Bioproduction of p -Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation

Abstract

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.