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Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12

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Author: Verhoef, S. · Ruijssenaars, H.J. · Bont, J.A.M.de · Wery, J.
Type:article
Date:2007
Institution: TNO Kwaliteit van Leven
Source:Journal of Biotechnology, 1, 132, 49-56
Identifier: 240246
doi: doi:10.1016/j.jbiotec.2007.08.031
Keywords: Biology · Biotechnology · Metabolic engineering · p-Hydroxybenzoate · Pseudomonas putida · Renewable feedstock · Solvent-tolerance · Cell culture · Fermentation · Glucose · Glycerol · Metabolites · Metabolic engineering · p-Hydroxybenzoate · Pseudomonas putida · Renewable feedstock · Solvent-tolerance · Aromatic compounds · 4 hydroxybenzoate 3 monooxygenase · 4 hydroxybenzoic acid · carbon · glucose · glycerol · para coumaric acid · phenylalanine ammonia lyase · solvent · tyrosine · article · bacterial strain · bacterium culture · chemostat · culture technique · enzyme activity · enzyme inactivation · fed batch fermentation · metabolic engineering · metabolite · nonhuman · nutrient · nutrient solution · priority journal · Pseudomonas putida · Pseudomonas putida s12 · reaction analysis · shake flask culture · steady state · Pseudomonas putida

Abstract

Pseudomonas putida strain S12palB1 was constructed that produces p-hydroxybenzoate from renewable carbon sources via the central metabolite l-tyrosine. P. putida S12palB1 was based on the platform strain P. putida S12TPL3, which has an optimised carbon flux towards l-tyrosine. Phenylalanine ammonia lyase (Pal) was introduced for the conversion of l-tyrosine into p-coumarate, which is further converted into p-hydroxybenzoate by endogenous enzymes. p-Hydroxybenzoate hydroxylase (PobA) was inactivated to prevent the degradation of p-hydroxybenzoate. These modifications resulted in stable accumulation of p-hydroxybenzoate at a yield of 11% (C-mol C-mol-1) on glucose or on glycerol in shake flask cultures. In a glycerol-limited fed-batch fermentation, a final p-hydroxybenzoate concentration of 12.9 mM (1.8 g l-1) was obtained, at a yield of 8.5% (C-mol C-mol-1). A 2-fold increase of the specific p-hydroxybenzoate production rate (qp) was observed when l-tyrosine was supplied to a steady-state C-limited chemostat culture of P. putida S12palB1. This implied that l-tyrosine availability was the bottleneck for p-hydroxybenzoate production under these conditions. When p-coumarate was added instead, qp increased by a factor 4.7, indicating that Pal activity is the limiting factor when sufficient l-tyrosine is available. Thus, two major leads for further improvement of the p-hydroxybenzoate production by P. putida S12palB1 were identified. © 2007 Elsevier B.V. All rights reserved.