Improving the Production of Riboflavin by Introducing a Mutant Ribulose 5-Phosphate 3-Epimerase Gene in Bacillus Subtilis

Background: Ribulose 5-phosphate (Ru5P) and guanosine 5'-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP (purine de novo synthesis pathway). As a product of the oxidative pentose phosphate pathway (OPPP), Ru5P can flow back into the Embden-Meyerhof-Parnas pathway (EMP) through the non-oxidative pentose phosphate pathway (NOPPP). Major enzymes of the NOPPP include transaldolase (Tal), transketolase (Tkt), and ribulose 5-phosphate 3-epimerase (Rpe), which catalyze the conversion of Ru5P into xylulose 5-phosphate (X5P). Thus, inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. However, there are no studies on the mutation or deletion of Rpe for improving riboflavin production. Results: We mutated the genomic copy of rpe, in the riboflavin-producing Bacillus subtilis BSLY. The resulting strain BSR produced 320.50±31.28 mg/L of riboflavin in a fermentation medium containing 40 g/L sucrose as the carbon source, representing a 5-fold increase over the parental strain. We also constructed an rpe nonsense mutation strain BSRN, which produced 365.69±27.53 mg/L riboflavin in fermentation medium. However, BSRN had a growth defect compared with BSR and BSLY, which was also present in media containing glucose, maltose, and fructose. The growth of BSRN was restored when mutant or wild-type rpe was overexpressed. Notably, BSR exhibited respective increases of the inosine and guanine titers by 163% and 40%. Furthermore, a 35.96-fold increase of inosine 5’-monophosphate (IMP) titer was detected in BSRN. The transcription levels of most OPPP, purine, and GTP synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were respectively 49% lower and 310% higher than in BSLY. The production of riboflavin was increased to 479.90±33.21 mg/L when ribA was expressed in BSR. The expression of zwf, gntZ, prs, and purF, respectively enhanced the riboflavin production by 48%, 31%, 26%, and 35%. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29±63.44 mg/L).Conclusions: This paper describes a mutation of the ribulose 5-phosphate 3-epimerase and its influence on riboflavin production. The results of qRT-PCR and HPLC analyses indicated that the rpe mutant showed a different pattern of purine metabolism, while the cells maintained generally normal levels of the transcription of genes related to PPP and purine de novel synthesis pathways. With the increased expression of genes in the OPPP, purine, and riboflavin synthesis pathways, the production of riboflavin was effectively enhanced, showing the potential for further engineering of this strain.