Rational Engineering of Escherichia coli for High-Level Production of Riboflavin.

Riboflavin is widely used as a food additive. Here, multiple strategies were used to increase riboflavin production in Escherichia coli LS31T. First, purR deletion and co-overexpression of fbp, purF, prs, gmk, and ndk genes resulted in an increase of 18.6% in riboflavin titer (reaching 729.7 mg/L). Second, optimization of reduced nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide ratio and respiratory chain activity in LS31T increased the titer up to 1020.2 mg/L. Third, the expression level of the guaC gene in LS31T was downregulated by ribosome binding site replacement, and the riboflavin production was increased by 10.6% to 658.5 mg/L. Then, all the favorable modifications were integrated together, and the resulting strain LS72T produced 1339 mg/L of riboflavin. Moreover, the riboflavin titer of LS72T reached 21 g/L in fed-batch cultivation, with a yield of 110 mg riboflavin/g glucose. To our knowledge, both the riboflavin titer and yield obtained in fed-batch fermentation are the highest ones among all the rationally engineered strains.