玉米赤霉烯酮降解酶基因zlhy-6在枯草芽孢杆菌中的表达  

Expression of Zearalenone Degrading Enzyme Gene zlhy-6 in Bacillus subtilis

作　　者：符浩东 张晨曦 李奕霏 郑永权[3] 刘阳[1,2] FU Haodong;ZHANG Chenxi;LI Yifei;ZHENG Yongquan;LIU Yang(Institute of Food Science and Technology,Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process/Ministry of Agriculture and Rural Affairs,Beijing 100193;School of Life Science and Engineering,Foshan University,Foshan,Guangdong 528231;Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193)

机构地区：[1]中国农业科学院农产品加工研究所/农业农村部农产品质量安全收贮运管控重点实验室,北京100193 [2]佛山科学技术学院生命科学与工程学院,广东佛山528231 [3]中国农业科学院植物保护研究所,北京100193

出　　处：《核农学报》2022年第5期885-894,共10页Journal of Nuclear Agricultural Sciences

基　　金：国家重点研发计划项目(2017YFC1600900);国家重点研发计划项目(2019YFC1604502)。

摘　　要：玉米赤霉烯酮(ZEN)是具有雌激素作用的次生代谢产物,可以被来源于Gliocladium roseum的内酯水解酶降解为无毒物质。为了实现玉米赤霉烯酮降解酶基因zlhy-6在枯草芽孢杆菌中的表达,获得不含抗生素抗性基因的食品级重组枯草芽孢杆菌,本研究采用一步克隆及重叠延伸PCR的方法构建了单启动子和包含HpaⅡ和P43双启动子的表达质粒,将质粒转化到枯草芽孢杆菌中,获得重组菌株168/pMA5-zlhy-6和168/pMA5-P43-zlhy。然后构建了重组整合载体amy-p43-zlhy,将zlhy-6基因整合到枯草芽孢杆菌168的基因组中。通过Cre/lox系统敲除抗生素抗性基因,获得整合了P43-zlhy表达盒的食品级重组枯草芽孢杆菌BZ-zlhy。将构建的3个重组菌株在37℃、pH值7.5条件下培养36 h,结果显示,双启动子表达载体重组菌株的最高酶活性为2.2 U·mL^(-1),是单启动子菌株的1.2倍。双启动子重组菌株表达的降解酶对ZEN(4μg·mL^(-1),30 min)的降解率为65.1%。重组菌株枯草芽孢杆菌BZ-zlhy表达水平最低(0.4 U·mL^(-1))。本研究实现了玉米赤霉烯酮降解酶在枯草芽孢杆菌中表达,同时构建了不含抗生素抗性基因的食品级重组枯草芽孢杆菌,为玉米赤霉烯酮降解酶的工业化生产奠定了基础,也为解决粮食储藏和饲料生产中的ZEN污染提供了思路。Zearalenone is a secondary metabolite with estrogen effect,which can be degraded into non-toxic substances by lactone hydrolase derived from Gliocladium roseum.In order to realize the expression of zearalenone degrading enzyme gene zlhy-6 in Bacillus subtilis,and obtain food-grade recombinant Bacillus subtilis without antibiotic resistance gene.The single-promoter and double-promoter expression plasmids containing HpaⅡand P43 were constructed by one-step cloning and overlap extension PCR.The plasmids were transformed into Bacillus subtilis to obtain recombinant strains 168/pMA5-zlhy-6 and 168/pMA5-P43-zlhy.Then,the recombinant integration vector amy-p43-zlhy was constructed to integrate the zlhy-6 gene into the genome of Bacillus subtilis 168.Then,the antibiotic resistance gene was knocked out by the Cre/lox system,while the food-grade recombinant Bacillus subtilis 168-zlhy with integrated of the BZ-zlhy expression cassette was generated.The three recombinant strains were cultured at 37℃ and pH 7.5 for 36 h.The results showed that the highest enzyme activity of the dual-promoter expression vector recombinant strain was 2.2 U·mL^(-1),which was 1.2 times that of the single-promoter strain.The degradation rate of zearalenone(4μg·mL^(-1),30 min)by the degrading enzyme expressed by the double-promoter recombinant strain was 65.1%.As for the recombinant strain Bacillus subtilis BZ-zlhy,it has the lowest expression level(0.4 U·mL^(-1)),which may be due to the low copy number of the target gene.In this study,zearalenone degrading enzyme was expressed in Bacillus subtilis,and a food grade recombinant Bacillus subtilis without antibiotic resistance gene was constructed.This is the basis of industrial production of zearalenone degrading enzyme,and also provides ideas for solving ZEN pollution in grain storage and feed production.

关 键 词：玉米赤霉烯酮 枯草芽孢杆菌 降解酶 整合表达 串联启动子 

分 类 号：Q78[生物学—分子生物学] Q936