纳豆芽孢杆菌men基因过表达菌株的构建及提高溶氧强化MK-7合成  

Enhancement of MK-7 synthesis by engineered strains of Bacillus subtilis natto that overexpress men genes and by increasing available dissolved oxygen

作　　者：李楚 王晗[2] 王丽[2] 王鹏[2] 郑之明[2] LI Chu;WANG Han;WANG Li;WANG Peng;ZHENG Zhiming(Institutes of Physical Science and Information Technology,Anhui University,Hefei 230601,China;Hefei Institutes of Physical Science,Chinese Academy of Sciences,Hefei 230031,China)

机构地区：[1]安徽大学物质科学与信息技术研究院,合肥230601 [2]中国科学院合肥物质科学研究院,合肥230031

出　　处：《应用与环境生物学报》2022年第6期1393-1399,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家重点研发计划“合成生物学”重点专项“智能工业菌株高效合成技术体系”(202103a06020003)资助。

摘　　要：近年来,维生素K2更多的生理功能被发现,除了促进凝血之外,还能预防骨质疏松症和心血管疾病,有望治疗肿瘤和帕金森症.纳豆芽孢杆菌是被美国食品药品管理局认证的一种益生菌,可以用来生物制备维生素K2,其主要的产物形式为人体内半衰期较长、生物利用度较高的MK-7,在规模化生物制备维生素K2中的应用潜力较大.本研究优化枯草芽孢杆菌的Sipizizen转化法,使其适用于纳豆芽孢杆菌的分子转化;构建甲萘醌合成途径所涉及的全部8个men基因的过表达载体,并考察其转化纳豆芽孢杆菌之后发酵MK-7的性能变化;筛选出对MK-7合成影响较大的3个酶,分别为异戊烯基转移酶(MenA)、1,4-二羟基-2-萘甲酰辅酶A合酶(MenB)和去甲基萘醌甲基转移酶(MenG).通过对menA、menB和menG组合过表达筛选出一株MK-7产率较高的改造菌株(BN-pABG).在5L发酵罐中,通过供氧调控进一步优化其合成MK-7的生理过程,改造菌株的终产量为62.21 mg/L,较出发菌提高了1.26倍.这些结果表明menA、menB和menG组合过表达后对纳豆芽孢杆菌合成MK-7具有较强的促进作用,同时供氧条件的优化也对MK-7的合成有提升效果.(图6表6参21)In recent years, researchers have discovered novel physiological functions of vitamin K2. In addition to promoting blood clotting, it can prevent osteoporosis and cardiovascular disease and is expected to treat some tumors and Parkinson’s disease. Bacillus subtilis natto is a probiotic that has been approved by the U.S.Food and Drug Administration for use as a bioproducer of vitamin K2. Its product’s main form is MK-7, which has a long half-life in the human body and high bioavailability. Bacillus subtilis natto displays great potential for large-scale biological preparation of vitamin K2. In this study, the Sipizizen method of Bacillus subtilis transformation was optimized to make it suitable for molecular transformation of Bacillus subtilis natto. Vectors for overexpression of all 8 genes involved in the menadione synthetic pathway were constructed, and changes in MK-7 fermentation yield after transformation of Bacillus subtilis natto were investigated. Three enzymes were found to exert significant effects on MK-7 synthesis, namely isopentenyltransferase(MenA), 1,4-dihydroxy-2-naphthoyl-CoA synthase(MenB), and nornaphthoquinone methyltransferase(MenG). A modified strain(BNpABG) with higher MK-7 productivity was obtained by concerted overexpression of menA, menB, and menG.In a 5 L bioreactor, MK-7 synthesis was further enhanced by optimizing oxygen supply. The final yield of MK-7from the modified strain was 62.21 mg/L, 1.26 times higher than that of the original strain. These results show that combined overexpression of menA, menB, and menG strongly promotes MK-7 synthesis by Bacillus subtilis natto, and optimizing the oxygen supply conditions also promotes more robust MK-7 synthesis.

关 键 词：纳豆芽孢杆菌 Sipizizen转化法优化 甲萘醌途径 过表达载体构建 men基因 

分 类 号：TQ924[轻工技术与工程—发酵工程]