工程菌-酵母耦合高密度发酵生产乳糖-N-二糖  

Production of lacto-N-biose by coupled high-density fermentation with engineered strain-yeast

作　　者：李竹英 张洪涛 李嘉怡 张子梦 韩成 许淳 LI Zhuying;ZHANG Hongtao;LI Jiayi;ZHANG Zimeng;HAN Cheng;XU Chun(Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与发酵工业》2025年第6期120-126,共7页Food and Fermentation Industries

基　　金：国家博士后特别资助项目(2014T70472);中国博士后科学基金项目(2012M20996);江苏省博士后基金项目(1301011B);解放军后勤保障部开放课题项目(ZZCWS21J2001)。

摘　　要：乳糖-N-二糖(lacto-N-biose,LNB)是母乳寡糖(human milk oligosaccharides,HMOs)的主要组成部分,也是双歧因子候选者之一,但是目前缺少经济的大规模合成技术。该研究利用蔗糖磷酸化酶和LNB磷酸化酶的非依赖能荷再生优势,以酿酒酵母为能源供给细胞,建立了双菌共培养高效合成LNB的方法。首先构建了E.coli JM109(DE3)/pET28a-GalT1、E.coli JM109(DE3)/pET28a-GalT2、E.coli JM109(DE3)/pET28a-BLUSP的工程菌,筛选出合成二磷酸尿苷葡萄糖(uridine diphosphate glucose,UDP-Glc)产量高达10.5 g/L的最优菌株E.coli JM109(DE3)/pET28a-GalT1。然后与构建的工程菌E.coli JM109(DE3)/pET28a-SPase、E.coli JM109(DE3)/pET28a-GalE和E.coli JM109(DE3)/pET28a-BiGalHexNAcP四菌耦合发酵,LNB产量为6.78 g/L。最后构建了共表达菌株E.coli JM109(DE3)(含有质粒pRSFDuet1-SPase-GalT1和pETDuet1-GalE-BiGalHexNAcP),外源添加辅因子尿苷一磷酸(uridine monophosphate,UMP)(浓度为20 mmol/L),将其与酵母耦合发酵,LNB最高产量为28.66 g/L,与四菌株耦合发酵相比,产量提高了323%。这是目前关于LNB产量最高的报道。研究结果为低成本生产乳糖-N-二糖提供了新的技术路线。Lacto-N-biose(LNB)is a major component of human milk oligosaccharides(HMOs)and one of the candidates for bifidus factors.However,there is a lack of economical large-scale synthesis technology.This study took advantage of the non-dependent energy-charge regeneration of sucrose phosphorylase and LNB phosphorylase,and established a dual-strain co-culture method for efficient LNB synthesis using Saccharomyces cerevisiae as the energy supply cell.Firstly,the engineered strains E.coli JM109(DE3)/pET28a-GalT1,E.coli JM109(DE3)/pET28a-GalT2 and E.coli JM109(DE3)/pET28a-BLUSP were constructed,and the optimal strain E.coli JM109(DE3)/ET28a-GalT1 with a yield up to 10.5 g/L of UDP-Glc was screened.Then it was coupled fermentation with the constructed engineered strains E.coli JM109(DE3)/pET28a-SPase,E.coli JM109(DE3)/pET28a-GalE and E.coli JM109(DE3)/pET28a-BiGalHexNAcP,and the LNB yield was 6.78 g/L.Finally,the co-expression strain E.coli JM109(DE3)(containing plasmids pRSFDuet1-SPase-GalT1 and pETDuet1-GalE-BiGalHexNAcP)was constructed with exogenous addition of cofactor UMP(at a concentration of 20 mmol/L)and coupled with yeast to fermentation,and the highest LNB yield was 28.66 g/L,which increased the yield by 323%compared to the four-strain coupled fermentation.This is the highest reported yield of LNB to date.The results of the study provide a new technological route for the low-cost production of LNB.

关 键 词：乳糖-N-二糖 蔗糖 蔗糖磷酸化酶 耦合发酵 母乳寡糖 

分 类 号：TS201.3[轻工技术与工程—食品科学]