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Exoprotoemics of pseudomonas putida KT2440 outer membrane vesicles (OMV) and vesicle-free secretome (VSF)

ABSTRACT: Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in Nature is typically attributed to fungi, growing evidence suggests that bacteria can also modify this complex biopolymer. However, the spatiotemporal mechanisms for this modification remain unclear. Improved understanding of this biological and extracellular process would aid in our collective knowledge of both carbon cycling and advanced microbial strategies to valorize lignin. In this data set, we examined the exoproteome of Pseudomonas putida KT2440 grown with on lignin-rich media to characterize the secretion of abundant outer membrane vesicles (OMVs). Proteomic analysis revealed that the exoproteome is 20% more sub-compartmentalized into OMVs and OMV protein cargo are 52% more dynamic in lignin-rich media as compared to lignin-free media.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Pseudomonas Putida Kt2440 (ncbitaxon:160488)

SUBMITTER: Gregg Beckham

PROVIDER: MSV000084506 | MassIVE | Fri Oct 25 12:38:00 BST 2019

SECONDARY ACCESSION(S): PXD016028

REPOSITORIES: MassIVE

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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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Exoprotoemics of pseudomonas putida KT2440 outer membrane vesicles (OMV) and vesicle-free secretome (VSF)

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