Enhancing the expression of novel α-amino acid aster acyltransferase via multiple strategies and its characterization

α-Amino acid ester acyltransferase (AET) is a dipeptide synthase that utilizes amino acid esters and amino acids as substrates. With its broad substrate spectrum, high specific activity, and ATP independence, it holds significant promise for various applications. However, achieving efficient AET expression remains a challenge. In this study, novel AET genes were identified through gene mining. A total of six AET genes were synthesized, and their expression levels were investigated; however, none exhibited satisfactory expression. Through the application of multiple strategies, including codon optimization, amino acid sequence modification, and promoter engineering, AET expression was significantly enhanced. The total activity of the final recombinant strain, Em2M1, reached 741.83 U/mL at 20 °C-tens of times higher than the original strain-and exceeded previously reported levels. The characteristic of novel AET from Elizabethkingia miricola was also investigated.