Pathway-specific tuning mechanisms in the metabolism of lignin and plastic aromatic carbons in Comamonas testosteroni
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ABSTRACT:
ORGANISM(S): Comamonas testosteroni
PROVIDER: GSE192852 | GEO | 2022/11/17
REPOSITORIES: GEO
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Project description:Widely reported discrepancies between metabolic flux and transcripts or enzyme levels in bacterial metabolism imply complex regulation mechanisms need to be considered, especially in new bacterial platforms for bioremediation and bioproduction. Comamonas testosteroni strains, which metabolize various natural and xenobiotic aromatic compounds, represent such platforms whose metabolic regulations are still unknown. Here, we identify analogous multi-level regulation mechanisms in the metabolism of two lignin-related (4-hydroxybenzoate and vanillate) and one plastic-related (terephthalate) aromatic compounds in C. testosteroni KF-1, a wastewater isolate. Transcription-level regulation controlled initial catabolism and cleavage of the compounds, but subsequent carbon fluxes in central carbon metabolism are governed by metabolite-level thermodynamic regulation. Quantitative 13C-fingerprinting of tricarboxylic acid cycle and cataplerotic reactions elucidate key carbon routing that is not evident from enzyme abundance changes. Therefore, as-needed transcriptional activation of aromatic catabolism is coupled with metabolic fine-tuning of central metabolism, thus delineating pathway candidates for different metabolic manipulations.
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Project description:The degradation of aromatic compounds comprises an important step in the removal of pollutants and re-utilization of plastics and other non-biological polymers. Here we set out to study Pseudomonas sp. strain phDV1, a gram-negative bacterium that was selected for its ability to degrade aromatic compounds. In order to understand how the aromatic compounds and their degradation products are reintroduced in the metabolism of the bacteria and the systematic/metabolic response of the bacterium to the new carbon source, the proteome of this strain was analysed in the presence of succinate, phenol and o-, m-, p-cresol as sole carbon source. We then applied label-free quantitative proteomics to monitor overall proteome remodeling during metabolic adaptation to different carbon sources. As a reference proteome, we grew the bacteria in succinate and then compared the respective proteomes of bacteria grown on phenol and different cresols. In total, we identified 2295 proteins; 1908 proteins were used for quantification between different growth conditions. We found that 70, 100, 150 and 155 proteins were significantly differentially expressed in cells were grown in phenol, o-, m- and p-cresol-containing medium, respectively. The carbon source affected the synthesis of enzymes related to aromatic compound degradation, and in particular, the enzyme involved in the meta-pathway of monocyclic aromatic compounds degradation. In addition, proteins involved in the production of polyhydroxyalkanoate (PHA), an attractive biomaterial, showed higher expression levels in the presence of monocyclic aromatic compounds.Our results provide for the first time comprehensive information on the proteome response of this strain to monocyclic aromatic compounds.
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Project description:Lignin is an abundant aromatic polymer formed in plants by coupling reactions of p-coumaryl, coniferyl, and sinapyl alcohols. Pseudomonas putida KT2440 (P. putida KT2440), a robust soil bacterium, naturally catabolizes many aromatic compounds derived from lignin as carbon and energy sources and has been extensively engineered to valorize guaiacyl (G) and p-hydroxyphenyl (H) lignin-derived aromatic species to valuable chemicals. However, only a few studies report the capacity of Pseudomonads to grow on syringyl (S) lignin-derived compounds. In this work, we demonstrate that P. putida KT2440 catabolizes syringate in the presence of an auxiliary energy source. Overexpression of vanAB, encoding a native Rieske non-heme iron monooxygenase, enabled catabolism of syringate and syringaldehyde without an additional carbon and energy source. In vitro biochemical characterization of VanAB confirmed that this enzyme O-demethylates syringate to 3-O-methylgallate and, subsequently, 3-O-methylgallate to gallate, albeit 4- and 55-fold less efficiently than vanillate, respectively. Transcriptomics and proteomics experiments demonstrate that the galADBC transcripts and enzymes required for gallate catabolism are induced when vanAB is overexpressed during cultivation in syringate. Further, we show that that the endogenous protocatechuate-3,4-dioxygenase PcaHG ring-opens gallate to produce 2-pyrone-4,6-dicarboxylic acid (PDC), a promising bio-based chemical. Constitutive overexpression of vanAB and pcaHG along with galA deletion enabled PDC production from SA at a 70% (mol/mol) yield. Furthermore, leveraging the LigAB dioxygenase from Sphingobium sp. SYK-6 and VanAB O-demethylase from Pseudomonas sp. HR199 enabled simultaneous conversion of monomers with S-, G-, and H-type functionality to PDC at 82% (mol/mol) yield. Overall, this study elucidates a native pathway for catabolism of syringyl lignin-derived compounds in P. putida KT2440 and demonstrates the potential of this robust host for biological funneling of substrates derived from all three canonical monolignols.
