生物转化合成苯乳酸工程菌的培养条件优化

本研究旨在优化重组大肠杆菌Escherichia coli BL21 (DE3) harboring pRSF-aad-ldh10-fdh菌株的培养条件,获得高密的供生物转化苯丙氨酸为苯乳酸的细胞。实验考察了摇瓶发酵培养基碳源、氮源种类和浓度,3 L发酵罐中转速和通气量及恒速补料、DO-stat和pH-stat等不同分批补料策略对菌体密度的影响。结果表明,当碳源为4 g/L葡萄糖,氮源为24 g/L安琪酵母浸粉FM802,细胞干重最大可达9.24 g/L;当转速为400 r/min和通气量为1.5 vvm时,细胞干重最大可达10.18 g/L;以4 g/(L·h)恒速流加葡萄糖时,细胞干重最大可达13.71 g/L。本研究还对工程菌酶表达的诱导条件进行了优化,菌体培养2 h后,添加终浓度为0.08 mmol/L IPTG诱导剂,在25℃下诱导培养14 h所得细胞有利于生物转化。底物苯丙氨酸浓度为60 g/L,转化为苯丙酮酸的转化率为50.2%,转化为苯乳酸的转化率为35.2%。

Optimization of culture conditions for phenyllactic acid-producing engineering bacteria

The aim of this study was to optimize the culture conditions of recombinant Escherichia coli BL21(DE3) harboring pRSF-aad-ldh10-fdh and to obtain high-cell density for the biotransformation of phenyllactic acid from phenylalanine. The effects of the type and concentration of carbon source and nitrogen source in the shake flask, the rotational speed, aeration and different batch feeding strategies such as constant feed, DO-stat and pH-stat in the 3 L fermenter on the cell density were investigated. The results showed that when the carbon source was 4 g/L glucose and the nitrogen source was 24 g/L Angel Yeast extract FM802, the dry cell weight was up to 9.24 g/L;when the rotational speed was 400 r/min and the aeration was 1.5 vvm, the dry cell weight reached to 10.18 g/L. When the glucose was fed at a constant rate of 4 g/L/h, the dry cell weight was 13.71 g/L. Furthermore, the induction conditions of the key enzyme were also optimized. When the cells of recombinant E. coli BL21(DE3) harboring pRSF-aad-ldh10-fdh were cultivated in the optimized medium for 2 h, the IPTG was added to a final concentration with 0.08 mmol/L at 25 ℃ for 14 h. Then the cells were collected to transform 60 g/L substrate phenylalanine into intermediate product phenylpyruvic acid and target product phenyllactic acid, with a conversion rate of 50.2% and 35.2%, respectively.