Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p-coumarate

Eugene Kuatsjah, Christopher W. Johnson, Davinia Salvachúa, Allison Z. Werner, Michael Zahn, Caralyn J. Szostkiewicz, Christine A. Singer, Graham Dominick, Ikenna Okekeogbu, Stefan J. Haugen, Sean P. Woodworth, Kelsey J. Ramirez, Richard J. Giannone, Robert L. Hettich, John E. McGeehan, Gregg T. Beckham

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.

Original languageEnglish
Pages (from-to)31-42
Number of pages12
JournalMetabolic Engineering
Volume70
DOIs
StatePublished - Mar 2022

Funding

This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. EK, AZW, RJG, RLH, JEM, and GTB acknowledge funding from The Center for Bioenergy Innovation , a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science . Funding was provided to CWJ, DS, CJS, CAS, SJH, SPW, KJR, and GTB by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office . MZ and JEM were supported by Research England through the Expanding Excellence in England (E3) scheme. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. We thank the Diamond Light Source for beamtime (proposal MX-23269) and the staff of beamline I03 for supporting automated data collection. Erika Erickson is thanked for the construction of pEE003. Veda S. Boorla and Costas D. Maranas are thanked for helpful discussions. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. EK, AZW, RJG, RLH, JEM, and GTB acknowledge funding from The Center for Bioenergy Innovation, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Funding was provided to CWJ, DS, CJS, CAS, SJH, SPW, KJR, and GTB by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. MZ and JEM were supported by Research England through the Expanding Excellence in England (E3) scheme. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. We thank the Diamond Light Source for beamtime (proposal MX-23269) and the staff of beamline I03 for supporting automated data collection. Erika Erickson is thanked for the construction of pEE003. Veda S. Boorla and Costas D. Maranas are thanked for helpful discussions.

Keywords

  • Aromatic catabolism
  • Flavoprotein
  • Metabolic engineering
  • Muconic acid
  • Paenibacillus sp. JJ-1b
  • Para-hydroxybenzoate hydroxylase
  • PobA
  • PraI
  • Pseudomonas putida KT2440

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