维生素B12生物合成领域的十年回顾与技术进展

doi:10.13560/j.cnki.biotech.bull.1985.2025-0032

生物技术通报 ›› 2025, Vol. 41 ›› Issue (11): 62-74.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0032

维生素B₁₂生物合成领域的十年回顾与技术进展

张纪娇¹^,²(), 王会颖², 房欢²^,³^,⁴^,⁵(), 张大伟¹^,²^,³^,⁴^,⁵^,⁶()

^1.大连工业大学食品学院，大连 116034
^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，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.天津科技大学生物工程学院，天津 300457

收稿日期:2025-01-08 出版日期:2025-11-26 发布日期:2025-12-09
通讯作者: 房欢，男，博士，副研究员，研究方向：维生素B₁₂生物合成；E-mail: fang_h@tib.cas.cn
张大伟，男，博士，研究员，研究方向：微生物代谢工程与合成生物学；E-mail: zhang_dw@tib.cas.cn
作者简介:张纪娇，女，硕士研究生，研究方向：维生素B₁₂生物合成；E-mail: zhangjijiao@tib.cas.cn
基金资助:
中国科学院战略性先导科技专项(XDC0110200);国家自然科学基金项目(22178372)

A Decade Review and Technological Advances in the Field of Vitamin B ₁₂ Biosysthesis

ZHANG Ji-jiao¹^,²(), WANG Hui-ying², FANG Huan²^,³^,⁴^,⁵(), ZHANG Da-wei¹^,²^,³^,⁴^,⁵^,⁶()

^1.School of Food Science, Dalian University of Technology, Dalian 116034
^2.Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308
^3.University of Chinese Academy of Sciences, Beijing 100049
^4.State Key Laboratory of Engineering Biology for Low-Carbon Synthesis, Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308
^5.National Center of Technology Innovation for Synthetic Biology, Tianjin 300308
^6.College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457

Received:2025-01-08 Published:2025-11-26 Online:2025-12-09

摘要/Abstract

摘要：

维生素B₁₂是人体、动物及多种微生物必需的小分子化合物，广泛参与一碳代谢、同型半胱氨酸的甲基化及脂肪酸代谢等生理过程。近年来，随着微生物学与代谢工程的快速发展，微生物发酵法合成维生素B₁₂的研究取得了显著进展。本文首先简要介绍了维生素B₁₂合成途径，然后回顾了近十年几种主要合成维生素B₁₂的微生物，包括工业生产菌株Pseudomonasdenitrificans、Sinorhizobium meliloti、Ensifer adhaerens、Propionibacterium freudenreichii和近几年新出现的人工菌种异源合成维生素B₁₂方面的研究进展，探讨了这些微生物的代谢工程策略、发酵工艺以及在工业化生产中的应用潜力。此外，还分析了当前研究的挑战与未来发展方向，如基因工程改造与代谢调控策略在提高维生素B₁₂生产中的应用。通过对这些微生物的合成途径的深入了解，有望为工业规模维生素B₁₂生产提供新的理论依据和技术支持。

关键词: 维生素B₁₂, 微生物, 代谢工程, 生物合成途径, 工业生产菌株

Abstract:

Vitamin B₁₂ is an essential small molecule compound for humans, animals and many microorganisms, which is widely involved in physiological processes such as one-carbon metabolism, homocysteine methylation and fatty acid metabolism. In recent years, with the development of microbiology and metabolic engineering, the research on microbial fermentation synthesis of vitamin B₁₂ has made remarkable progress. In this paper, we first briefly introduce the vitamin B₁₂ synthesis pathway, and then review several major microorganisms synthesizing vitamin B₁₂ in the last decade, including the industrial production strain Pseudomonas denitrificans, Sinorhizobium meliloti, Ensifer adhaerens, and Propionibacterium freudenreichii, and newly emerged artificial strains in recent years for heterologous synthesis of vitamin B₁₂. The article discusses the metabolic engineering strategies, fermentation processes and the potential application of these microorganisms in industrial production. In addition, current research challenges and future directions are analyzed, such as the application of genetic engineering modifications and metabolic regulation strategies in enhancing vitamin B₁₂ production. The in-depth understanding of the synthetic pathways of these microorganisms is expected to provide new theoretical basis and technical support for industrial-scale vitamin B₁₂ production.

Key words: vitamin B₁₂, microorganisms, metabolic engineering, biosynthetic pathways, industrial production strains

张纪娇, 王会颖, 房欢, 张大伟. 维生素B₁₂生物合成领域的十年回顾与技术进展[J]. 生物技术通报, 2025, 41(11): 62-74.

ZHANG Ji-jiao, WANG Hui-ying, FANG Huan, ZHANG Da-wei. A Decade Review and Technological Advances in the Field of Vitamin B ₁₂ Biosysthesis[J]. Biotechnology Bulletin, 2025, 41(11): 62-74.

