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

Abstract

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.

Increasing Yield of 2,3,5,6-Tetramethylpyrazine in Baijiu Through Saccharomyces cerevisiae Metabolic Engineering

“…After knocking out the BDH1 gene, the relative expression level of the BDH1 gene and the enzyme activity of the BDH decreased, which resulted in the pyruvate flux was successfully redirected toward acetoin pathway (Bae et al, 2016). With the accumulation of acetoin, the TTMP produced during the fermentation of Baijiu has also increased, which is consistent with the previous research conclusions (Hao et al, 2013;Zhang et al, 2019;Zhong et al, 2020).…”

“…First, acetoin is used as a substrate to react with ammonium (or ammonia) to form α-hydroxyimine (Xiao et al, 2014). Subsequently, α-hydroxyimine is converted to 2-amino-3-butanone, which condense to form TTMP spontaneously (Xu et al, 2018;Zhang et al, 2019). It has been reported that the content of TTMP also increased with a gradually increased concentration of acetoin, suggesting that acetoin is a precursor of TTMP (Rizzi, 1988;Chen et al, 2010;Zhu et al, 2010;Meng et al, 2020).…”

Effects of Tetragenococcus halophilus and Candida versatilis on the production of aroma‐active and umami‐taste compounds during soy sauce fermentation

“…precursors addition, enzyme identification, are difficult and time-consuming to elucidate the forming pathways of these compounds. [13][14][15][16][17] Microbes,…”

Construction of a third NAD+de novobiosynthesis pathway

“…E. coli DMP overexpresses two genes encoding Tdh and SpNox. Tdh is an NAD + -dependent L-threonine-3-dehydrogenase that oxidizes L-threonine to (2S)-2-amino-3-oxobutanoate, which then spontaneously undergoes decarboxylation and dimerization to yield DMP 38 ; SpNox is an NADH oxidase (H2O forming) for NAD + recycling 39 . Compared with E. coli DMP-Con (E. coli DMP with empty pAB1s), the NAD(H) levels of the new strain E. coli C3N-DMP increased 2.7-fold, and DMP production was 3.8-fold higher (Fig.…”