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

• Published in: Applied and Environmental Microbiology Vol. 75; no. 4; pp. 931 - 936
• Main Authors: Verhoef, Suzanne, Wierckx, Nick, Westerhof, R.G. Maaike, de Winde, Johannes H, Ruijssenaars, Harald J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.02.2009; American Society for Microbiology (ASM)
• Subjects: Ammonia; Bacteria; Bacteriology; Biological and medical sciences; Biotechnology; Carboxy-Lyases - genetics; Carboxy-Lyases - metabolism; Culture Media - chemistry; Decanoic Acids - chemistry; Fermentation; Fundamental and applied biological sciences. Psychology; Glucose; Glucose - metabolism; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; Microbiology; Phenylalanine Ammonia-Lyase - genetics; Phenylalanine Ammonia-Lyase - metabolism; Physiology and Biotechnology; Polystyrenes - metabolism; Pseudomonas putida - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Solvents; Water; Pseudomonadales; Water; Production; Tolerance; Pseudomonadaceae; Bacteria; Glucose; Fermentation; Pseudomonas putida; Solvent
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.02186-08

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.