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The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose

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Author: Nijkamp, K. · Luijk, N. van · Bont, J.A.M. de · Wery, J.
Type:article
Date:2005
Institution: TNO Kwaliteit van Leven TNO Voeding
Source:Applied Microbiology and Biotechnology, 2, 69, 170-177
Identifier: 238792
doi: doi:10.1007/s00253-005-1973-7
Keywords: Nutrition · Biotechnology · Biosynthesis · Enzymes · Genes · Glucose · Glycerol · Metabolism · Organic acids · Toxic materials · Cinnamic acid · Phenylalanine ammonia lyase · Pseudomonas putida · Rhodosporidium toruloides · Strain · 3 fluorophenylalanine · Antimetabolite · Cinnamic acid · Glucose · Glycerol · Phenylalanine · Phenylalanine ammonia lyase · Solvent · Amino acid synthesis · Bacterial strain · Bacterium culture · Bacterium mutant · Bioaccumulation · Bioavailability · Controlled study · Gene construct · Gene overexpression · Genetic code · High throughput screening · Host · Metabolite · Nonhuman · Pseudomonas putida · Quantum yield · Randomization · Rhodosporidium toruloides · Yeast · 3-Deoxy-7-Phosphoheptulonate Synthase · Catalysis · Cinnamates · Drug Tolerance · Fermentation · Genetic Engineering · Genetic Vectors · Glucose · Mutation · Phenylalanine · Phenylalanine Ammonia-Lyase · Prephenate Dehydratase · Pseudomonas putida · Solvents · Bacteria (microorganisms) · Pseudomonas putida · Rhodosporidium toruloides

Abstract

A Pseudomonas putida S12 strain was constructed that efficiently produced thefine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine availability was the main bottleneck in cinnamic acid production, which could not be overcome by the overexpressing enzymes of the phenylalanine biosynthesis pathway. A successful approach in abolishing this limitation was the generation of a bank of random mutants and selection on the toxic phenylalanine anti-metabolite m-fluoro-phenylalanine. Following high-throughput screening, a mutant strain was obtained that, under optimised culture conditions, accumulated over 5 mM of cinnamic acid with a yield (Cmol%) of 6.7%. © Springer-Verlag 2005.