Modular Engineering of the Flavin Pathway in Escherichia coli for Improved Flavin Mononucleotide and Flavin Adenine Dinucleotide Production.

In this work, modular engineering of Escherichia coli was peformed to improve flavin production and the conversion ratio of riboflavin (RF) to FMN/FAD. The RF operon and the bifunctional RF kinase/FAD synthetase were divided into two separate modules. The two modules were expressed at different levels to produce RF: ribF ratios ranging from 2:20 to 7:5. The best strain respectively produced 324.1 and 171.6 mg/L of FAD and FMN in shake flask fermentation, and the titers reached 1899.3 and 872.7 mg/L in a fed-batch process. Furthermore, error-prone PCR (epPCR) of the E. coli ribF gene was performed. The highest FMN production of the best mutant reached 586.1 mg/L in shake flask cultivation. Moreover, this mutant produced 1017.5 mg/L FMN with a greatly reduced proportion of FAD in fermenter culture. To the best of our knowledge, this is the highest production of FAD and FMN in a microbial fermentation process reported to date.