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-ribF(5) 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-ribF(5) and anti-guaC(3) 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-ribF(5) and anti-guaC(3) 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.

Automated summary

This study successfully enhanced riboflavin production in Escherichia coli by fine-tuning the expression levels of ribF, purA, and guaC genes using synthetic regulatory small RNAs. The best strain achieved the highest yield of riboflavin by optimizing promoters and co-expressing different sRNAs, leading to a significant increase in riboflavin titer compared to the base strain. Overall, this is the first study to simultaneously regulate multiple genes to enhance riboflavin synthesis, resulting in a record-high yield among reported strains.