Abstract: In this study, the de novo synthesis pathway of 2’-FL was established in Escherichia coli BL21star(DE3). The β-galactosidase gene (lacZ) and the UDP-glucose lipid carrier transferase gene (wcaJ) were knocked out by the CRISPR/Cas9 system. The effects of various exogenous α-1,2-fucosyltransferases on 2’-FL synthesis were investigated. Subsequently, the transcriptional levels of genes involved in the synthesis pathway were regulated, and the fermentation conditions were optimized. The results showed that after fermentation for 72 h, a 2’-FL concentration of 0.34 g/L was obtained in shake flasks with overexpression of the de novo synthesis pathway genes in E. coli BL21star (DE3), and the concentration of 2’-FL was increased to 2.12 g/L by knockout of the lacZ and wcaJ genes. Expression of α-1,2-fucosyltransferase Wcfb from Bacteroides fragilis in shake flasks resulted in the highest concentration of 4.12 g/L. Under the optimized fermentation conditions: 28 ℃ and 0.2 mmol/L final isopropyl-β-D-thiogalactoside (IPTG), the 2’-FL concentration was increased to 5.01 and 31.2 g/L in a shake flask and in a 5 L fermenter under fed-batch condition, respectively.

Key words: 2’-fucosyllactose; CRISPR/Cas9; α-1,2-fucosyltransferase; biosynthesis; optimization of fermentation conditions