Background: 2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide (HMO) in human milk and has important physiological functions. The market demand of 2'-FL is continuing to grow, but high production cost has limited its availability. To solve the dilemma, biosynthesis of 2'-FL has been proposed and is considered the most promising pathway for massive production. α-1,2-Fucosyltransferase is one of the key elements involved in its biosynthesis, but the limited intracellular accumulation and unstable properties of α-1,2-fucosyltransferases when expressed in host strains have become a major hurdle for the effective biosynthesis of 2'-FL.
Results: A combinatorial engineering strategy of synergic modification of ribosome binding site, fusion peptide and enzyme gene was leveraged to enhance the soluble expression of α-1,2-fucosyltransferases and promote enzyme activity. The preferable combination was to employ an optimized ribosome binding site region to drive 3 × FLAG as a fusion partner along with the α-1,2-fucosyltransferase for expression in Escherichia coli (DE3) PlySs, and protein yield and enzyme activity were remarkably improved by 11.51-fold and 13.72-fold, respectively.
Conclusion: After finely tuning the synergy among different elements, the abundant protein yield and high enzyme activity confirmed that the drawbacks of heterologous expression in α-1,2-fucosyltransferase had been properly addressed. A suitable external environment further drives the efficient synthesis of α-1,2-fucosyltransferases. To our knowledge, this is the first report of a systematic and effective modification of α-1,2-fucosyltransferase expression, which could potentially serve as a guideline for industrial application. © 2022 Society of Chemical Industry.
Keywords: biocatalysis; heterologous expression; protein engineering; α-1,2-fucosyltransferase.
© 2022 Society of Chemical Industry.