产3-羟脯氨酸重组菌的构建及发酵优化

Construction of Recombinant Escherichia coli Strains Producing cis-3-Hydroxyproline and Preliminary Optimization of the Fermentation Conditions

顺式-3-羟基-L-脯氨酸(顺式-3-羟脯氨酸)可用于合成多种抗癌药物,具有重要的商业价值,目前大多通过添加IPTG来诱导表达脯氨酸-3-羟化酶,采用两步法生物合成顺式-3-羟脯氨酸。作者通过目的基因优化设计,引入强启动子色氨酸串联启动子(Ptrp2)来避免异源表达时的诱导剂使用,构建重组质粒p ES-Ptrp2-P3H,成功构建了重组大肠杆菌BL21(DE3)/p ET21a-Ptrp2-P3H,优化后的脯氨酸-3-羟化酶基因(P3H)改变了168个碱基,GC含量由原来的64.83%降低到49.31%。该菌在初步优化培养基(葡萄糖1 g/d L,甘油0.125 g/d L,胰蛋白胨1.6 g/d L,(NH_4)_2SO_40.5 g/d L,K_2HPO_40.1 g/d L,NaCl 0.2 g/d L,FeSO_41 mmol/L,MgSO_40.5 g/d L,CaCl_20.015 g/d L,脯氨酸10 g/L,pH 7.5)上能一步法原位合成顺式-3-羟脯氨酸,摇瓶发酵24 h,产量0.8 g/L,比优化前提高一倍以上,为进一步开展顺-3-羟脯氨酸产业化提供了依据。

The cis-3-hydroxy-L-proline can be used to synthesize many anticancer drugs with important commercial value.At present,cis-3-hydroxyproline is produced with IPTG-inducible recombinants in two steps.The subject of this study is to construct a recombinant with high proline-3-hydroxylase activity and generate cis-3-hydroxyproline without adding IPTG which is expensive and toxic.We constructed the recombinant plasmid pES-Ptrp2-P3H and transformed E.coli BL21 （DE3） successfully.Compared with the original gene sequence of proline 3-hydroxylase,168 nucleotides were changed and the GC percentage was reduced from 64.83 % to 49.31%.The preliminarily optimized medium was 1 g/dL glucose,0.125 g/dL glycerinum,1.6 g/dL tryptone,0.5 g/dL （NH4）2SO4,0.1 g/dL K2HPO4,0.2 g/dL NaC1,1 mmol/L FeSO4,0.5 g/dL MgSO4,0.015 g/dL CaC12,10 g/L L-proline,and pH valve at 7.5.At shaking flask level,cis-3-hydroxyproline was accumulated to about 0.8 g/L in 24 h,which was one times higher than before.It provides the basis for the industrialization of cis-3-hydroxyproline bioproduction.