摘要
丁香酚是一种绿色生物农药,主要用于防治番茄灰霉病、葡萄霜霉病和马铃薯晚疫病等主要的农业病害.目前丁香酚是从丁香和罗勒等植物中提取制备,其规模和产能受到限制,还存在废渣排放影响生态环境的问题.本研究采用合成生物学和模块化工程策略,设计构建工程大肠杆菌,以期微生物从头合成丁香酚.将丁香酚异源合成途径分成3个模块,分别是上游的阿魏酸合成模块、中游的松柏醇合成模块和下游的丁香酚合成模块,对每个模块进行不同来源基因的组合筛选和表达优化.利用His标签、敲除甲基供体合成途径中的全局调控基因metJ,优化了上游模块(PcTAL-HpaBC-AtCOMT),减少了中间产物咖啡酸的积累,提高了阿魏酸合成.筛选了4CL和醇脱氢酶基因来源,研究了表达模式,获得最优的中游模块是双顺反子表达At4CL1-AtCCR1,单顺反子表达YahK,松柏醇产量最高.对3个酰基转移酶基因和4个丁香酚合成酶基因进行组合筛选,获得最优的下游模块是单顺反子PhCFAT和GdlEGS2,丁香酚合成最多.最后,在酪氨酸高产底盘中表达丁香酚合成途径,对初始葡萄糖、酵母提取物和诱导剂浓度等进行优化.在适宜的培养基和发酵条件下,丁香酚的产量为114.67mg/L.本文采用模块化代谢工程策略优化丁香酚异源合成途径,改造底盘菌株,首次实现了微生物从头合成丁香酚,具有重要应用前景.
Eugenol is a green biological pesticide that is mainly used to control agricultural diseases,such as tomato gray mold,grape downy mildew,and potato late blight.Since eugenol has been extracted only from cloves,basil,and other plants so far,its production rate is poor and is unfriendly to the environment in terms of waste residue discharge.In this study,in order to synthesize eugenol de novo using microorganisms,synthetic biology and modular engineering strategies were used for designing,constructing,and engineering Escherichia coli.The eugenol heterogeneous synthesis pathway was divided into three modules,namely the upstream module for ferulic acid synthesis,midstream module for coniferyl alcohol synthesis,and downstream module for eugenol synthesis.Combinatorial screening of enzymes from different sources in these modules and expression optimization of each module were performed.By using His-tag and knocking out the global regulatory gene metJ,the upstream module(PcTAL-HpaBC-AtCOMT)was optimized to reduce the accumulation of intermediate caffeic acid and increase the synthesis of ferulic acid.4CL and alcohol dehydrogenase genes were screened,and their expression patterns were studied.The optimal midstream module was the one having a bicistronic expression of At4CL1-AtCCR1 and monocistronic expression of YahK,which maximized the yield of coniferyl alcohol.Combinatorial screening of three acyltransferase genes and four eugenol synthase genes resulted in the most eugenol synthesis,with monocistronic PhCFAT and GdlEGS2. The eugenol synthesis pathway was expressed in a tyrosine-over-producing chassis. By optimizing the initial concentration of glucose,yeast extract,and inducer,the eugenol yield reached 114.67mg/L within a suitable medium and fermentation conditions. By modularly optimizing the eugenol heterogeneous synthesis pathway and transforming the chassis strains,the first de novo synthesis of eugenol by microorganisms was realized herein with a promising application prospect.
作者
赵广荣
曹嘉誉
马雅婷
邱泽天
李嘉
Zhao Guangrong;Cao Jiayu;Ma Yating;Qiu Zetian;Li Jia(School of Chemical Engineering and Technology,Tianjin University,Tianjin 300350,China;Frontier Science Center for Synthetic Biology and Key Laboratory of System Bioengineering(Ministry of Education),Tianjin 300350,China)
出处
《天津大学学报（自然科学与工程技术版）》
EI
CAS
CSCD
北大核心
2022年第7期728-736,共9页
Journal of Tianjin University：Science and Technology
基金
广东省重点领域研发计划资助项目(2020B0303070002)。
关键词
丁香酚
大肠杆菌
代谢工程
合成生物学
eugenol
Escherichia coli
metabolic engineering
synthetic biology
