Engineering Bacillus subtilis J46 for efficient utilization of galactose through adaptive laboratory evolution

Efficient utilization of galactose by microorganisms can lead to the production of valuable bio-products and improved metabolic processes. While Bacillus subtilis has inherent pathways for galactose metabolism, there is potential for enhancement via evolutionary strategies. This study aimed to boost galactose utilization in B. subtilis using adaptive laboratory evolution (ALE) and to elucidate the genetic and metabolic changes underlying the observed enhancements. The strains of B. subtilis underwent multiple rounds of adaptive laboratory evolution (approximately 5000 generations) in an environment that favored the use of galactose. This process resulted in an enhanced specific growth rate of 0.319 ± 0.005 h −1 , a significant increase from the 0.03 ± 0.008 h −1 observed in the wild-type strains. Upon selecting the evolved strain BSGA14, a comprehensive whole-genome sequencing revealed the presence of 63 single nucleotide polymorphisms (SNPs). Two of them, located in the coding sequences of the genes araR and glcR, were found to be the advantageous mutations after reverse engineering. The strain with these two accumulated mutations, BSGALE4, exhibited similar specific growth rate on galactose to the evolved strain BSGA14 (0.296 ± 0.01 h −1 ). Furthermore, evolved strain showed higher productivity of protease and β-galactosidase in mock soybean biomass medium. ALE proved to be a potent tool for enhancing galactose metabolism in B. subtilis . The findings offer valuable insights into the potential of evolutionary strategies in microbial engineering and pave the way for industrial applications harnessing enhanced galactose conversion. Key Points Following adaptive laboratory evolution in a minimal medium with galactose serving as the selective pressure, the B. subtilis BSGA14 strain was successfully developed, exhibiting a specific growth rate of 0.319 ± 0.005 h −1 . Among SNPs after resequencing, mutation in the araR gene enhances the uptake of galactose in Bacillus subtilis , leading to increased galactose utilization. The nonsense mutation in the glcR gene, which is involved in carbon catabolite repression (CCR) and sugar phosphorylation, influences galactose metabolism in B. subtilis .