An Alkylpyrazine Synthesis Mechanism Involving L-Threonine-3-Dehydrogenase Describes the Production of 2,5-Dimethylpyrazine and 2,3,5-Trimethylpyrazine by Bacillus subtilis

Alkylpyrazines are important contributors to the flavor of traditional fermented foods. Here, we studied the synthesis mechanisms of 2,5-dimethylpyrazine (2,5-DMP) and 2,3,5-trimethylpyrazine (TMP). Substrate addition, whole-cell catalysis, stable isotope tracing experiments, and gene manipulation revealed that L-threonine is the starting point involving L-threonine-3-dehydrogenase (TDH) and three uncatalyzed reactions to form 2,5-DMP. TDH catalyzes the oxidation of L-threonine. The product of this reaction is L-2-amino-acetoacetate, which is known to be unstable and can decarboxylate to form aminoacetone. It is proposed that aminoacetone spontaneously converts to 2,5-DMP in a pH-dependent reaction, via 3,6-dihydro-2,5-DMP. 2-Amino-3-ketobutyrate coenzyme A (CoA) ligase (KBL) catalyzes the cleavage of L-2-amino-acetoacetate, the product of TDH, into glycine and acetyl-CoA in the presence of CoA. Inactivation of KBL could improve the production of 2,5-DMP. Besides 2,5-DMP, TMP can also be generated by Bacillus subtilis 168 by using L-threonine and D-glucose as the substrates and TDH as the catalytic enzyme. IMPORTANCE Despite alkylpyrazines' contribution to flavor and their commercial value, the synthesis mechanisms of alkylpyrazines by microorganisms remain poorly understood. This study revealed the substrate, intermediates, and related enzymes for the synthesis of 2,5-dimethylpyrazine (2,5-DMP), which differ from the previous reports about the synthesis of 2,3,5,6-tetramethylpyrazine (TTMP). The synthesis mechanism described here can also explain the production of 2,3,5-trimethylpyrazine (TMP). The results provide insights into an alkylpyrazine's synthesis pathway involving L-threonine-3-dehydrogenase as the catalytic enzyme and L-threonine as the substrate.