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

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.