文献类型：期刊文献

中文题名：重组大肠杆菌多基因串联表达合成反式-4-羟基-L-脯氨酸

英文题名：Co-expression of multiple genes for the synthesis of trans-4-hydroxy-L-proline in Escherichia coli

作者：盛花开[1];衣玉兰[1];李志敏[1];叶勤[1]

机构：[1]华东理工大学生物工程学院生物反应器工程国家重点实验室,上海200237

年份：2016

卷号：26

期号：1

起止页码：81

中文期刊名：生物技术

外文期刊名：Biotechnology

收录：北大核心:【北大核心2014】；CSCD:【CSCD_E2015_2016】；

语种：中文

中文关键词：重组大肠杆菌;重叠PCR;无缝组装;反式-4-羟基-L-脯氨酸

外文关键词：recombinant E. coli, overlap PCR, seamless assembly, Hyp

摘要：[目的]得到表达多个基因的重组大肠杆菌,以期实现葡萄糖为碳源生产反式-4-羟基-L-脯氨酸。[方法]以菌液为模板,PCR得到不同来源的proB、proA、proC、p4h基因,重叠PCR串联相邻基因,所得片段通过无缝组装与p ET-28a载体连接,并在大肠杆菌BL21中表达,筛选阳性表达菌株,电泳及测序验证,重组菌在30 ml MCG培养基摇瓶中发酵,分光光度法和HPLC检测产量。[结果]0.5 mmol/L IPTG诱导24 h,5组串联基因成功在大肠杆菌中表达,并以葡萄糖为碳源,获得产物反式-4-羟基-L-脯氨酸,培养基不添加L-脯氨酸时E.coli BL21/p ET-28a-BAHHbs、E.coli BL21/p ET-28a-HBACkp和E.coli BL21/p ET-28a-HBAmg产量达到0.28 g/L、0.16 g/L和0.29 g/L,添加4 mmol/L的L-脯氨酸时,分别为0.64 g/L、0.55 g/L和0.74 g/L。[结论]proB、proA、proC及p4h基因成功在大肠杆菌中表达,5株重组菌在30℃摇瓶中诱导表达24 h得到产物反式-4-羟基-L-脯氨酸,其中E.coli BL21/p ET-28a-HBAmg产量达到0.74 g/L,为今后在发酵罐中生产奠定基础。
[ Objective] Recombinant Escherichia coli BL21 strains was constructed and was expected to produce trans -4 -hy- droxy- L- proline （Hyp） using glucose as substrate. [Methods]proB,proA,proC,p4h genes was amplified by PCR with different bacterium solution as template. Overlap PCR was used to connect adjacent gene fragments ,then cloned to pET -28a by seamless assembly and transformed into E. coli BL21. Recombinant strains were cultivated in MCG shake flaks and production was detected by spectrophotometry and HPLC. [ Results] Five strains had been expressed under the induction of 0.5 mmol/L IPTG. Hyp was obtained using glucose as substrate, and the production of E. coli BL21/pET- 28a- BAHHbs, E. coli BL21/pET -28a- HBACkp,E. coli BL21/pET-28a-HBAmg was 0.28 g/L,0.16 g/L,0.29 g/L without L- proline and 0.64 g/L, 0.55 g/L ,0.74 g/L with 4mmol/L L- proline in medium. [ Conclusion ]proB ,proA ,proC and p4h genes were successfully ex- pressed in E. coli BL21. Hyp could be produced by recombinant strains induced in 30℃ shakers for 24 h ours. The production of E. coli BL21/pET -28a -HBAmg reached 0.74 g/L, which was prepared for the fermenter production.