钝齿棒杆菌溶氧诱导型启动子的筛选及其功能验证  

Screening of dissolved oxygen induced promoters in Corynebacterium crenatum and functional verification

作　　者：张博慧[1] 徐美娟[1] 饶志明[1] 许正宏[2] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,无锡214122 [2]江南大学药学院制药工程实验室,无锡214122

出　　处：《微生物学报》2013年第9期933-942,共10页Acta Microbiologica Sinica

基　　金：教育部新世纪优秀人才计划(NCET-10-0459);国家"973项目"(2012CB725202);国家"863计划"(2012AA022102;2011AA02A211);国家自然科学基金(21276110);中央高校基本科研业务费专项资金(JUSRP51306A;JUSRP1009);高等学校博士学科点专项科研基金(20110093120001);江苏高校优势学科建设工程项目~~

摘　　要：【目的】筛选钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5内源溶氧诱导型启动子,验证其在不同溶氧条件下受溶氧调控的功能。【方法】以本课题组前期利用双向电泳结合质谱鉴定技术分离鉴定出的高低供氧条件下钝齿棒杆菌SYPA5-5胞内的显著差异蛋白(27个)为基础,结合棒杆菌基因组信息分析高低溶氧状态下显著差异蛋白中单位拷贝的表达量,以其中单位拷贝表达量高的蛋白为目的蛋白,克隆目的蛋白编码基因的上游启动子,分别替换棒杆菌-大肠杆菌穿梭表达质粒pDXW-8的外源tac启动子,在大肠杆菌和钝齿棒杆菌中表达报告基因gfp和cat,并检测其表达效率。【结果】筛选出高溶氧上调蛋白转酮醇酶和延胡索酸水化酶作为目的蛋白,克隆其启动子区命名为P-tkt和P-fum。在重组大肠杆菌和重组钝齿棒杆菌中,P-tkt在高溶氧条件下CAT活性是低溶氧条件下CAT活性的2.8和3.2倍。该结果在5 L发酵罐中得到验证。【结论】启动子P-tkt为首次筛选到的钝齿棒杆菌内源性高溶氧诱导型启动子,适用于高溶氧条件发酵生产氨基酸的菌株中,有利于在高供氧条件下提高棒杆菌中氨基酸的合成代谢能力。[ Objective ] Purpose of this work was to screen promoters which were induced by dissolved oxygen in Corynebacterium crenatum SYPAS-5. [ Methods] Based on the genomic information of Corynebacterium, we identified target proteins which were the highest expression level of one copy of 27 known proteins from our earlier study. Based on the upstream sequence of the coding gene sequences, we amplified two promoters, named P-tkt and P-fum. The tac promoter of recombinant pDXW-8-cat and pDXW-8-gfp were replaced by the new promoters, and the recombinant plasmids were transformed into E. coli JM109 and C. crenatum SYPAS-5 respectively. At different dissolved oxygen level, we compared the function of promoters by the expression of chloramphenicol acetyltransferase （CAT） and green fluorescent protein （GFP）. [ Results] Results show that we identified 2 target proteins, which were transketolase and fumarate hydratase. At the high dissolved oxygen level the CAT activity of E. coli JM109/pDXW-Ptkt-cat and C. crenatum SYPAS- 5/pDXW-Ptkt-cat were 3. 032 U/mg and 1. 987 U/rag, which were 2.8-fold and 3.2-fold than that in the low dissolved oxygen, respectively. We got similar results by using a 5-L fermentor. [ Conclusion] The P-tkt promoter induced by high dissolved oxygen was suitable for fermentation to produce amino acids and beneficial to improve ability of synthesis and metabolism of amino acids at high dissolved oxygen level.

关 键 词：钝齿棒杆菌 供氧差异 诱导型 启动子 

分 类 号：Q933[生物学—微生物学]