Enhanced biosynthesis of 2’-Fucosyllactose in Escherichia coli through α-1,2-fucosyltransferase mining and pathway optimization

Abstract

2’-Fucosyllactose (2’-FL), the most abundant human milk oligosaccharides (HMOs), plays crucial biological roles in regulation of intestinal microbiota, inhibition of pathogen adhesion, and immune enhancement. It holds considerable market potential in the pharmaceutical and food industries. Compared to chemical route, microbial synthesis using engineered strains, such as Escherichia coli, offers a more economical and efficient production route and currently represents the industrial standard for 2’-FL manufacturing. Notably, α-1,2-fucosyltransferase (FucT2) serves as the key rate-limiting enzyme in the 2’-FL biosynthetic pathway. Therefore, enhancing FucT2 activity is essential for improving 2’-FL titer. In this study, we explored and characterized an efficient FucT2 from Helicobacter himalayensis (H.him-FucT2), which demonstrated high catalytic activity in the synthesis of 2’-FL. Comparative analysis confirmed that H.him-FucT2 exhibits superior activity to FucT2 from Helicobacter pylori. Upon expression in an engineered Escherichia coli capable of de novo 2’-FL biosynthesis, the initial titer of 2’-FL reached 0.22 g/L. To further enhance the titer of 2’-FL, we adopted a dual-module bypass gene knockout strategy to redirect metabolic flux. Specifically, metabolic bypass genes (pfkA, pfkB, nudD, nudK, lacZ, and wcaJ) were systematically knocked out using the CRISPR/Cas9 system. Subsequently, through the composite application of global regulation and fermentation condition optimization, the titer of 2’-FL was further increased by 13.7 times, which reached 11.02 g/L with fed-batch fermentation.

Keywords

Human milk oligosaccharides / 2’-Fucosyllactose / α-1,2-fucosyltransferase / Escherichia coli

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Funding

the National Key Research and Development Program of China(2021YFC2102000)

the Postgraduate Research & Practice Innovation Program of Jiangsu Province(KYCX22_2376)

RIGHTS & PERMISSIONS

Jiangnan University