Bioproduction of p-hydroxystyrene from glucose by the solvent-tolerant bacterium Pseudomonas putida S12 in a two-phase water-decanol fermentation.
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.
Verhoef
Suzanne
S
TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Julianalaan 67, 2628 BC Delft, The Netherlands. suzanne.verhoef@tno.nl
Wierckx
Nick
N
Westerhof
R G Maaike
RG
de Winde
Johannes H
JH
Ruijssenaars
Harald J
HJ
D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry*; D003652:Decanoic Acids / Q000737:chemistry*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D010650:Phenylalanine Ammonia-Lyase / Q000235:genetics / Q000378:metabolism; D011137:Polystyrenes / Q000378:metabolism*; D016958:Pseudomonas putida / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D003652:Decanoic Acids; D011137:Polystyrenes; D011994:Recombinant Proteins; C047732:hydroxystyrene-styrene; D002262:Carboxy-Lyases; C087194:phenylacrylic acid decarboxylase; D010650:Phenylalanine Ammonia-Lyase; D005947:Glucose