Table_1_Effects of Alkali Stress on the Growth and Menaquinone-7 Metabolism of Bacillus subtilis natto.DOCX

Menaquinone-7 (MK-7) is an important vitamin K2, synthesized from the menaquinone parent ring and seven isoprene side chains. Presently, the synthesis of MK-7 stimulated by environmental stress primarily focuses on oxygen stress, while the effect of alkali stress is rarely studied. Therefore, this study researched the effects of alkali stress on the fermentation performance and gene expression of Bacillus subtilis natto. The organism’s growth characteristics, biomass, sporogenesis, MK-7 biosynthesis, and gene expression were analyzed. After a pH 8.5 stress adaptation treatment for 0.5 h and subsequent fermentation at pH 8.5, which promoted the growth of the strain and inhibited the spore formation rate. In addition, biomass was significantly increased (P < 0.05). The conversion rate of glycerol to MK-7 was 1.68 times higher than that of the control group, and the yield of MK-7 increased to 2.10 times. Transcriptomic analysis showed that the MK-7 high-yielding strain had enhanced carbon source utilization, increased glycerol and pyruvate metabolism, enhanced the Embden-Meyerhof pathway (EMP), tricarboxylic acid (TCA) circulation flux, and terpenoid biosynthesis pathway, and promoted the accumulation of acetyl-CoA, the side-chain precursor of isoprene. At the same time, the up-regulation of transketolase increased the metabolic flux of the pentose phosphate (HMP) pathway, which was conducive to the accumulation of D-erythrose 4-phosphate, the precursor of the menadione parent ring. This study’s results contribute to a better understanding of the effects of environmental stress on MK-7 fermentation by Bacillus subtilis natto and the molecular regulatory mechanism of MK-7 biosynthesis.

甲基萘醌-7（Menaquinone-7，MK-7）是一类重要的维生素K2（vitamin K2），其由甲基萘醌母环与七个异戊二烯侧链合成得到。目前，针对环境胁迫（environmental stress）诱导MK-7合成的研究主要聚焦于氧化胁迫（oxygen stress），而碱胁迫（alkali stress）的相关影响鲜有报道。因此，本研究探讨了碱胁迫对纳豆芽孢杆菌（Bacillus subtilis natto）发酵性能与基因表达的调控效应。研究分析了该菌株的生长特性、生物量、芽孢形成能力、MK-7生物合成水平以及基因表达情况。经pH 8.5胁迫适应处理0.5 h后，于pH 8.5条件下进行发酵，可促进菌株生长并抑制芽孢形成速率。此外，菌株生物量显著提升（P < 0.05）；甘油转化为MK-7的转化率较对照组提升1.68倍，MK-7产量提升至对照组的2.10倍。转录组学分析结果显示，高产MK-7菌株的碳源利用效率增强，甘油与丙酮酸代谢水平提升，糖酵解途径（Embden-Meyerhof pathway，EMP）、三羧酸循环（tricarboxylic acid cycle，TCA）通量以及萜类生物合成通路均得到强化，同时促进了异戊二烯侧链前体乙酰辅酶A（acetyl-CoA）的积累。与此同时，转酮醇酶（transketolase）的上调表达提升了戊糖磷酸途径（pentose phosphate pathway，HMP）的代谢通量，这有利于甲基萘醌母环前体D-赤藓糖-4-磷酸（D-erythrose 4-phosphate）的积累。本研究结果有助于更深入地理解环境胁迫对纳豆芽孢杆菌发酵生产MK-7的影响，以及MK-7生物合成的分子调控机制。