Dataset Information

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

ABSTRACT: Lignin modifications and the exoproteome of three aromatic catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. Samples were digested with trypsin, then analyzed by LC-MS/MS. Data was searched with MSGF+ and abundances inferred using PNNL's DMS and MTS Processing pipeline

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Pseudomonas Putida Kt2440 (ncbitaxon:160488) Amycolatopsis Sp. Atcc 39116 (ncbitaxon:385957) Rhodococcus Jostii Rha1 (ncbitaxon:101510)

SUBMITTER: Gregg T. Beckham

PROVIDER: MSV000084524 | MassIVE | Fri Nov 01 10:28:00 GMT 2019

SECONDARY ACCESSION(S): PXD016114

REPOSITORIES: MassIVE

ACCESS DATA

Publications

Outer membrane vesicles catabolize lignin-derived aromatic compounds in <i>Pseudomonas putida</i> KT2440.

Salvachúa Davinia D Werner Allison Z AZ Pardo Isabel I Michalska Martyna M Black Brenna A BA Donohoe Bryon S BS Haugen Stefan J SJ Katahira Rui R Notonier Sandra S Ramirez Kelsey J KJ Amore Antonella A Purvine Samuel O SO Zink Erika M EM Abraham Paul E PE Giannone Richard J RJ Poudel Suresh S Laible Philip D PD Hettich Robert L RL Beckham Gregg T GT

Proceedings of the National Academy of Sciences of the United States of America 20200403 17

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 also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine ligni ...[more]

PMID: 32245809

Similar Datasets

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.

Project description:Strains of Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimizing the conversion of biomass to fuels and chemicals. The functioning of CCR in a P. putida strain, P. putida M2, capable of growing on both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 was able to co-utilize sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis of these co- cultivations revealed that glucose exerted stronger repression compared to xylose on the proteins involved in aromatic catabolism. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc mediated by CRISPRi led to faster growth and uptake of both glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda protein and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that the levels of CrcY and CrcZ homologs in M2, previously identified as sRNAs involved in CCR in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).

Dataset Information

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440

Publications

Outer membrane vesicles catabolize lignin-derived aromatic compounds in <i>Pseudomonas putida</i> KT2440.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure