调控质膜稳态提高枯草芽孢杆菌积累四烯甲萘醌MK-4  

Regulation of plasma membrane homeostasis to increase the accumulation of menaquinone-4 in Bacillus subtilis

作　　者：原攀红 吕雪芹 刘延峰 李江华 刘龙[1,2] 堵国成 YUAN Panhong;LYU Xueqin;LIU Yanfeng;LI Jianghua;LIU Long;DU Guocheng(Science Center for Future Foods,Jiangnan University,Wuxi 214122,China;Key Laboratory of Carbohydrate Chemistry and Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学,未来食品科学中心,江苏无锡214122 [2]糖化学与生物技术教育部重点实验室(江南大学),江苏无锡214122

出　　处：《食品与发酵工业》2021年第18期1-7,共7页Food and Fermentation Industries

基　　金：国家自然科学基金(31871784,32021005);山东省重大项目(2019JZZY011002)。

摘　　要：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.

关 键 词：枯草芽孢杆菌 MK-4 细胞膜的完整性 细胞膜的流动性 细胞膜的渗透性 

分 类 号：TQ924[轻工技术与工程—发酵工程]