基于耦合发酵策略的乳酰-N-三糖Ⅱ模块化合成研究  

Modular synthesis of lacto-N-trioseⅡbased on coupling fermentation strategy

作　　者：黎玉 李忠霞 邓明超 王志杰 周文 Wengang CHAI 张洪涛[1] LI Yu;LI Zhongxia;DENG Mingchao;WANG Zhijie;ZHOU Wen;Wengang CHAI;ZHANG Hongtao(Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China;BYHEALTH Institute of Nutrition&Health,Guangzhou 510000,China;Glycosciences Laboratory,Faculty of Medicine Imperial College London,Harrow HA13UJ,U.K)

机构地区：[1]糖化学与生物技术教育部重点实验室,江南大学生物工程学院,江苏无锡214122 [2]汤臣倍健营养健康研究院,广东广州510000 [3]Glycoscience Laboratory,Faculty of Medicine Imperial,College London,Harrow HA13UJ,U.K

出　　处：《食品与发酵工业》2024年第15期16-24,共9页Food and Fermentation Industries

基　　金：汤臣倍健营养科学研究基金开放课题项目(TY202101096);解放军后勤保障部开放课题项目(ZZCWS21J2001);国家博士后特别资助项目(2014T70472);国家博士后基金项目(2012M20996)。

摘　　要：母乳寡糖在调节婴儿肠道菌群和保障婴儿健康方面都有积极的作用。乳酰-N-三糖Ⅱ(lacto-N-trioseⅡ,LNTⅡ)是母乳寡糖的核心结构单元。目前合成LNTⅡ的方法存在价格昂贵、操作繁琐等缺点。该研究以乳糖和N-乙酰氨基葡萄糖为底物用三菌株耦合发酵策略合成LNTⅡ。首先构建用于合成N-乙酰氨基葡萄糖-1-磷酸(N-acetylglucosamine-1-phosphate,GlcNAc-1-P)模块的Escherichia coli JM109(DE3)/pET28a-nahK工程菌,优化发酵条件后,GlcNAc-1-P最高产量为16.88 g/L,转化率为70.05%。然后构建分别包含agx1、agx2、pmglmU基因的3株工程菌,并筛选出E.coli JM109(DE3)/pET28a-agx1,其合成尿苷二磷酸乙酰氨基葡萄糖(uridine diphosphate acetylglucosamine,UDP-GlcNAc)的能力最优,在优化的生产体系中UDP-GlcNAc的最大产量为24.78 g/L。最后构建工程菌E.coli JM109(DE3)/pET28a-lgtA并与E.coli JM109(DE3)/pET28a-nahK、E.coli JM109(DE3)/pET28a-agx1耦合发酵合成LNTⅡ,通过优化三菌株耦合发酵体系,LNTⅡ产量达到3.03 g/L,比优化前提高近4倍。该文基于三菌株耦合发酵策略实现了LNTⅡ的合成,这种策略具有通用性强、简便快捷和成本低廉的优点,将为母乳寡糖的规模化生产提供一种新方法。Human milk oligosaccharides(HMOs)play an important role in regulating intestinal flora and protecting infant health.Lacto-N-trioseⅡ(LNTⅡ)is the core structural unit of HMOs.Hitherto,the developing methods for LNTⅡsynthesis are expensive and complicated.In this work,lactose and N-acetylglucosamine were used as substrate to synthesize LNTⅡby the three-strain coupling fermentation strategy.In order to synthesize N-acetylglucosamine-1-phosphate(GlcNAc-1-P),the engineering bacteria Escherichia coli JM109(DE3)/pET28a-nahK was constructed,the highest yield of GlcNAc-1-P was 16.88 g/L and the conversion rate was 70.05%using optimizing the fermentation conditions.Then three engineering strains containing agx1,agx2,and pmglmU genes were constructed,respectively.The selected E.coli JM109(DE3)/pET28a-agx1 had the highest ability to synthesize uridine diphosphate acetylglucosamine(UDP-GlcNAc),the maximum yield of UDP-GlcNAc was 24.78 g/L with the optimized fermentation system.The engineering strain E.coli JM109(DE3)/pET28a-lgtA was constructed and coupled with E.coli JM109(DE3)/pET28a-nahK and E.coli JM109(DE3)/pET28a-agx1 for producing LNTⅡ.The final optimized yield of LNTⅡreached to 3.03 g/L,which was nearly 4 times higher than that before optimization.Based on the three-strain coupling fermentation strategy,this project realized the synthesis of LNTⅡThis strategy has the advantages of strong versatility,simplicity and low cost,which will provide a new method for the large-scale production of HMOs.

关 键 词：LNTⅡ 耦合发酵 母乳寡糖 UDP-GLCNAC GlcNAc-1-P 

分 类 号：TS201.2[轻工技术与工程—食品科学] TS205.5[轻工技术与工程—食品科学与工程]