通过解除磷酸糖胁迫提高枯草芽孢杆菌N-乙酰氨基葡萄糖合成能力  

Improving N-acetylglucosamine synthesis ability of Bacillus subtilis by relieving phosphosugar stress

作　　者：房峻 黄子洋 刘延峰 李江华 堵国成 吕雪芹 刘龙 FANG Jun;HUANG Ziyang;LIU Yanfeng;LI Jianghua;DU Guocheng;LYU Xueqin;LIU Long(Key Laboratory of Carbohydrate Chemistry and Biotechnology,Jiangnan University,Wuxi 214122,China;Science Center for Future Foods,Jiangnan University,Wuxi 214122,China)

机构地区：[1]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 [2]江南大学,未来食品科学中心,江苏无锡214122

出　　处：《食品与发酵工业》2023年第6期27-34,共8页Food and Fermentation Industries

基　　金：国家重点研发计划(2018YFA0900504);国家自然基金委创新群体项目(32021005)。

摘　　要：N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)在食品、保健品以及药品领域有着广泛的应用。该课题组前期构建了一株具有较高GlcNAc合成效率的枯草芽孢杆菌底盘细胞FMIK,由于GlcNAc前体的过量积累引起了磷酸糖胁迫,限制了FMIK的GlcNAc产量。因此,该文首先通过实施荧光定量PCR验证磷酸糖胁迫对于glcR-ywpJ操纵子转录水平影响,并通过凝胶迁移率确定GlcR与P_(glcR)的结合能力;然后,利用易错PCR构建P_(glcR-ep)突变体库,设计并使用基于荧光检测的高通量筛选系统,鉴定得到P_(glcR)与GlcR结合的关键区域;最后,在FMIK菌株的基础上对筛选得到的P_(glcR)与GlcR的结合关键区域进行敲除,得到工程菌株FMIK-m4。该菌株有效降低了GlcNAc前体浓度,解除了磷酸糖胁迫导致的二次生长现象,且摇瓶内GlcNAc产量提高至18.21 g/L。N-acetylglucosamine(GlcNAc)is widely used in the fields of food,nutrition and drugs.In previous studies,our team constructed a chassis cell of Bacillus subtilis with high synthesis efficiency of GlcNAc through metabolic regulation.However,the excessive accumulation of GlcNAc precursor caused phosphosugar stress,which limited the yield of GlcNAc.In this study,the effect of phosphate stress on the transcription level of glcR-ywpJ operon was verified by qRT-PCR;the binding ability between GlcR and P_(glcR)was determined by EMSA.Then,a P_(glcR-ep)mutant library was constructed by error prone PCR,and a high-throughput screening system based on fluorescence detection was designed to identify the key regions of P_(glcR)binding to GlcR.Finally,on the basis of FMIK strain,the key binding regions between P_(glcR)and GlcR were knocked out,and the engineering strain FMIK-m4 was obtained.This strain effectively reduced the concentration of GlcNAc precursor,relieved the secondary growth phenomenon caused by phosphate sugar stress,and increased the yield of GlcNAc in the shake flask to 18.21 g/L.

关 键 词：枯草芽孢杆菌 N-乙酰氨基葡萄糖 磷酸糖胁迫 转录因子 磷酸酶 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]