Title: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440

Abstract

Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36 h was increased nearly 70%more » with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72 h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24 h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72 h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). In conclusion, these results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals.« less

Authors:

Johnson, Christopher W. ^[1]; Abraham, Paul E.  ^[2];

• Search DOE PAGES for author "Abraham, Paul E."
• Search DOE PAGES for ORCID "0000-0003-2685-9123"

Linger, Jeffrey G. ^[1]; Khanna, Payal ^[1]; Hettich, Robert L. ^[2]; Beckham, Gregg T. ^[1]

---

+ Show Author Affiliations

1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

Publication Date:

Wed May 31 00:00:00 EDT 2017

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office

OSTI Identifier:

1376605

Alternate Identifier(s):

OSTI ID: 1372629

Report Number(s):

NREL/JA-5100-68933
Journal ID: ISSN 2214-0301

Grant/Contract Number:  

AC05-00OR22725; AC36-08GO28308

Resource Type:

Accepted Manuscript

Journal Name:

Metabolic Engineering Communications

Additional Journal Information:

Journal Volume: 5; Journal Issue: C; Journal ID: ISSN 2214-0301

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

60 APPLIED LIFE SCIENCES; Carbon catabolite repression; Catabolite repression control; Crc; Pseudomonas putida KT2440; ciscis-Muconate; Muconic acid; Lignin valorization; 09 BIOMASS FUELS; carbon catabolite repression; catabolite repression control; ciscis-muconate; muconic acid; lignin valorization