图/表 4

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微生物 Microorganism	主要策略 Main strategy	产量 Yield	参考文献Reference
Pseudomonas denitrificans	响应面法优化培养基中麦芽糖浆、玉米浆、甜菜碱	198.80 mg/L	［27］
	代谢组学分析培养基中添加甜菜碱对维生素B₁₂合成作用	（58.61 ± 3.21） mg/L	［28］
	表达透明颤菌vgb基因	比产物合成速率提高52%	［29］
	调节氧气供应改变细胞形态	（239.7 ± 8） mg/L	［30］
	筛选强启动子表达维生素B₁₂合成途径关键基因cobA	75.5 mg/L	［31］
	过表达b12fla基因的突变株提高维生素B₁₂产量	比野生型提高8.09%	［32］
Sinorhizobium meliloti	开发基于Cas12k的染色体整合与转录调控工具，整合hemA、hemB、hemC、hemD、cobA，阻断cysG	92 mg/L	［9］
	以ARTP诱变为核心，结合基因改造（过表达关键突变基因），实现维生素B₁₂产量阶梯式提升	104.54 mg/L	［33］
	挖掘木糖诱导启动子，表达hemA	提高11%	［14］
	基于核糖开关的维生素B₁₂高通量筛选技术与ARTP联合应用	（156±4.2） mg/L	［34］
	开发正向响应的维生素B₁₂高通量筛选技术，筛选紫外诱变菌种	10%菌株维生素B₁₂产量提高	［35］
Ensifer adhaerens	ARTP诱变、流式细胞仪分选	110.25 mg /L	［36］
	转录组挖掘内源启动子，精准表达维生素B₁₂合成基因cobSV、cobQ和cobW	171.2 mg/L	［37］
	敲除西罗血红素合成途径基因cysG、弱化血红素合成途径基因hemE	（114.17±5.77） mg/L	［38］
	通过动力学分析不同碳源（麦芽糖、蔗糖、葡萄糖、果糖）对发酵的影响，发现蔗糖为最佳碳源，可显著提升维生素B₁₂产量	115 mg/L	［39］
	比较转录组分析不同维生素B₁₂合成菌株，过表达维生素B₁₂合成途径基因cobA、cobT	（245.6±4.36） mg/L	［40］
Propionibacterium freudenreichii	发酵过程中控制丙酸生成量和DMBI补料策略	58.8 mg/L	［41］
	扩展床吸附生物反应器中以玉米秸秆水解液为碳源分批补料发酵联产维生素B₁₂和丙酸	47.6 mg/L	［42］
	采用半连续发酵工艺，膜分离菌体使腺苷钴啉醇酰胺与DMBI反应，非原位合成维生素B₁₂	（56.76±3.86） mg/L	［43］
	使用大豆液态酸性蛋白渣进行发酵，Plackett-Burman实验和响应面法优化培养基配方	0.6 mg/g cells	［44］
	Plackett-Burman实验和响应面法优化培养基配方	（8.32±0.02） mg/L	［45］
	丙酸作碳源，微好氧发酵	184 μg/g DCW	［16］
	核糖体工程调节细菌基础代谢	单位细胞维生素B₁₂产量提高5.2倍	［46］
Escherichia coli	染色体上整合前体模块基因hemOBCD、钴吸收基因cbiMNQO，3个质粒上分别表达HBA合成基因cobAIGJMFKLH、钴（II）啉酸a，c-二酰胺合成基因cobNSTW、腺苷咕啉醇酰胺磷酸合成基因cobR、cobA、cbiP、pduX、cobD、cbiB，sRNA弱化hemF、hemG，敲除乙酸合成基因ackA-pta、敲除乳酸合成基因ldhA、敲除endA	307 μg/g DCW	［17］
	筛选不同来源cobB，双顺反正优化cobN表达，精准调节cobS、cobT表达，筛选不同来源的腺苷咕啉醇酰胺磷酸合成基因组合，正交实验优化培养基中碳氮比	530.29 μg/g DCW	［47］
	敲除metE构建维生素B₁₂营养缺陷型菌株，调节metH表达优化生物量和维生素B₁₂合成	13.2 μg/L	［48］
	开发标准化基因编辑工具，将维生素B₁₂合成途径基因整合在染色体上，构建无质粒菌株。筛选腺苷咕啉醇酰胺磷酸合成基因组合，将途径基因以单、多顺反子表达，改变途径基因染色体整合位点	1.49 mg/L	［49］
	多元模块化代谢工程优化钴（II）啉酸a，c-二酰胺、腺苷咕啉醇酰胺磷酸合成模块，添加不同种类有机氮到培养基中，5 L反应器放大	2.89 mg/L	［50］
	通过基因工程（整合异源基因、表达vgb基因、引入ED途径改造碳代谢）与发酵优化（单因素及Taguchi法优化培养基），提升大肠杆菌合成维生B₁₂的产量，最终在5 L发酵罐中放大培养时产量达到21.09 mg/L	21.09 mg/L	［51］

