Improving riboflavin production by knocking down ribF, purA and guaC genes using synthetic regulatory small RNA

Riboflavin is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. The down-regulation of expression levels of ribF, purA and guaC genes involved in the downstream or branch reactions of riboflavin biosynthesis pathway could direct more carbon flux to riboflavin accumulation. In this study, we made an attempt to fine-tune the expression levels of the 3 genes by using synthetic regulatory small RNA to enhance riboflavin production in Escherichia coli. Firstly, each of the 3 genes was knocking down by using 5 different sRNAs, respectively, and a highest increase of 50.2 % in riboflavin titer was achieved by using anti-ribF₅ sRNA. Then this sRNA was further co-expressed with 5 anti-purA and 5 anti-guaC sRNAs to simultaneously knocking down 2 or 3 genes. Co-expression of anti-ribF₅ and anti-guaC₃ led to the highest riboflavin production of 1091.3 mg/L, which was further increased by 97.6 % compared to the base strain. Finally, the expression levels of anti-ribF₅ and anti-guaC₃ were further fine-tuned by using 4 different promoters. The best strain WY40, in which the two sRNAs were respectively expressed by P_J23100 and P_J23107 promoter, produced 1454.5 mg/L riboflavin with an increase of 163.4 % compared to the base strain. To our knowledge, it's the first study to enhance riboflavin synthesis by simultaneously regulating the expression levels of ribF, purA and guaC genes, which led to a highest yield of 0.147 g/g glucose among all reported riboflavin-producing strains.