Construction of a plasmid-free Escherichia coli strain for lacto-N-neotetraose biosynthesis

Abstract

Lacto-N-neotetraose (LNnT), with promising bioactive properties, is one of the most significant nonfucosylated human milk saccharides. Recently, its synthesis by microbial fermentation has attracted great attention. However, most recent studies have focused on the use of plasmid for high-level LNnT production, which is undesirable for industrial applications. Therefore, this study aimed to construct a plasmid-free recombinant strain to biosynthesize LNnT. First, a de novo synthesis pathway for LNnT was constructed in Escherichia coli. Then, the ribosome binding site (RBS) and expression pattern of β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactosyltransferase (LgtB) were optimized. Different fusion peptides and enzyme assembly scaffolds were also verified for LNnT enhancement, which resulted in an LNnT titer of 1.70 g/L. Furthermore, a clustered regularly interspaced short palindromic repeat-mediated interference (CRISPRi) system was introduced to downregulate the competitive pathway, thereby enhancing the supply of two precursors (UDP-N-acetylglucosamine and UDP-galactose). Finally, a plasmid-free engineered strain was developed via genome integration of lgtA and lgtB coupled with enzyme assembly scaffolds, which produced 23.73 g/L of LNnT in a 3-L bioreactor. These findings provide insights into the plasmid-free recombinant E. coli strain construction for the efficient LNnT biosynthesis.

Keywords

Lacto-N-neotetraose / Escherichia coli / RBS optimization / Fusion protein / Enzyme assembly / CRISPRi

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Funding

Key Research and Development Program of China(2022YFC2104903); National Natural Science Foundation of China(31930085)