食品与发酵工业 >

2026 , Vol. 52 >Issue 5: 1 - 9

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.043152

研究报告

代谢工程改造枯草芽孢杆菌高效生产维生素K2

  • 郭凤鸣 ,
  • 朱启尧 ,
  • 杜丽平 ,
  • 付刚 ,
  • 张大伟
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  • 1(天津科技大学 生物工程学院,天津,300457)
    2(中国科学院 天津工业生物技术研究所,天津,300308)
    3(中国科学院大学 生命科学学院,北京,100049)
    4(低碳合成工程生物学全国重点实验室,中国科学院工业生物技术研究所,天津,300308)
    5(国家合成生物技术创新中心,天津,300308)
    6(大连工业大学 生物工程学院,辽宁 大连,116034)
第一作者:硕士研究生(杜丽平教授、付刚副研究员和张大伟研究员为共同通信作者,E-mail:dlp123@tust.edu.cn;fu_g@tib.cas.cn;zhang_dw@tib.cas.cn)

收稿日期: 2025-04-28

  修回日期: 2025-07-05

  网络出版日期: 2026-03-25

基金资助

国家重点研发计划项目(2020YFA0907800);天津市科技计划项目(24ZYCGYS00690);中国科学院国际合作局全球共性挑战专项计划项目(306GJHZ2023019GC)

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Metabolic engineering of Bacillus subtilis for efficient production of vitamin K2

  • GUO Fengming ,
  • ZHU Qiyao ,
  • DU Liping ,
  • FU Gang ,
  • ZHANG Dawei
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  • 1(School of Bioengineering, Tianjin University of Science & Technology, Tianjin 300457, China)
    2(Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China)
    3(University of Chinese Academy of Sciences College of Life Science, Beijing 100049, China)
    4(National Key Laboratory of Low Carbon Synthetic Engineering Biology, Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China)
    5(National Innovation Center for Synthetic Biology, Tianjin 300308, China)
    6(School of Bioengineering, Dalian Polytechnic University, Dalian 116034, China)

Received date: 2025-04-28

  Revised date: 2025-07-05

  Online published: 2026-03-25

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摘要

七烯甲萘醌(menaquinone-7,MK-7)是维生素K2的重要活性形式,在促进骨骼健康、预防心血管疾病和治疗神经退行性疾病方面具有广泛的应用前景。该研究选用枯草芽孢杆菌168(Bacillus subtilis 168)作为底盘菌株,采用多模块代谢工程策略系统优化MK-7的生物合成途径。首先,敲除竞争性分支代谢途径的关键基因,包括异分支酸-丙酮酸水解酶基因(dhbB)、芳香族氨基酸合成基因(trpE/aroH)和细菌磷酸酶转移系统(phosphotransferase system,PTS)中的葡萄糖转运蛋白基因(ptsG),以减少前体分流。随后,通过过表达NADH脱氢酶基因(ndh)和末端氧化酶基因(sdhCAB),增强电子传递链效率,促进电子传递。进一步,引入根癌农杆菌(Agrobacterium tumefaciens)的异源基因dxs,并过表达内源限速酶基因hepS,以提高甲基赤藓糖醇磷酸(methylerythritol 4-phosphate,MEP)途径的通量。最终,改造后的高产菌株S13在摇瓶发酵中MK-7产量达到(123.90±6.52)mg/L。在3 L发酵罐中进行放大验证后,产量进一步提升至242.38 mg/L,为MK-7的工业化微生物发酵生产奠定了基础。

关键词： 维生素K2; 七烯甲萘醌; 枯草芽孢杆菌; 代谢工程; 生物合成

本文引用格式

郭凤鸣 , 朱启尧 , 杜丽平 , 付刚 , 张大伟 . 代谢工程改造枯草芽孢杆菌高效生产维生素K2[J]. 食品与发酵工业, 2026 , 52(5) : 1 -9 . DOI: 10.13995/j.cnki.11-1802/ts.043152

Abstract

Menaquinone-7 (MK-7), an important bioactive form of vitamin K2, exhibits broad application prospects in promoting bone health, preventing cardiovascular diseases and treating neurodegenerative disorders.In this study, Bacillus subtilis 168 was selected as the chassis strain, and a multi-module metabolic engineering strategy was employed to optimize the MK-7 biosynthetic pathway systematically.Initially, key genes in competing branch metabolic pathways, including the isochorismate-pyruvate lyase gene (dhbB), aromatic amino acid biosynthesis genes (trpE/aroH), and the phosphotransferase system (PTS) glucose transporter gene (ptsG), were knocked out to minimize precursor diversion.Subsequently, the efficiency of the electron transport chain was enhanced by overexpressing the NADH dehydrogenase gene (ndh) and the terminal oxidase gene (sdhCAB), thereby facilitating electron transfer.Furthermore, the heterologous dxs gene from Agrobacterium tumefaciens was introduced, and the endogenous rate-limiting enzyme gene hepS was overexpressed to increase the flux of the methylerythritol 4-phosphate (MEP) pathway.Ultimately, the engineered high-yielding strain S13 achieved an MK-7 titer of (123.90±6.52) mg/L in shake-flask fermentation.Upon scale-up validation in a 3 L fermenter, the production further increased to 242.38 mg/L, laying a solid foundation for the industrial-scale microbial fermentation of MK-7.

Key words： vitamin K2; menaquinone-7; Bacillus subtilis; metabolic engineering; biosynthesis

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