拟杆菌遗传操作工具箱：从传统方法到合成生物学前沿

doi:10.13560/j.cnki.biotech.bull.1985.2025-1001

摘要/Abstract

摘要：

拟杆菌属（Bacteroides）作为人体肠道微生物组中极为丰富且功能重要的共生菌群，在营养代谢、免疫调节及疾病发生中发挥着关键作用。然而，由于该属细菌具有严格厌氧生长的特性及活跃的限制-修饰系统等内在因素，长期以来，针对其的研究面临遗传工具稀缺、操作效率低下等技术瓶颈。本文系统回顾了拟杆菌遗传操作工具箱的发展历程，总结了这些工具在功能基因验证、代谢途径解析及活菌疗法开发等方面的具体应用。文章首先概述了拟杆菌遗传操作所需的基本元件；继而介绍了靶向已知基因的编辑方法，包括同源重组、CRISPR-Cas系统以及基因回补表达系统等；进而阐述了全基因组范围内筛选新功能基因的策略，如转座子诱变等；结合具体案例，分析了这些技术在解析拟杆菌致病机制与代谢调控网络等方面的应用；最后对当前面临的挑战进行展望，指出多位点编辑、群落水平操作等未来发展方向。本综述旨在为研究人员提供一套系统而实用的遗传操作策略，以推动拟杆菌功能机制的深入研究及其在合成生物学领域的应用拓展。

关键词: 拟杆菌, 人类肠道共生菌, 遗传操作, 基因编辑

Abstract:

Bacteroides,as an extremely abundant and functionally important commensal bacterial group in the human gut microbiome, play key roles in nutrient metabolism, immune regulation, and disease development. However, due to its strict anaerobic nature and active restriction-modification systems, genetic manipulation of Bacteroides has long been hampered by limited tools and low efficiency. This article systematically reviews the development of the genetic manipulation toolbox for Bacteroides and summarizes the specific applications of these tools in functional gene validation, metabolic pathway elucidation, and the development of live bacterial therapeutics. The article first outlines the essential components required for genetic manipulation in Bacteroides. It then introduces targeted gene-editing methods, including homologous recombination, CRISPR-Cas systems, and genetic complementation systems. Subsequently, it describes strategies for genome-wide screening of novel functional genes, such as transposon mutagenesis. Through concrete examples, it analyzes the application of these technologies in deciphering the pathogenic mechanisms and metabolic regulatory networks of Bacteroides. Finally, it discusses current challenges and future directions, including multi-site editing and community-level manipulation. This review aims to provide researchers with a systematic and practical set of genetic manipulation strategies to advance in-depth studies of Bacteroides’ functional mechanisms and its applications in the field of synthetic biology.

Key words: Bacteroides, human gut commensal bacteria, genetic manipulation, gene editing

陈玲嫣, 李伟勋, 逄欣欣, 高翔, 焦绪瑶. 拟杆菌遗传操作工具箱：从传统方法到合成生物学前沿[J]. 生物技术通报, 2026, 42(2): 51-64.

CHEN Ling-yan, LI Wei-xun, PANG Xin-xin, GAO Xiang, JIAO Xu-yao. Bacteroides Genetic Manipulation Toolbox： From Conventional Methods to the Frontiers of Synthetic Biology[J]. Biotechnology Bulletin, 2026, 42(2): 51-64.

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类型 Type	名称/系统 Name/System	诱导剂 Inducer	动态范围 Dynamic range	来源/策略 Source/Strategy	参考文献 References
组成型启动子	P_BT1311			内源启动子，基于σ因子	［23］
	P1、P_cfiA、P_cepA、P_cfxA			内源启动子	［29］
	P_AM1-P_AM4		20倍	在P_BT1311特定位点插入26 bp序列	［33］
	P_BfP1E6及其变体		3×10⁴倍	源自噬菌体基因组，通过点突变获得	［34］
诱导型启动子	鼠李糖诱导系统	鼠李糖	104倍	由转录激活因子RhaR介导	［33］
	甘露聚糖诱导系统	甘露聚糖	100倍	基于多糖利用位点启动子	［35］
	葡聚糖诱导系统	葡聚糖	3-5倍	基于多糖利用位点启动子	［36］
	P_BT3324启动子	硫酸软骨素	60倍	由杂合双组分系统BT3334控制	［37］
	P_BT0268启动子	阿拉伯半乳聚糖	29倍	由杂合双组分系统BT0267控制	［38］
	IPTG诱导系统	IPTG	8倍和22倍	基于大肠杆菌LacI系统改造	［33］
	P1T_DP启动子	脱水四环素		将tetO2操纵子插入P1启动子，并与RBS组合优化	［39］
	胆汁酸诱导系统	胆汁酸	超过400倍	将操纵子插入P_cfxA启动子，用P_BT1311驱动调节基因	［40］

