Enhancing riboflavin production in Bacillus subtilis via guanine transporter-mediated regulation of the purine biosynthesis pathway

Abstract

During riboflavin biosynthesis in Bacillus subtilis, GTP acts as a precursor supplied by the purine biosynthesis pathway. This study revealed that GTP biosynthesis is tightly regulated by intracellular guanine levels via a guanine-sensing riboswitch, limiting the final riboflavin yield. Using resting cell transformation assays, we confirmed that the transporters NupG and PbuO mediate guanine efflux and influx, respectively. Overexpression of nupG in the original strain BR reduced intracellular guanine levels, resulting in enhanced riboflavin production. These findings indicate that NupG-mediated intracellular guanine efflux could deregulate riboswitch-induced repression of the pur operon. This deregulation increased metabolic flux through the de novo purine biosynthesis pathway and elevated the synthesis of precursor GTP. As a result, engineered strain BR-02 achieved a riboflavin titer of 1,508.22 mg/L, a 15.3% increase over the original strain BR. This study highlights that transporter engineering is an effective metabolic engineering strategy to overcome purine feedback repression in riboflavin biomanufacturing, offering a new rationale for designing microbial cell factories.

Graphical abstract

Keywords

Bacillus subtilis / Riboflavin / Feedback repression / Metabolic engineering

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Jiangnan University