Novel Mutations in PRPP Synthetase to Deregulate the Purine Pathway and Its Effect on Riboflavin Production in Bacillus Subtilis

Abstract

Riboflavin, also known as Vitamin B2, is a molecule critical for both human and animal health. Its biosynthesis is fed by two pathways, the pentose phosphate pathway and the tightly regulated purine pathway. In an effort to debottleneck riboflavin biosynthesis, this work reports on the characterization and attempted deregulation of the purine pathway through novel mutations designed in the allosteric site of PRPP synthetase in an industrially relevant riboflavin producing strain of B. subtilis. Here we identify a PRPP synthetase mutant, Q141S, that demonstrates increased resistance to inhibition by GDP with a 3 fold increase of the Ki over the WT enzyme along with a higher maximum enzyme rate (Q141S Vmax of 0.596 nmol∙min-1∙mL-1 vs WT Vmax of 0.322 nmol∙min-1∙mL-1 ) when assayed in vitro. When tested in vivo, strains overexpressing this mutant displayed higher levels of uracil and guanosine along with an unexpected buildup of the penultimate precursor molecules in riboflavin synthesis, DMRL and ORL. These molecules were observed on the order of 6 and 8 fold higher than the control strain on average.