Engineering riboflavin-overproducing Bacillus subtilis via pathway gene overexpression

Overexpression of genes at key metabolic nodes frequently enhances the yield of target products. However, this often leads to widespread issues with growth defect and plasmid instability. This study utilizes the riboflavin over-producer Bacillus subtilis U3 as a model to systematically investigate the impact of overexpressing riboflavin operon genes on strain growth and plasmid structural instability. Overexpression of genes in rib operon enhances target product yields by 13.2 % in U3, but the engineered strains suffer growth defects and plasmid instability. Frameshift mutations in the ribD gene were found to significantly reduce the loss of operon gene fragments by 16.7 %. Furthermore, this study investigates the application of the Respiration Activity Monitoring System (RAMOS) in evaluating the growth of metabolically engineered strains. RAMOS serves as a medium optimization tool and pre-fermentation screening platform. Specifically, guanine supplementation increased biomass by 11.1 % in zwf-overexpressing strains, whereas histidine, uracil, and tryptophan supplementation could improve the biomass of purF-overexpressing strain by 71.1 %. This approach provides a methodological framework for resolving growth defects in metabolic engineering.