Novel Mode Engineering for β-Alanine Production in Escherichia coli with the Guide of Adaptive Laboratory Evolution

The strategy of anaerobic biosynthesis of beta-alanine by Escherichia coli (E. coli) has been reported. However, the low energy production under anaerobic condition limited cell growth and then affected the production efficiency of beta-alanine. Here, the adaptive laboratory evolution was carried out to improve energy production of E. coli lacking phosphoenolpyruvate carboxylase under anaerobic condition. Five mutants were isolated and analyzed. Sequence analysis showed that most of the consistent genetic mutations among the mutants were related with pyruvate accumulation, indicating that pyruvate accumulation enabled the growth of the lethal parent. It is possible that the accumulated pyruvate provides sufficient precursors for energy generation and CO2 fixing reaction catalyzed by phosphoenolpyruvate carboxykinase. B0016-100BB (B0016-090BB, recE::FRT, mhpF::FRT, ykgF::FRT, mhpB:: mhpB *, mhpD:: mhpD *, rcsA:: rcsA *) was engineered based on the analysis of the genetic mutations among the mutants for the biosynthesis of beta-alanine. Along with the recruitment of glycerol as the sole carbon source, 1.07 g/L beta-alanine was generated by B0016-200BB (B0016-100BB, aspA::FRT) harboring pET24a-panD-AspDH, which was used for overexpression of two key enzymes in beta-alanine fermentation process. Compared with the starting strain, which can hardly generate beta-alanine under anaerobic condition, the production efficiency of beta-alanine of the engineered cell factory was significantly improved.

Automated summary

The study reports an adaptive laboratory evolution approach to enhance energy production in E. coli lacking phosphoenolpyruvate carboxylase under anaerobic conditions, leading to improved beta-alanine production efficiency. Mutants with genetic mutations related to pyruvate accumulation were isolated and analyzed, indicating that pyruvate accumulation enabled growth and energy generation. This approach resulted in significant improvement in beta-alanine production efficiency compared to the starting strain.