Optimized Biosynthetic Pathway for Nonnatural Amino Acids: An Efficient Approach for L‐2‐Aminobutyric Acid Production

ABSTRACT L‐2‐Aminobutyric acid (L‐2‐ABA) is a nonnatural chiral α‐amino acid which is widely used in various chiral pharmaceuticals and medical intermediates. Currently, the microbial metabolic engineering approach to enable Escherichia coli to produce L‐2‐ABA autonomously exists the problem of low synthesis efficiency, limiting its large‐scale application. In this study, we successfully constructed a strain of E. coli that can produce L‐2‐ABA efficiently via multi‐pathway transformation. Firstly, the growth defect of the start strain was restored by the help of screening transcriptional regulators. To maximize the accumulation of L‐2‐ABA, enhancements were made to the main synthetic pathways as well as cofactor systems and energy supply. Subsequently, transport proteins associated with osmotic stress tolerance were modified to improve adaptability of the strain during fermentation. Ultimately, the titer of L‐2‐ABA reached 42.14 g/L through the final strain ABAT38 in a 5‐liter bioreactor, with a productivity of 0.40 g/L/h and a glucose conversion of 0.39 g/g, which exceeded the highest levels reported before. The strategies proposed in this study contribute to the production of L‐2‐ABA. At the same time, it has reference significance for the biosynthesis of related nonnatural amino acids with phosphoenolpyruvate as the intermediate metabolite.