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

DOI: 10.1016/j.ymben.2021.12.010

Authors: Eugene Kuatsjah (tional Renewable Energy Laboratory; Oak Ridge National Laboratory) , Christopher W. Johnson (National Renewable Energy Laboratory) , Davinia Salvachúa (National Renewable Energy Laboratory) , Allison Z. Werner (National Renewable Energy Laboratory; Oak Ridge National Laboratory) , Michael Zahn (University of Portsmouth) , Caralyn J. Szostkiewicz (National Renewable Energy Laboratory) , Christine A. Singer (National Renewable Energy Laboratory) , Graham Dominick (National Renewable Energy Laboratory) , Ikenna Okekeogbu (Oak Ridge National Laboratory) , Stefan J. Haugen (National Renewable Energy Laboratory) , Sean P. Woodworth (National Renewable Energy Laboratory) , Kelsey J. Ramirez (National Renewable Energy Laboratory) , Richard J. Giannone (Oak Ridge National Laboratory) , Robert L. Hettich (Oak Ridge National Laboratory) , John E. Mcgeehan (University of Portsmouth) , Gregg T. Beckham (National Renewable Energy Laboratory; Oak Ridge National Laboratory)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Metabolic Engineering , VOL 34

State: Published (Approved)
Published: January 2022
Diamond Proposal Number(s): 23269

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.

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

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography

Added On: 04/01/2022 11:42

Discipline Tags:

Biotechnology Biochemistry Catalysis Chemistry Structural biology Engineering & Technology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)