초록
<P><B>Abstract</B></P> <P>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 <I>E</I>. <I>coli</I> 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 <I>E</I>. <I>coli</I> BL15 could efficiently convert the renewable substrate glucose to acetoin, one precursor of TMP, with 68.4g/L by fed-batch fermentation, the record of acetoin production using <I>E</I>. <I>coli</I>. Thereafter, the preferred ammonium salt (the other precursor) and pH values of the initial solution were investigated for the production of TMP, respectively. Finally, 16.1g/L TMP was achieved using diammonium hydrogen phosphate within 40min under an initial pH value of 7.5.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A suitable acetoin gene cluster was selected to generate the recombinant <I>E</I>. <I>coli</I>. </LI> <LI> Expression of water forming NADH oxidase was regulated for cofactor rebalancing. </LI> <LI> Fed-batch fermentation was optimized to produce 68.4g/L acetoin. </LI> <LI> Tetramethylpyrazine of 16.1g/L was converted from acetoin fermentation broth in 40min. </LI> </UL> </P>