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

Abstract:

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 NZ9000 6 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 2.5 μg/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.

Key words: 2'-fucosyllactose, human milk oligosaccharides, Lactococcus lactis, metabolic engineering, microbial whole-cell synthesis, homologous recombination, fermentation optimization