Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

Davinia Salvachúa; Thomas Rydzak; Raquel Auwae; Annette De Capite; Brenna A. Black; Jason T. Bouvier; Nicholas S. Cleveland; Joshua R. Elmore; Jay D. Huenemann; Rui Katahira; William E. Michener; Darren J. Peterson; Holly Rohrer; Derek R. Vardon; Gregg T. Beckham; Adam M. Guss

doi:10.1111/1751-7915.13481

Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

Davinia Salvachúa, Thomas Rydzak, Raquel Auwae, Annette De Capite, Brenna A. Black, Jason T. Bouvier, Nicholas S. Cleveland, Joshua R. Elmore, Jay D. Huenemann, Rui Katahira, William E. Michener, Darren J. Peterson, Holly Rohrer, Derek R. Vardon, Gregg T. Beckham, Adam M. Guss

Synthetic Biology

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131 Scopus citations

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 β-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 compounds and lignin.

Original language	English
Pages (from-to)	290-298
Number of pages	9
Journal	Microbial Biotechnology
Volume	13
Issue number	1
DOIs	https://doi.org/10.1111/1751-7915.13481
State	Published - Jan 1 2020

Funding

Funding information Funding was provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office. This work was authored in part by Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. This work was also authored in part by Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. Funding was provided by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funders	Funder number
U.S. Department of Energy	DE-AC05-00OR22725, DE-AC36-08GO28308
Office of Energy Efficiency and Renewable Energy
Oak Ridge National Laboratory
National Renewable Energy Laboratory

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Salvachúa, D., Rydzak, T., Auwae, R., De Capite, A., Black, B. A., Bouvier, J. T., Cleveland, N. S., Elmore, J. R., Huenemann, J. D., Katahira, R., Michener, W. E., Peterson, D. J., Rohrer, H., Vardon, D. R., Beckham, G. T., & Guss, A. M. (2020). Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin. Microbial Biotechnology, 13(1), 290-298. https://doi.org/10.1111/1751-7915.13481

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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 β-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 compounds and lignin.",

author = "Davinia Salvach{\'u}a and Thomas Rydzak and Raquel Auwae and {De Capite}, Annette and Black, {Brenna A.} and Bouvier, {Jason T.} and Cleveland, {Nicholas S.} and Elmore, {Joshua R.} and Huenemann, {Jay D.} and Rui Katahira and Michener, {William E.} and Peterson, {Darren J.} and Holly Rohrer and Vardon, {Derek R.} and Beckham, {Gregg T.} and Guss, {Adam M.}",

note = "Publisher Copyright: {\textcopyright} 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.",

year = "2020",

month = jan,

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doi = "10.1111/1751-7915.13481",

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Salvachúa, D, Rydzak, T, Auwae, R, De Capite, A, Black, BA, Bouvier, JT, Cleveland, NS, Elmore, JR, Huenemann, JD, Katahira, R, Michener, WE, Peterson, DJ, Rohrer, H, Vardon, DR, Beckham, GT & Guss, AM 2020, 'Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin', Microbial Biotechnology, vol. 13, no. 1, pp. 290-298. https://doi.org/10.1111/1751-7915.13481

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T1 - Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

AU - Salvachúa, Davinia

AU - Rydzak, Thomas

AU - Auwae, Raquel

AU - De Capite, Annette

AU - Black, Brenna A.

AU - Bouvier, Jason T.

AU - Cleveland, Nicholas S.

AU - Elmore, Joshua R.

AU - Huenemann, Jay D.

AU - Katahira, Rui

AU - Michener, William E.

AU - Peterson, Darren J.

AU - Rohrer, Holly

AU - Vardon, Derek R.

AU - Beckham, Gregg T.

AU - Guss, Adam M.

PY - 2020/1/1

Y1 - 2020/1/1

N2 - 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 β-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 compounds and lignin.

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Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

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