Abstract: As a kind of prebiotics, 2'-fucosyllactose (2'-FL) can be added to infant formula, and is essential for the development of the intestinal flora and immune system of infants. The synthetic pathway of 2'-FL was established by heterologously co-expressing L-fucose kinase/GDP-L-fucose pyrophosphorylase gene fkp and α-1,2 fucosyltransferase gene futC in Escherichia coli BL21star (DE3), which were derived from Bacteroides fragilis and Helicobacter pylori respectively.Secondly, the β-galactosidase and UDP-glucose lipid carrier transferase genes lacZ and wcaJ were deleted through the CRISPR/Cas9 system to block the decomposition of intermediate metabolites and explore the influence of these genes on 2'-FL synthesis. The fermentation results showed that single deletion of lacZ gene promoted the yield of 2'-FL by 1.88 times, which had no significant effect on cell growth. After double mutation of genes lacZ and wcaJ, the yield of 2'-FL increased by 4.89 times, and the cell growth was not affected significantly. Under optimized shake flask fermentation, the fermentation conditions were determined as follows: a defined medium was used, the induction concentration of IPTG was 0.2 mmol/L, the induction temperature was 25 ℃, and the inoculation amount was 5%. Under these conditions, the yield of 2'-FL reached 1.44 g/L. This study confirmed that deletion of β-galactosidase and UDP-glucose lipid carrier transferase genes could significantly increase the yield of 2'-FL, which provides a reference for expanding its industrial production.
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