类型 Type	名称/系统 Name/System	诱导剂 Inducer	动态范围 Dynamic range	来源/策略 Source/Strategy	参考文献 References
组成型启动子	P_BT1311			内源启动子，基于σ因子	［23］
	P1、P_cfiA、P_cepA、P_cfxA			内源启动子	［29］
	P_AM1-P_AM4		20倍	在P_BT1311特定位点插入26 bp序列	［33］
	P_BfP1E6及其变体		3×10⁴倍	源自噬菌体基因组，通过点突变获得	［34］
诱导型启动子	鼠李糖诱导系统	鼠李糖	104倍	由转录激活因子RhaR介导	［33］
	甘露聚糖诱导系统	甘露聚糖	100倍	基于多糖利用位点启动子	［35］
	葡聚糖诱导系统	葡聚糖	3-5倍	基于多糖利用位点启动子	［36］
	P_BT3324启动子	硫酸软骨素	60倍	由杂合双组分系统BT3334控制	［37］
	P_BT0268启动子	阿拉伯半乳聚糖	29倍	由杂合双组分系统BT0267控制	［38］
	IPTG诱导系统	IPTG	8倍和22倍	基于大肠杆菌LacI系统改造	［33］
	P1T_DP启动子	脱水四环素		将tetO2操纵子插入P1启动子，并与RBS组合优化	［39］
	胆汁酸诱导系统	胆汁酸	超过400倍	将操纵子插入P_cfxA启动子，用P_BT1311驱动调节基因	［40］

基因编辑工具 Genome-editing tools	分子 Molecules	基因/蛋白质 Genes/Proteins	效果/机制 Effect/Mechanism	参考文献 References
同源重组	吲哚、吲哚硫酸盐（IS）	BT1492	敲除BT1492，阻断了色氨酸向吲哚的转化，降低尿毒症毒素（吲哚硫酸盐）的水平	［86］
同源重组	荚膜多糖（CPS）	CPS合成相关基因	删除不同CPS合成基因簇，揭示每种CPS赋予拟杆菌在多种生态位中定植与生存的能力	［87］
同源重组	硫酸酯酶、外膜囊泡（OMVs）	厌氧硫酸酯酶成熟酶（anSME）	anSME缺失菌株无法诱导小鼠发生结肠炎，证实OMVs与宿主免疫细胞之间的互作具有硫酸酯酶依赖性	［88］
CRISPR-Cas12	母乳寡糖（HMOs）	PUL33基因簇	GH33是介导唾液酸化HMOs利用的关键基因	［89］
同源重组	TGF-β、KGF-2	卵形拟杆菌的木聚糖调控系统	利用木聚糖调控系统实现了TGF-β和KGF-2等治疗分子的原位递送	［90-91］
异源表达	丁酸盐	丁酸盐生物合成途径相关基因	在B. thetaiotaomicron中异源表达丁酸盐合成途径，实现治疗浓度丁酸盐的合成，有助于维持肠道稳态	［92］
启动子替换、异源表达	草酸	草酸降解途径相关基因	表达草酸降解途径，将启动子替换为紫菜聚糖诱导型启动子，实现了工程菌在肠道中的可控定植并降解草酸，显著降低高草酸尿症水平	［93］
同源重组、异源表达	OmpA、SseB、H5N1、KGF-2	OmpA、SseB等基因	利用OMVs作为载体向黏膜有效递送疫苗抗原和治疗性蛋白，证明其作为黏膜生物制剂和递送平台的实用性和有效性	［94］

基因编辑工具 Genome-editing tools	分子 Molecules	基因/蛋白质 Genes/Proteins	效果/机制 Effect/Mechanism	参考文献 References
同源重组	吲哚、吲哚硫酸盐（IS）	BT1492	敲除BT1492，阻断了色氨酸向吲哚的转化，降低尿毒症毒素（吲哚硫酸盐）的水平	［86］
同源重组	荚膜多糖（CPS）	CPS合成相关基因	删除不同CPS合成基因簇，揭示每种CPS赋予拟杆菌在多种生态位中定植与生存的能力	［87］
同源重组	硫酸酯酶、外膜囊泡（OMVs）	厌氧硫酸酯酶成熟酶（anSME）	anSME缺失菌株无法诱导小鼠发生结肠炎，证实OMVs与宿主免疫细胞之间的互作具有硫酸酯酶依赖性	［88］
CRISPR-Cas12	母乳寡糖（HMOs）	PUL33基因簇	GH33是介导唾液酸化HMOs利用的关键基因	［89］
同源重组	TGF-β、KGF-2	卵形拟杆菌的木聚糖调控系统	利用木聚糖调控系统实现了TGF-β和KGF-2等治疗分子的原位递送	［90-91］
异源表达	丁酸盐	丁酸盐生物合成途径相关基因	在B. thetaiotaomicron中异源表达丁酸盐合成途径，实现治疗浓度丁酸盐的合成，有助于维持肠道稳态	［92］
启动子替换、异源表达	草酸	草酸降解途径相关基因	表达草酸降解途径，将启动子替换为紫菜聚糖诱导型启动子，实现了工程菌在肠道中的可控定植并降解草酸，显著降低高草酸尿症水平	［93］
同源重组、异源表达	OmpA、SseB、H5N1、KGF-2	OmpA、SseB等基因	利用OMVs作为载体向黏膜有效递送疫苗抗原和治疗性蛋白，证明其作为黏膜生物制剂和递送平台的实用性和有效性	［94］