Multi-strategy combinatorial regulation of β-alanine biosynthesis pathway in Bacillus subtilis
Authors: Yang Shaomei , Li Jiachang , Meng Rong , Shao Yunhang , Wang Anlong , Wang Zengjian , Xiong Peng , Gao Zhengquan 
Published: September 2022

Abstract
Background
β-Alanine is the only naturally occurring β-amino acid and widely used in chemical industry, medicine, food and feed fields. At present, it is more attractive and potential to construct a microbial cell factory through metabolic engineering to produce β-alanine by fermentation. However, the biosynthesis of β-alanine in Bacillus subtilis, a typical industrial model microorganism of food safety grade, has not been thoroughly explored.
Results
In this study, a total of six L-aspartate-α-decarboxylases (PanDs), including native and two mutant, two other bacterial-derived and one insect-derived, were overexpressed in B. subtilis, respectively. Among them, overexpressions of native PanD and mutant PanDE56S resulted in the greatest increase in β-alanine production, which were 108.14 ± 11.90 mg/L and 104.04 ± 3.11 mg/L after 48 h of fermentation, and 842% and 806% higher than that of the control strain, respectively. Meanwhile, the relative transcription levels of panD were increased by 21.3-fold and 23.9-fold, and the activities of PanD were increased by 43% and 80%. Then, 16 single-gene knockout strains were constructed to block the competing metabolic pathways that consumed phosphoenolpyruvate (PEP), pyruvate, L-glutamate (Glu) and L-aspartate (Asp). Among them, knockouts of ptsG, fbp, ydaP, yhfS, mmgAand pckA, increased β-alanine production by 8.8%, 16.4%, 15.1%, 13.2%, 9.8% and 10.4%, respectively. Subsequently, a multigene knockout strain was constructed that simultaneously knocked out these six genes, resulting in a 40.1% increase in β-alanine production. Furthermore, 10 single-gene suppressor strains were constructed through clustered regularly interspaced short palindromic repeat interference (CRISPRi) technology to inhibit other related competing metabolic pathways, and found that inhibiting the expression of glmS, accB and accA, increased the β-alanine production by 18.4%, 18.1% and 17.7%, respectively. However, the expression of the dCas9 protein resulted in a 13.6% decrease in β-alanine production. Finally, strains expressing endogenous pfkA and aspB, and heterologous ppc were constructed respectively, and found that expression of PEP carboxylase PPC increased the production of β-alanine by 81.7% to 236.15 ± 19.94 mg/L.
Conclusions
Overexpressions of native PanD and mutant PanDE56S could significantly boost the decarboxylation of Asp to generate β-alanine, which was the key step to limit the excessive synthesis of β-alanine. Genes ptsG, fbp, ydaP, yhfS, mmgA, pckA, and genes glmS, accB, accAwere knockout and repression targets, respectively, that facilitated the β-alanine synthesis. Moreover, introduction of heterologous oxaloacetate anaplerotic reaction mediated by PPC could significantly improve the β-alanine synthesis. In conclusion, this study adopted multiple strategies to investigate the de novo biosynthetic pathway of β-alanine in B. subtilis for the first time, providing a solid foundation for constructing the high-yielding strain of β-alanine in the future.