产顺式-4-羟脯氨酸枯草芽孢杆菌工程菌的构建及发酵优化

Construction and optimization of fermentation of Bacillus subtilis cis-4-hydroxyproline engineering bacteria

顺式-4-羟脯氨酸(CHOP)是一种重要的手性结构物质,可广泛用于药物以及香料制造。本研究为开发CHOP的生物合成方法,将来源于中华根瘤菌(Sinorhizobium meliloti)的L-脯氨酸-4-羟化酶在枯草芽孢杆菌WB800N中实现了成功表达,构建获得了能够以L-脯氨酸为底物合成CHOP的工程菌株,进而对该菌株的发酵条件以及发酵组分进行了优化研究。结果表明:工程菌株的最适发酵温度为25℃,最适诱导剂浓度为1 mmol/L,最适发酵p H为5;在发酵体系中加入5 mmol/L硫酸亚铁和2.5 g/L L-脯氨酸时最有利于合成CHOP。在最优发酵体系下发酵60 h后,顺式-4-羟脯氨酸的产量可以达到120.2 mg/L。本文成功的构建了羟脯氨酸生产菌株枯草杆菌WB800 p HT-P4H,而研究结果为枯草杆菌工程菌发酵生产顺式-4-羟铺氨酸提供了基础。

Cis- 4- hydroxyproline （CHOP）is an important chiral structure material, which can be widely used in pharmaceuticals and perfume manufacturing.In this study,in order to develop the new way to produce the cis-4-hydroxyproline by biological method,L-proline-4-hydroxylase geue derived from Sinorhizobiam meliloti was first successfully expressed in Bacillus subtilis WB800N, which converts L-proline to cis-4-hydroxyproline. The fermentation conditions and fermentation components of the strain were optimized. The results showed that adding 5 mmol/L sulfuric acid ferrous in Bacillus subtilis fermentation were favourable for the formation of cis- 4 - hydroxyproline. The optimum fermentation temperature of cis- 4 - hydroxyproline was 25 -C .The optimum concentration of inducing agent and pH were 1 mmoL/L and 5, respectively. After induction,the optimum substrate L-proline concentration was 2.5 g/L. After 60 h of Bacillus subtilis ferment cis-4- hydroxyproline,cis-4-hydroxyproline concentration of 120.2 mg/L was achieved. This study construct a strain of Bacillus subtilis WB800N pHT-P4H to produce CHOP,and microbial fermentation by Bacillus subtilis WB800N is a potential approach for cis-4-hydroxy-L-proline production by fermentation.