A combinational optimization method for efficient synthesis of tetramethylpyrazine by the recombinant Escherichia coli

2,3,5,6-Tetramethylpyrazine (TMP) is a functional chemical applied for curing cardiovascular and cerebrovascular diseases, and also a famous compound for adding food flavours. In this study, a combinational optimization method was used for realizing the efficient production of TMP. First, a combined engineering way was used to generate a recombinant strain designated E. coli BL15, which contained the selection of acetoin biosynthetic gene cluster and fine tuning of NADH oxidase expression. Second, medium formulation and aeration rate were optimized, and E. coli BL15 could efficiently convert the renewable substrate glucose to acetoin, one precursor of TMP, with 68.4 g/L by fed-batch fermentation, the record of acetoin production using E. coli. Thereafter, the preferred ammonium salt (the other precursor) and pH values of the initial solution were investigated for the production of TMP, respectively. Finally, 16.1 g/L TMP was achieved using diammonium hydrogen phosphate within 40 min under an initial pH value of 7.5.