Combinatorial metabolic engineering and tolerance evolving of Escherichia coli for high production of 2′-fucosyllactose

2 '-Fucosyllactose (2 '-FL) is an important functional ingredient of advanced infant formula. Here, Escherichia coli MG1655 was engineered for achieving high 2 '-FL production. The expressions of 2 '-FL synthesis pathway genes were finely regulated with single or multi copies according to rate-limiting enzyme diagnosis. On this basic, the branch pathway genes were deleted, and the overexpression of the 2 '-FL efflux protein SetA and the fructose-1,6-bisphosphatase GlpX were tuned. The resulting strain produced 46.06 +/- 1.28 g/L 2 '-FL in a 5-L fermenter. Furtherly, adaptive laboratory evolution was conducted. A rpoC gene mutation was obtained which could improve the cell tolerance and the 2 '-FL production up to 61.06 +/- 1.93 g/L, with the highest productivity of 1.70 g/L/h among E. coli strains by now. Taken together, this work provides a combinatorial strategy to improve 2 '-FL accumulation including rational fine-tuning pathway genes expressions and irrational adaptive laboratory evo-lution. This study should be helpful for constructing high level 2 '-FL producers.

Automated summary

In this study, E. coli MG1655 was engineered to achieve high production of 2'-FL. The expression of 2'-FL synthesis pathway genes was finely regulated and branch pathway genes were deleted. The resulting strain produced 46.06 g/L of 2'-FL in a 5-L fermenter. Through adaptive laboratory evolution, a mutation in the rpoC gene was obtained, leading to improved cell tolerance and 2'-FL production of up to 61.06 g/L. This work provides a strategy for improving 2'-FL accumulation and has important implications for constructing high-level 2'-FL producers in the future.