Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Collaborative Innovation Center for Mountain Ecology & Agro-Bioengineering (CICMEAB), College of Life Sciences/Institute of Agro-Bioengineeringering, Guizhou University, Guiyang, 550025, Guizhou, China.
BMC Biotechnol. 2022 Jul 4;22(1):18. doi: 10.1186/s12896-022-00748-4.
Tetramethylpyrazine (TTMP) is a flavoring additive that significantly contributes to the formation of flavor compounds in soybean-based fermented foods. Over recent years, the application of TTMP in the food industry and medicine has been widely investigated. In addition, several methods for the industrial-scale production of TTMP, including chemical and biological synthesis, have been proposed. However, there have been few reports on the synthesis of TTMP through amino acid metabolic flux. In this study, we investigated genetic alterations of arginine metabolic flux in solid-state fermentation (SSF) of soybeans with Bacillus subtilis (B.subtilis) BJ3-2 to enhance the TTMP yield.
SSF of soybeans with BJ3-2 exhibited a strong Chi-flavour (a special flavour of ammonia-containing smelly distinct from natto) at 37 °C and a prominent soy sauce-like aroma at 45 °C. Transcriptome sequencing and RT-qPCR verification showed that the rocF gene was highly expressed at 45 °C but not at 37 °C. Moreover, the fermented soybeans with BJ3-2ΔrocF (a rocF knockout strain in B. subtilis BJ3-2 were obtained by homologous recombination) at 45 °C for 72 h displayed a lighter color and a slightly decreased pH, while exhibiting a higher arginine content (increased by 14%) than that of BJ3-2. However, the ammonia content of fermented soybeans with BJ3-2ΔrocF was 43% lower than that of BJ3-2. Inversely, the NH content in fermented soybeans with BJ3-2ΔrocF was increased by 28% (0.410 mg/kg). Notably, the TTMP content in fermented soybeans with BJ3-2ΔrocF and BJ3-2ΔrocF + Arg (treated with 0.05% arginine) were significantly increased by 8.6% (0.4617 mg/g) and 18.58% (0.504 mg/g) respectively than that of the BJ3-2.
The present study provides valuable information for understanding the underlying mechanism during the TTMP formation process through arginine metabolic flux.
四甲基吡嗪（TTMP）是一种调味添加剂，可显著促进大豆发酵食品中风味化合物的形成。近年来，TTMP 在食品工业和医药领域的应用得到了广泛的研究。此外，已经提出了几种 TTMP 的工业规模生产方法，包括化学合成和生物合成。然而，很少有报道通过氨基酸代谢通量合成 TTMP。在这项研究中，我们通过枯草芽孢杆菌（B.subtilis）BJ3-2 固态发酵（SSF）大豆来研究精氨酸代谢通量的遗传改变，以提高 TTMP 的产量。
BJ3-2 固态发酵大豆在 37°C 时表现出强烈的 Chi 风味（一种不同于纳豆的氨味独特的气味），在 45°C 时表现出明显的酱油香气。转录组测序和 RT-qPCR 验证表明，rocF 基因在 45°C 时高度表达，但在 37°C 时不表达。此外，BJ3-2ΔrocF（通过同源重组获得的枯草芽孢杆菌 BJ3-2 中的 rocF 敲除菌株）发酵的大豆在 45°C 下发酵 72 小时后颜色较浅，pH 值略有下降，而精氨酸含量（增加了 14%）高于 BJ3-2。然而，BJ3-2ΔrocF 发酵大豆中的氨含量比 BJ3-2 低 43%。相反，BJ3-2ΔrocF 发酵大豆中的 NH3 含量增加了 28%（0.410mg/kg）。值得注意的是，BJ3-2ΔrocF 和 BJ3-2ΔrocF+Arg（用 0.05%精氨酸处理）发酵的大豆中的 TTMP 含量分别显著增加了 8.6%（0.4617mg/g）和 18.58%（0.504mg/g）比 BJ3-2。
本研究为通过精氨酸代谢通量了解 TTMP 形成过程中的潜在机制提供了有价值的信息。
