Screening and identification of genes involved in β-alanine biosynthesis in Bacillus subtilis

β-alanine is the only naturally occurring β-amino acid, which is widely used in medicine, food, and feed fields, and generally produced through synthetic biological methods based on engineered strains of Escherichia coli or Corynebacterium glutamicum. However, the β-alanine biosynthesis in Bacillus subtilis, a traditional industrial model microorganism of food safety grade, has not been thoroughly explored. In this study, the native l-aspartate-α-decarboxylase was overexpressed in B. subtilis 168 to obtain an increase of 842% in β-alanine production. A total of 16 single-gene knockout strains were constructed to block the competitive consumption pathways to identify a total of 6 genes (i.e., ptsG, fbp, ydaP, yhfS, mmgA, and pckA) involved in β-alanine synthesis, while the multigene knockout of these 6 genes obtained an increased β-alanine production by 40.1%. Ten single-gene suppression strains with the competitive metabolic pathways inhibited revealed that the inhibited expressions of genes glmS, accB, and accA enhanced the β-alanine production. The introduction of heterologous phosphoenolpyruvate carboxylase increased the β-alanine production by 81.7%, which was 17-fold higher than that of the original strain. This was the first study using multiple molecular strategies to investigate the biosynthetic pathway of β-alanine in B. subtilis and to identify the genetic factors limiting the excessive synthesis of β-alanine by microorganisms.