Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation
Bioproduction of p-Hydroxystyrene from Glucose by the Solvent-Tolerant Bacterium Pseudomonas putida S12 in a Two-Phase Water-Decanol Fermentation
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DOI：
10.1128/aem.02186-08
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发表时间：
2009-02-15
影响因子：
4.4
通讯作者：
Ruijssenaars, Harald J.
中科院分区：
文献类型：
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作者：
Verhoef, Suzanne;Wierckx, Nick;Ruijssenaars, Harald J.
两个耐溶剂的恶臭假单胞菌S12菌株，最初设计用于苯酚和对香豆酸生产，被设计用于从葡萄糖有效生产对羟基苯乙烯。这是通过引入分别编码L-苯丙氨酸/L-酪氨酸氨裂解酶和p-香豆酸脱羧酶的基因pal和pdc建立的。这些酶允许中心代谢物L-酪氨酸通过对香豆酸转化为对羟基苯乙烯。通过灭活编码阿魏酰辅酶A合成酶的fcs基因来防止对香豆酸中间体的降解。选择表现最好的菌株并以补料分批模式培养，导致以6.7%的产率（每C-mol葡萄糖C-mol对羟基苯乙烯）和0.4mM h（-1）的最大体积生产率形成4.5mM对羟基苯乙烯。在该浓度下，由于对羟基苯乙烯的毒性，生长和生产完全停止。通过在补料分批培养期间应用第二相1-癸醇来提取对羟基苯乙烯，克服了产物毒性。这导致最大体积生产率（0.75 mM h（-1））增加两倍，最终总对羟基苯乙烯浓度为21 mM，与单相补料分批培养相比提高了四倍。对羟基苯乙烯在水相中的最终浓度为1.2mM，而在1-癸醇相中得到的浓度为147 mM（17.6克升（-1））。因此，生产低价值化合物苯酚的恶臭假单胞菌S12菌株被成功地改变用于生产有毒的增值化合物对羟基苯乙烯。
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.