Site-directed mutagenesis of bifunctional riboflavin kinase/FMN adenylyltransferase via CRISPR/Cas9 to enhance riboflavin production.

ABSTRACT: Vitamin B₂ is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D,

SUBMITTER: Fu B 

PROVIDER: S-EPMC11047187 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature