Riboflavin (vitamin B2) is an essential nutrient required for human health, serving as the precursor to metabolic coenzymes FAD and FMN. Microbial fermentation using Bacillus subtilis has become the predominant industrial production method due to the organism’s rapid growth, high yield potential, and well-characterized genetics.
During riboflavin biosynthesis, GTP serves as a critical precursor supplied by the de novo purine biosynthesis pathway. However, this pathway is subject to stringent regulation through multiple mechanisms including the PurR repressor protein, a guanine-sensing riboswitch in the pur operon 5’-UTR, and feedback inhibition by purine nucleotides.
The research team identified NupG and PbuO as transporters responsible for purine and nucleoside transport. Through resting cell transformation assays and fermentation studies, they confirmed that NupG mediates guanine efflux while PbuO facilitates guanine influx. Strain BR-02, which overexpresses nupG, exhibited intracellular guanine levels of only 0.509 μmol/g DCW compared to 0.962 in the parental strain—a 47% reduction. This decrease relieved riboswitch-mediated repression of the pur operon, enhanced metabolic flux through the de novo purine biosynthesis pathway, and elevated GTP precursor availability.
The engineered strain achieved a riboflavin titer of 1,508.22 mg/L, representing a 15.3% increase over the original strain. Importantly, the modification maintained normal energy charge levels and had minimal impact on cell growth and glucose consumption.
The study provides a scientifically grounded and efficient engineering framework for regulating intracellular metabolite concentrations to mitigate feedback inhibition effects in microbial cell factories.
The work entitled “Enhancing riboflavin production in Bacillus subtilis via guanine transporter-mediated regulation of the purine biosynthesis pathway” was published on Systems Microbiology and Biomanufacturing (published on Mar. 30, 2026).
DOI: 10.1007/s43393-026-00452-w