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Bioproduction of p-hydroxystyrene from glucose by the solvent-tolerant bacterium Pseudomonas putida S12 in a two-phase water-decanol fermentation

Author: Verhoef, S. · Wierckx, N. · Westerhof, R.G.M. · Winde, J.H.de · Ruijssenaars, H.J.
Type:article
Date:2009
Institution: TNO Kwaliteit van Leven
Source:Applied and Environmental Microbiology, 4, 75, 931-936
Identifier: 241379
doi: doi:10.1128/AEM.02186-08
Keywords: Biology · Biotechnology · Amino acids · Biochemical engineering · Encoding (symbols) · Gene encoding · Glucose · Phenols · Solvents · Strain · 1-Decanol · Ammonia lyase · Bio productions · Concentration of · Decanol · Decarboxylase · Efficient productions · Fed batches · Fed-batch cultivations · Hydroxystyrene · L tyrosines · P-coumaric acids · Product toxicities · Pseudomonas putida · Second phase · Synthetase · Volumetric productivities · Water phase · Toxicity · alcohol · bacterium · bioengineering · enzyme activity · fermentation · gene expression · glucose · metabolite · polymer · toxicity · Carboxy-Lyases · Culture Media · Decanoic Acids · Fermentation · Glucose · Phenylalanine Ammonia-Lyase · Polystyrenes · Pseudomonas putida · Recombinant Proteins · Bacteria (microorganisms) · Pseudomonas putida

Abstract

Two solvent-tolerant Pseudomonas putida S12 strains, originally designed for phenol and p-coumarate production, were engineered for efficient production of p-hydroxystyrene from glucose. This was established by introduction of the genes pal and pdc encoding L-phenylalanine/L-tyrosine ammonia lyase and p-coumaric acid decarboxylase, respectively. These enzymes allow the conversion of the central metabolite L-tyrosine into p-hydroxystyrene, via p-coumarate. Degradation of the p-coumarate intermediate was prevented by inactivating the fcs gene encoding feruloyl-coenzyme A synthetase. The best-performing strain was selected and cultivated in the fed-batch mode, resulting in the formation of 4.5 mM p-hydroxystyrene at a yield of 6.7% (C-mol of p-hydroxystyrene per C-mol of glucose) and a maximum volumetric productivity of 0.4 mM h-1. At this concentration, growth and production were completely halted due to the toxicity of p-hydroxystyrene. Product toxicity was overcome by the application of a second phase of 1-decanol to extract p-hydroxystyrene during fed-batch cultivation. This resulted in a twofold increase of the maximum volumetric productivity (0.75 mM h-1) and a final total p-hydroxystyrene concentration of 21 mM, which is a fourfold improvement compared to the single-phase fed-batch cultivation. The final concentration of p-hydroxystyrene in the water phase was 1.2 mM, while a concentration of 147 mM (17.6 g liter-1) was obtained in the 1-decanol phase. Thus, a P. putida S12 strain producing the low-value compound phenol was successfully altered for the production of the toxic value-added compound p-hydroxystyrene. Copyright © 2009, American Society for Microbiology. All Rights Reserved.