Riboflavin (RF) and its active forms, the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), have been extensively used in the food, feed and pharmaceutical industries. Modern commercial production of riboflavin is based on microbial fermentation, but the established genetically engineered production strains are facing new challenges due to safety concerns in the food and feed additives industry. High yields of flavin mononucleotide and flavin adenine dinucleotide have been obtained using whole-cell biocatalysis processes. However, the necessity of adding expensive precursors results in high production costs. Consequently, developing microbial cell factories that are capable of efficiently producing flavin nucleotides at low cost is an increasingly attractive approach. The biotechnological processes for the production of RF and its cognate cofactors are reviewed in this article.

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.