乳酸乳球菌生产2'-岩藻糖基乳糖的途径构建及发酵培养基优化  

Synthetic Pathway Construction of Producing 2'-fucosyllactose by Lactococcus lactis and Optimization of Fermentation Medium

作　　者：程亚楠 张文聪 周圆[2] 孙雪 李玉[1] 李庆刚 CHENG Ya-nan;ZHANG Wen-cong;ZHOU Yuan;SUN Xue;LI Yu;LI Qing-gang(College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457;College of Pearl River,Tianjin University of Finance and Economics,Tianjin 301811)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津财经大学珠江学院,天津301811

出　　处：《生物技术通报》2023年第9期84-96,共13页Biotechnology Bulletin

基　　金：国家自然科学基金面上项目(31871741)。

摘　　要：2’-岩藻糖基乳糖(2’-fucosyllactose, 2’-FL)是一种含量最丰富的人乳寡糖,微生物全细胞合成是生产2’-FL的重要方法。乳酸乳球菌(Lactococcus lactis)作为一种可直接用于乳制品的食品级微生物,目前还没有用于生产人乳寡糖的报道。首先,在两种常用L. lactis底盘NZ3900和NZ9000中利用含有基因fkp、futC、lacF的重组质粒pNZ8148-2f构建2’-FL补救合成途径,在添加10 g/L岩藻糖和5 g/L乳糖的发酵培养基中,测得2’-FL的摇瓶产量分别为0.16 g/L与0.4 g/L,结合对二者生长状况等综合分析,选取NZ9000作为优势底盘。然后,在NZ9000中构建2’-FL从头合成途径,利用同源重组技术并借助L. lactis中特有的敲除整合载体p NZ5319,在敲除非必需基因upp的同时将基因manA、manB、gmd和wcaG整合到染色体上,借助载体pNZ8148-1将基因manC、futC和lacF以质粒的形式引入到细胞中,同时利用不同启动子P32、Pnis对酶的表达水平进行组合调控。获得最优菌株NZ9000 6,在添加20 g/L葡萄糖和5 g/L乳糖的发酵培养基中,测得2’-FL的摇瓶产量为0.28 g/L。最后,在发酵培养基中对氯化高铁血红素、胰蛋白胨和酵母粉的浓度进行了优化,在添加2.5μg/mL氯化高铁血红素、15 g/L胰蛋白胨、6 g/L酵母粉时2’-FL的摇瓶产量达到1.58 g/L,为相关报道的较高水平。该研究证实了L. lactis合成2’-FL的可行性,为2’-FL的生产提供了新思路。2'-fucosyllactose(2'-FL)is the most abundant human milk oligosaccharide.Microbial fermentation is an important method to produce 2'-FL.Lactococcus lactis(L.lactis),a food safety grade strain,can be directly used in dairy products,has not been reported for the production of human milk oligosaccharides.First,the 2'-FL salvage synthesis pathway was constructed by using a recombinant plasmid pNZ8148-2f containing gene fkp,futC,lacF in two usual L.lactis chassis NZ3900 and NZ9000.In the fermentation medium with 10 g/L fucose and 5 g/L lactose,the yields of 2'-FL in flasks were 0.16 g/L and 0.4 g/L,respectively.Combined with the growth assay,NZ9000 was considered as a better chassis.Second,2'-FL de novo synthesis pathway was constructed in NZ9000,i.e.,using homologous recombination technology and the unique knockout integration vector pNZ5319 in L.lactis,integrating the gene manA,manB,gmd and wcaG to the chromosome while knocking out unnecessary gene upp,and introducing the gene manC,futC and lacF to the cell by vector pNZ8148-1.Meanwhile,different promoter P32 and Pnis were used to optimize the expressions of the pathway enzymes.The best strain NZ90006 was obtained with a 2'-FL production of 0.28 g/L in flask fermentation medium with 20 g/L glucose and 5 g/L lactose.Finally,the concentrations of hemin,tryptone and yeast extract were optimized in the fermentation medium.With the addition of 2.5μg/mL methemoferin chloride,15 g/L tryptone,and 6 g/L yeast extract in the fermentation medium,the final 2'-FL production reached 1.58 g/L which was relatively high among the reported results.Thus,the feasibility of 2'-FL synthesis by L.lactis was proved,which provides a new idea for the production of 2'-FL.

关 键 词：2’-岩藻糖基乳糖 人乳寡糖 乳酸乳球菌 代谢工程 微生物全细胞合成 同源重组 发酵优化 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]