微生物 Microorganism	主要策略 Main strategy	产量 Yield	参考文献Reference
Pseudomonas denitrificans	响应面法优化培养基中麦芽糖浆、玉米浆、甜菜碱	198.80 mg/L	［27］
	代谢组学分析培养基中添加甜菜碱对维生素B₁₂合成作用	（58.61 ± 3.21） mg/L	［28］
	表达透明颤菌vgb基因	比产物合成速率提高52%	［29］
	调节氧气供应改变细胞形态	（239.7 ± 8） mg/L	［30］
	筛选强启动子表达维生素B₁₂合成途径关键基因cobA	75.5 mg/L	［31］
	过表达b12fla基因的突变株提高维生素B₁₂产量	比野生型提高8.09%	［32］
Sinorhizobium meliloti	开发基于Cas12k的染色体整合与转录调控工具，整合hemA、hemB、hemC、hemD、cobA，阻断cysG	92 mg/L	［9］
	以ARTP诱变为核心，结合基因改造（过表达关键突变基因），实现维生素B₁₂产量阶梯式提升	104.54 mg/L	［33］
	挖掘木糖诱导启动子，表达hemA	提高11%	［14］
	基于核糖开关的维生素B₁₂高通量筛选技术与ARTP联合应用	（156±4.2） mg/L	［34］
	开发正向响应的维生素B₁₂高通量筛选技术，筛选紫外诱变菌种	10%菌株维生素B₁₂产量提高	［35］
Ensifer adhaerens	ARTP诱变、流式细胞仪分选	110.25 mg /L	［36］
	转录组挖掘内源启动子，精准表达维生素B₁₂合成基因cobSV、cobQ和cobW	171.2 mg/L	［37］
	敲除西罗血红素合成途径基因cysG、弱化血红素合成途径基因hemE	（114.17±5.77） mg/L	［38］
	通过动力学分析不同碳源（麦芽糖、蔗糖、葡萄糖、果糖）对发酵的影响，发现蔗糖为最佳碳源，可显著提升维生素B₁₂产量	115 mg/L	［39］
	比较转录组分析不同维生素B₁₂合成菌株，过表达维生素B₁₂合成途径基因cobA、cobT	（245.6±4.36） mg/L	［40］
Propionibacterium freudenreichii	发酵过程中控制丙酸生成量和DMBI补料策略	58.8 mg/L	［41］
	扩展床吸附生物反应器中以玉米秸秆水解液为碳源分批补料发酵联产维生素B₁₂和丙酸	47.6 mg/L	［42］
	采用半连续发酵工艺，膜分离菌体使腺苷钴啉醇酰胺与DMBI反应，非原位合成维生素B₁₂	（56.76±3.86） mg/L	［43］
	使用大豆液态酸性蛋白渣进行发酵，Plackett-Burman实验和响应面法优化培养基配方	0.6 mg/g cells	［44］
	Plackett-Burman实验和响应面法优化培养基配方	（8.32±0.02） mg/L	［45］
	丙酸作碳源，微好氧发酵	184 μg/g DCW	［16］
	核糖体工程调节细菌基础代谢	单位细胞维生素B₁₂产量提高5.2倍	［46］
Escherichia coli	染色体上整合前体模块基因hemOBCD、钴吸收基因cbiMNQO，3个质粒上分别表达HBA合成基因cobAIGJMFKLH、钴（II）啉酸a，c-二酰胺合成基因cobNSTW、腺苷咕啉醇酰胺磷酸合成基因cobR、cobA、cbiP、pduX、cobD、cbiB，sRNA弱化hemF、hemG，敲除乙酸合成基因ackA-pta、敲除乳酸合成基因ldhA、敲除endA	307 μg/g DCW	［17］
	筛选不同来源cobB，双顺反正优化cobN表达，精准调节cobS、cobT表达，筛选不同来源的腺苷咕啉醇酰胺磷酸合成基因组合，正交实验优化培养基中碳氮比	530.29 μg/g DCW	［47］
	敲除metE构建维生素B₁₂营养缺陷型菌株，调节metH表达优化生物量和维生素B₁₂合成	13.2 μg/L	［48］
	开发标准化基因编辑工具，将维生素B₁₂合成途径基因整合在染色体上，构建无质粒菌株。筛选腺苷咕啉醇酰胺磷酸合成基因组合，将途径基因以单、多顺反子表达，改变途径基因染色体整合位点	1.49 mg/L	［49］
	多元模块化代谢工程优化钴（II）啉酸a，c-二酰胺、腺苷咕啉醇酰胺磷酸合成模块，添加不同种类有机氮到培养基中，5 L反应器放大	2.89 mg/L	［50］
	通过基因工程（整合异源基因、表达vgb基因、引入ED途径改造碳代谢）与发酵优化（单因素及Taguchi法优化培养基），提升大肠杆菌合成维生B₁₂的产量，最终在5 L发酵罐中放大培养时产量达到21.09 mg/L	21.09 mg/L	［51］

维生素B₁₂生物合成领域的十年回顾与技术进展

A Decade Review and Technological Advances in the Field of Vitamin B ₁₂ Biosysthesis

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