Title: Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

Abstract

ABSTRACT Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds ( p- coumarate and ferulate) mixture, 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 revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p -coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+ p -coumarate. Themore » increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+ p -coumarate) condition compared to when repression was absent ( p -coumarate or glucose only). IMPORTANCE A newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.« less

Authors:

Shrestha, Shilva  ^[1];

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• Search DOE PAGES for ORCID "0000-0001-5062-3634"

Awasthi, Deepika ^[1]; Chen, Yan ^[1]; Gin, Jennifer ^[1]; Petzold, Christopher J. ^[1]; Adams, Paul D. ^[2]; Simmons, Blake A. ^[1]; Singer, Steven W.  ^[1];

• Search DOE PAGES for author "Singer, Steven W."
• Search DOE PAGES for ORCID "0000-0002-4229-8314"

Nikel, ed., Pablo Ivan

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+ Show Author Affiliations

1. Joint BioEnergy Institute, Emeryville, California, USA, Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
2. Joint BioEnergy Institute, Emeryville, California, USA, Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

Publication Date:

Tue Oct 31 00:00:00 EDT 2023

Sponsoring Org.:

USDOE

OSTI Identifier:

2001020

Resource Type:

Published Article

Journal Name:

Applied and Environmental Microbiology

Additional Journal Information:

Journal Name: Applied and Environmental Microbiology Journal Volume: 89 Journal Issue: 10; Journal ID: ISSN 0099-2240

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

English