利用蔗糖产尿苷二磷酸葡萄糖的大肠杆菌构建  

Construction of Escherichia coli producing uridine diphosphate glucose from sucrose

作　　者：姚睿 赵丽婷 陈磊 李由然 顾正华[1,3] 石贵阳 丁重阳[1,2,3] YAO Rui;ZHAO Liting;CHEN Lei;LI Youran;GU Zhenghua;SHI Guiyang;DING Zhongyang(National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;Key laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;Schoo1 of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学,粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [2]江南大学,糖化学与生物技术教育部重点实验室,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2023年第22期9-18,共10页Food and Fermentation Industries

基　　金：轻工业技术与工程国家一流学科计划项目(LITE2018-22)。

摘　　要：尿苷二磷酸葡萄糖(uridine diphosphate glucose,UDP-glucose)不仅是细胞壁合成的前体物质,也是多种糖供体的重要前体物质。代谢工程可赋予微生物利用廉价易获得的原料生产特定化学物质的能力。该研究采用代谢工程技术改造大肠杆菌(Escherichia coli)蔗糖利用和UDP-葡萄糖合成的代谢路径,在E.coli BL21(DE3)中以蔗糖为唯一碳源,在实现细胞生长的同时促进UDP-葡萄糖的生产。通过在E.coli BL21(DE3)中共表达蔗糖通透酶基因(cscB)、果糖激酶基因(cscK)、蔗糖合酶基因(susy),确定了高拷贝数质粒(pMD19-T)和启动子串联策略(P_(csc)-P_(T7))是合成UDP-葡萄糖的最优策略,成功构建了能够利用蔗糖产UDP-葡萄糖的工程菌株ESBK09。此外,为了进一步增加菌株的稳定性,将cscB基因整合到E.coli BL21(DE3)基因组中构建工程菌株EPT03,该菌株被证明能够利用蔗糖生产UDP-葡萄糖。构建具有蔗糖利用能力的工业菌株将促进蔗糖作为碳源的使用,有助于石油化工经济向生物经济的过渡,为今后利用UDP-葡萄糖生产葡萄糖基化生物分子提供通用性底盘微生物菌株,也为后续利用UDP-葡萄糖合成相关大分子物质的代谢网络的构建提供了关键的一环。Uridine diphosphate glucose(UDP-glucose) is not only a precursor for cell wall synthesis,but also an important precursor for many sugar donors.It provides a variety of sugar donors for the synthesis of oligosaccharides,polysaccharides,glycosides and other bioactive macromolecules,and realize the diversification of their structures.Metabolic engineering has the ability to produce specific chemicals from cheap and readily available raw materials.In this study,Escherichia coli BL21(DE3) with clear genetic background was selected as the starting strain,the metabolic pathway of sucrose utilization and UDP-glucose synthesis in E.coli was modified by metabolic engineering technology.E.coli BL21(DE3) used sucrose as the only carbon source in order to achieve one-step synthesis from sucrose to UDP-glucose in vivo,and the hydrolyzed product fructose can be consumed for cell growth at the same time.By introducing sucrose permease(CscB),fructokinase(CscK) from E.coli ATCC 13281 non-phosphoenolpyruvate-dependent phosphotransferase system(non-PEP-PTS system) and sucrose synthase(SuSy) from Nitrosospira multiformis,a new pathway of UDP-glucose synthesis using sucrose as substrate was designed and constructed.On this basis,with the help of systematic metabolic engineering strategies,high copy number plasmid(pMD19-T) and promoter tandem strategy(P_(csc)-P_(T7)) were identified as the optimal strategy for UDP-glucose synthesis,the engineering strain ESBK09 which can produce UDP-glucose from sucrose was constructed successfully.In addition,in order to further increase the stability of the strain,the cscB gene was integrated into the genome of E.coli BL21(DE3) to construct the engineered strain EPT03,the recombinant strain EPT03 grew well in sucrose medium,with an OD_(600) of about 4.8 and a sucrose consumption rate of about 70%.The strain was shown to be able to produce UDP-glucose from sucrose.The utilization of sucrose is achieved through chromosome integration and plasmid construction,so that the E.coli strains unable to use suc

关 键 词：代谢工程 大肠杆菌 UDP-葡萄糖 蔗糖合酶 从头合成途径 

分 类 号：TS201.3[轻工技术与工程—食品科学]