Deregulation of Purine Pathway in Bacillus Subtilis and Its Use in Riboflavin Biosynthesis

Purine nucleotides are essential metabolites for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and biosynthesis of several amino acids and riboflavin. As a tightly regulated pathway, purine biosynthesis is regulated at transcriptional and metabolic levels at different steps of the pathway. It seems reasonable to redirect metabolic flux toward purine biosynthesis by deregulation of purine pathway, which may improve GTP supply and riboflavin production. To eliminate transcriptional repression, the pur operon repressor PurR and the 5’-UTR of the operon containing the guanine riboswitch were disrupted. Quantitative RT-PCR analysis revealed that the relative transcription levels of purine genes were up-regulated to a great extent. Furthermore, site-directed mutagenesis was successfully introduced into PRPP amidotransferase (encoded by purF) to remove feedback inhibition by intracellular metabolites based on homologous alignment and analysis. Overexpression of mutant purF (aspartic acid²⁹³, lysine³¹⁶, and serine⁴⁰⁰) significantly increased its activity and triggered a strong refractory effect of PRPP amidotransferase on purine nucleotides mediated inhibition. And the nucleotides ATP and GTP exerted a marked stimulatory effect on in vitro PRPP amidotransferase activity at the lower purine nucleotide concentrations assayed. Consequently, the engineered strains had also increased intracellular metabolite concentrations from purine pathway. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to enhance the production of riboflavin in the triple mutant strain 4-fold (826.52 mg/L). Based on the deregulation of purine pathway by eliminating transcriptional repression and feedback inhibition, an extended application was recommended for the yield of products directly stemming from purine pathway in B. subtilis.