view that GL2 acts as a negative regulator of anthocyanin biosynthesis, gl2-1D seedlings accumulated less whereas gl2-3 seedlings accumulated more anthocyanins in response to sucrose. Gene expression analysis indicated that expression of late, but not early, biosynthesis genes in the flavonoid pathway was dramatically reduced in gl2-1D but elevated in gl2-3 mutants. Further analysis showed that expression of some MBW component genes involved in the regulation of late biosynthesis genes was reduced in gl2-1D but elevated in gl2-3 mutants, and chromatin immunoprecipitation results indicated that some MBW component genes are targets of GL2. We also showed that GL2 functions as a transcriptional repressor. Altogether, these results indicate that GL2 negatively regulates anthocyanin biosynthesis in Arabidopsis by directly repressing the expression of some MBW component genes.« less
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