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Optimization of the solvent-tolerant Pseudomonas putida S12 as host for the production of p-coumarate from glucose

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Author: Nijkamp, K. · Westerhof, R.G.M. · Ballerstedt, H. · Bont, J.A.M.de · Wery, J.
Type:article
Date:2007
Institution: TNO Kwaliteit van Leven
Source:Applied Microbiology and Biotechnology, 3, 74, 617-624
Identifier: 239860
doi: doi:10.1007/s00253-006-0703-0
Keywords: Biology · Biotechnology · Degradation · Feeding · Genes · Genetic engineering · Glucose · Mutagenesis · Optimization · Production control · Solvents · Cellular L-tyrosine · Molar ratio · p-coumarate · Solvent-tolerant Pseudomonas putida · Bacteria · cinnamic acid · glucose · para coumaric acid · phenylalanine · tyrosine · amino acid · bacterium · biodegradation · degradation · enzyme · glucose · metabolite · optimization · organic acid · article · controlled study · feeding · host · mutant · nonhuman · Pseudomonas putida · Cinnamates · Coenzyme A Ligases · Coumaric Acids · Fermentation · Gene Deletion · Glucose · Phenylalanine · Pseudomonas putida · Tyrosine · Pseudomonas putida

Abstract

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular L-tyrosine availability, and formation of the by-product cinnamate via L-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l-1) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1. © 2006 Springer-Verlag.