维生素MK-4在食品、保健和制药工业中被广泛用于膳食补充剂和药物治疗。为了获得1株四烯甲萘醌MK-4的高产菌株,对细胞膜的稳态进行了系统的分析和优化。首先,对出发菌株Bacillus subtilis BC04细胞膜的完整性相关的基因进行整合分析,发现膜蛋白相关基因srfAD对MK-4的积累有重要的影响;然后对流动性、渗透性相关基因进行整合分析,找到了影响MK-4积累的基因ypkP和fadR。通过组合优化,确定优势菌株为BF14,其整合了基因srfAD、ypkP,敲除了基因fadR。BF14在40 ℃条件下,摇瓶发酵7 d,产量达到(213.80±2.16) mg/L,比优化前提高了19.51%。基于上述结果,在3 L发酵罐中进行发酵条件优化,确定了初始葡萄糖质量浓度为10 g/L,补料维持葡萄糖质量浓度4~10 g/L的分批发酵工艺,最终MK-4的产量达到(249.65±2.34)mg/L,较摇瓶水平提高了16.77%,生产强度为2.08 mg/(L·h)。研究表明,优化细胞膜的稳态可以有效提高菌株生产MK-4的能力,该成果为进一步提升工业水平MK-4发酵性能奠定了基础。
Vitamin menaquinone-4 (MK-4) is widely used in dietary supplements and pharmaceutical therapies in the food, healthcare, and pharmaceutical industries. To get a high MK-4 yield strain, the homeostasis of cell membrane was systematically analyzed and optimized. Firstly, the integrity-related genes of the cell membrane were integrated and analyzed in the original strain Bacillus subtilis BC04, indicating that the membrane protein-related gene srfAD had an important influence on the accumulation of MK-4. Then, the genes related to fluidity and permeability were analyzed, suggesting that ypkP and fadR played a significant role in the accumulation of MK-4. The optimal strain was identified as BF14 by combinatorial optimization, including integration of gene srfAD and ypkP, and knockout of the gene fadR. The yield of BF14 reached (213.80±2.16) mg/L after shake-flask fermentation at 40 ℃ for 7 days, an increase by 19.51% compared with that before optimization. Based on the above results, the fermentation conditions were optimized in a 3L fermenter, and a batch fermentation process was determined with initial glucose concentration of 10 g/L and feeding to maintain glucose concentration of 4-10 g/L. The final MK-4 yield reached (249.65±2.34) mg/L, an increase by 16.77% compared with the level of shaking flask, and the production intensity was 2.08 mg/(L·h). The results showed that optimizing the cell membrane homeostasis of B. subtilis could effectively improve the MK-4 yield, and could lay a foundation for further improving the fermentation performance of MK-4 at the industrial level.
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