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

作　　者：姚雪娜 张震宇[1,2,3] 孙付保[1,2,3] 陈昶[1,2,3] 黄建华[1,2,3] 沈松[1,2,3] YAO Xuena ZHANG Zhenyu SUN Fubao CHEN Chang HUANG Jianhua SHEN Song(School of Biotechnology,Jiangnan University,Wuxi 214122,China Key Laboratory of Industrial Biotechnology, Jiangnan University,Wuxi 214122, China Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Wuxi 214122, China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [3]江南大学糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2017年第3期243-251,共9页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(30800018;30970058);江苏省自然科学基金项目(BK2012554);高等学校博士学科点专项科研基金项目(200802951036);工业生物技术教育部重点实验室主任基金项目(KLIB-ZR200801)

摘　　要：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.

关 键 词：脯氨酸-3-羟化酶 顺式-3-羟脯氨酸 密码子优化 重组菌构建 发酵优化 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]