1株产顺式-4-羟基-L-脯氨酸基因工程菌的构建及发酵初步优化

Construction of cis-4-hydroxy-L-proline producing recombinant E. coli and preliminary optimization of its fermentation conditions

顺式-4-羟基-L-脯氨酸是一种可用于合成多种药物和香料的手性结构物质。通过对L-脯氨酸顺式-4-羟化酶基因的优化设计,并引入色氨酸串联启动子,构建出1株能表达L-脯氨酸顺式-4-羟化酶的重组大肠杆菌JM109/p EHC4,从而可以将游离的L-脯氨酸转化为顺式-4-羟基-L-脯氨酸。对该菌株进行摇瓶发酵优化,得出优选培养基为(g/L):葡萄糖10,甘油10,蛋白胨10,NaCl 6,FeSO_4·7H_2O 0.278,L-抗坏血酸0.528,(NH_4)_2SO_45,K_2HPO41,MgSO_40.2,CaCl_20.015,L-脯氨酸4。在该条件下发酵12 h,顺式-4-羟基-L-脯氨酸的产量达到657.08mg/L,比优化前提高了3.76倍;在24 h时产量达到1 582.75 mg/L。研究结果为顺式-4-羟基-L-脯氨酸的微生物转化法生产提供了基础。

cis-4-hydroxy-L-proline is a valuable chiral building block for the organic synthesis of pharmaceuticals and perfume. By optimizing the target gene and introducing a tryptophan tandem promoter recombinant strain expressing the L-proline cis-4-hydroxylases,E. coli JM109 / pEHC4,was constructed. Through the expression of L-proline cis-4-hydroxylases,free L-proline was converted to cis-4-hydroxy-L-proline. The optimized medium composition contained glucose 10 g / L,glycerol 10 g / L,peptone 10 g / L,NaCl 6 g / L,FeSO4·7H2O 0. 278 g / L,L-ascorbate 0. 528 g / L,（ NH4）2SO45 g / L,K2HPO41 g / L,MgSO40. 2 g / L,CaCl20. 015 g / L,and L-proline 4 g / L. Finally,after fermentation under the optimal conditions for 12 h,cis-4-hydroxy-L-proline production reached 657. 08 mg / L,which was 3. 76 times higher than that before fermentation,and then reached 1 582. 75 mg / L after 24 h. Microbial transformation by L-proline cis-4-hydroxylases is a potential approach for cis-4-hydroxy-L-proline production.