启动子串联及改造提高FAD为辅基的葡萄糖脱氢酶在Bacillus subtilis中的表达  

Promoter tandem and transformation for FAD-conjugated glucose dehydrogenase expression in Bacillus subtilis

作　　者：张玲[1] 林荣 宋祖坤 王男 杨海麟[1] ZHANG Ling;LIN Rong;SONG Zukun;WANG Nan;YANG Hailin(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University, Wuxi 214122,China;Suzhou Suncadia Biopharmaceuticals CO.,Ltd,Suzhou 215000,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]苏州盛迪亚生物医药有限公司,江苏苏州215000

出　　处：《食品与发酵工业》2019年第8期15-21,共7页Food and Fermentation Industries

基　　金：江苏省产学研(BY2016022-40);国家轻工技术与工程一流学科自主课题资助(2018-23)

摘　　要：以黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)为辅基的葡萄糖脱氢酶(glucose dehydrogenase with FAD,FAD-GDH,EC 1.1.99.10),与辅基结合紧密,催化效率高,是临床检测血糖指标的新型诊断用酶。将Burkholderia cepacia的FAD-GDH基因(gdh)构建含单启动P_(HpaⅡ)的穿梭质粒p MA5-1,在蛋白酶缺陷型菌株Bacillus subtilis WB600中表达。为了获得该酶的高效表达,采用启动子串联及改造策略考察产酶情况。将4种启动子(P_(amyQ’),P_(43),P_(gsiB),P_(opuaa))分别与质粒上自带的启动子P_(HpaⅡ)串联,结果表明P_(HpaⅡ)-P_(amyQ’)串联组合获得的FAD-GDH胞内酶活最高,为2 497 U/L,是串联前单启动子的2.7倍。为了减少发酵过程中,葡萄糖和甘油对产酶的抑制作用,在串联组合的基础上删去P_(amyQ’)启动子中与碳代谢调控蛋白结合的cre位点,使胞内产酶水平提高至3 626 U/L,说明cre位点的去除能够减少碳代谢产物对启动子转录的抑制。本研究为新型诊断用酶FAD-GDH的菌种改造和工业化生产应用提供参考与借鉴。Glucose dehydrogenase (FAD-GDH,EC1.1.99.10),conjugated tightly with flavin adenine dinucleotide,is a novel diagnostic enzyme for the clinical detection of blood glucose.A protease-defective strain Bacillus subtilis WB600 was used as a host to construct a shuttle plasmid pMA5-1 containing a single promoter PHpaⅡ for expression of FAD-GDH gene ( gdh ) from Burkholderia cepacia.The use of promoters in series and transformation strategies to investigate enzyme production.Four promoters (PamyQ',P43,PgsiB,Popuaa ) were ligated with the promoter PHpaⅡ on the plasmid,respectively.The results showed that the intracellular enzymatic activity of FAD-GDH was highest with PHpaⅡ-P amyQ' tandem,which was 2 497 U/L.In order to reduce the inhibitory effect of glucose and glycerol on enzyme production during fermentation,on the basis of tandem combination,the cre sites binding to carbon metabolism regulatory proteins in PamyQ ' promoter were deleted,and the intracellular enzyme production level was increased to 3 626 U/L,indicating that the removal of cre sites can reduce the inhibition of carbon metabolism products on promoter transcription.This study provides a reference for genetic modification and industrial production of a new diagnostic enzyme (FAD-GDH).

关 键 词：黄素腺嘌呤二核苷酸(FAD) 葡萄糖脱氢酶 枯草芽孢杆菌 串联启动子 cre位点 

分 类 号：Q78[生物学—分子生物学]