Title: Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

Abstract

Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic-catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole-cell biocatalyst for funnelling lignin-derived compounds to value-added products, including its native carbon storage product, medium-chain-length polyhydroxyalkanoates (mcl-PHA). In this work, a series of metabolic engineering targets to improve mcl-PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p-coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in ..beta..-oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl-PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain - which contains all the genetic modifications detailed above - demonstrated a 53% and 200% increase in mcl-PHA titre (g l-1) and a 20% and 100% increase in yield (g mcl-PHA per g cell dry weight) from p-coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl-PHA production from aromatic compoundsmore » and lignin.« less

Authors:

Salvachúa, Davinia ^[1]; Rydzak, Thomas  ^[2];

• Search DOE PAGES for author "Rydzak, Thomas"
• Search DOE PAGES for ORCID "0000-0002-5176-3222"

Auwae, Raquel ^[2]; De Capite, Annette ^[2]; Black, Brenna A. ^[1]; Bouvier, Jason T.  ^[2];

• Search DOE PAGES for author "Bouvier, Jason T."
• Search DOE PAGES for ORCID "0000-0002-2047-1067"

Cleveland, Nicholas S. ^[1]; Elmore, Joshua R.  ^[2];

• Search DOE PAGES for author "Elmore, Joshua R."
• Search DOE PAGES for ORCID "0000-0003-4750-9640"

Huenemann, Jay D. ^[2]; Katahira, Rui ^[1]; Michener, William E. ^[1]; Peterson, Darren J. ^[1]; Rohrer, Holly ^[1]; Vardon, Derek R. ^[1]; Beckham, Gregg T. ^[1]; Guss, Adam M.  ^[2]

• Search DOE PAGES for author "Guss, Adam M."
• Search DOE PAGES for ORCID "0000-0001-5823-5329"

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+ Show Author Affiliations

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

Publication Date:

Fri Aug 30 00:00:00 EDT 2019

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 Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office

OSTI Identifier:

1559676

Alternate Identifier(s):

OSTI ID: 1562862

Report Number(s):

NREL/JA-5100-74637
Journal ID: ISSN 1751-7915

Grant/Contract Number:  

AC05-00OR22725; AC36‐08GO28308

Resource Type:

Accepted Manuscript

Journal Name:

Microbial Biotechnology (Online)

Additional Journal Information:

Journal Name: Microbial Biotechnology (Online); Journal Volume: 13; Journal Issue: 1; Journal ID: ISSN 1751-7915

Publisher:

Wiley

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; metabolic engineering; Pseudomonas putida; microbial conversion; lignin; PHA; hydrocarbon fuels; 09 BIOMASS FUELS