Direct evolution of riboflavin kinase significantly enhance flavin mononucleotide synthesis by design and optimization of flavin mononucleotide riboswitch

Flavin mononucleotide (FMN) is the active form of riboflavin. It has a wide range of application scenarios in the pharmaceutical and food additives. However, there are limitations in selecting generic high-throughput screening platforms that improve the properties of enzymes. First, the biosensor in response to FMN concentration was constructed using the FMN riboswitch and confirmed the function of this sensor. Next, the FMN binding site of the sensor was saturated with a mutation that increased its fluorescence range by approximately 127%. Then, the biosensor and the base editing system based on T7RNAP were combined to construct a platform for rapid mutation and screening of riboflavin kinase gene ribC mutants. The mutants screened using this platform increased the yield of FMN by 8-fold. These results indicate that the high-throughput screening platform can rapidly and effectively improve the activity of target enzymes, and provide a new route for screening industrial enzymes.