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Project description:Aromatic compounds are an important renewable source of commodity chemicals traditionally produced from fossil fuels. Aromatics derived from plant lignin can potentially be converted into commodity chemicals through depolymerization followed by microbial funneling of monomers and low molecular weight oligomers. This study investigates the catabolism of the b-5 linked aromatic dimer dehydrodiconiferyl alcohol (DC-A) by the bacterium Novosphingobium aromaticivorans. We used genome wide screens to identify candidate genes involved in DC-A catabolism. Subsequent in vivo and in vitro analyses of these candidates elucidated a catabolic pathway composed of four required gene products and several partially redundant dehydrogenases that convert DC-A to aromatic monomers that can be funneled into the central aromatic metabolic pathway of N. aromaticivorans. Specifically, a newly identified γ-formaldehyde lyase, PcfL, opens the phenylcoumaran ring to form a stilbene and formaldehyde. A lignostilbene dioxygenase, LsdD, then cleaves the stilbene to generate the aromatic monomers, vanillin and 5-formylferulate (5-FF). We also show that an aldehyde dehydrogenase FerD oxidizes 5-FF before it is decarboxylated by LigW, yielding ferulic acid. We found that some enzymes involved in b-5 catabolism pathway can act on multiple substrates and that some steps in the pathway can be mediated by multiple enzymes, providing new insights into the robust flexibility of aromatic catabolism in N. aromaticivorans. We performed a comparative genomic analysis to predict that key enzymes in the newly discovered b-5 aromatic catabolic pathway are common among Sphingomonads.
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Project description:To determine the wood degradation mechanism and its key genes of Lenzites gibbosa, we sequenced 15 transcriptomes of mycelial samples under woody environments at 3, 5, 7, and 11 d (D3, D5, D7, and D11) and non-woody environments (CK). All the transcripts were annotated as much as possible in eight databases to determine their function. The key genes and biological processes, relating to wood degradation, were predicted and screened. A total of 2069 differentially expressed genes (DEGs) were obtained in ten differential groups. Comparing wood with non-wood treatment conditions, the key genes were those participating in oxidation-reduction process, they were oxidoreductases and peroxidases genes, and their regulators genes; these genes mainly focused on the three biological processes of carbohydrate metabolism, lignin catabolism, and secondary metabolites biosynthesis, transport and catabolism. The mostly enriched subcategories in molecular function were oxidoreductase activity, peroxidase activity, and heme binding in GO annotation. One cellulose and hemicellulose degradation pathway and seven pathways related to lignin-derived aromatic compounds degradation or late lignin degradation were found. In conclusion, during the process of L. gibbosa growing on wood, gene expression at the transcriptional level indicated that lignin catabolism and hyphal growth were promoted, but the metabolism of carbon and carbohydrates including cellulose in lignocellulose in overall trend was inhibited to some extent. The results have important reference value for the study of degradation mechanism of wood white rot.
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