CRISPR/Cas9编辑系统在微生物天然产物研究中的应用

doi:10.12211/2096-8280.2023-110

摘要/Abstract

摘要：

微生物作为天然产物的巨大宝库，一直以来都是研究人员挖掘和开发新的活性化合物的重要来源。目前，利用基因编辑工具发现、生物合成和代谢调控天然产物的研究方法受到该领域研究者的广泛关注。CRISPR/Cas9遗传编辑系统以其独特的灵活靶向优势克服了其他遗传编辑方法常见的对序列同源或位点限制，简化了实验步骤，提高了实验效率，促进了天然产物研究领域的发展。本文主要介绍CRISPR/Cas9系统在微生物天然产物发现、生物合成和工程改造方面的应用，分别从CRISPR/Cas9系统的发展、天然产物生物合成基因簇的克隆和遗传编辑、天然产物结构衍生化和代谢调节、沉默天然产物基因簇的激活这几个方面阐述CRISPR/Cas9系统在微生物天然产物研究领域的优势。最后，针对CRISPR/Cas9系统无法克服的重组效率和宿主适应性问题提供了可行的解决思路。相信随着合成生物学和信息技术的发展，越来越多的与CRISPR/Cas9系统相关的遗传操作工具和方法会被开发，将不断推动天然产物领域的发展进步。

关键词: CRISPR/Cas9, 天然产物, 微生物, 合成生物学, 异源表达, 结构衍生化, 启动子工程, 代谢工程

Abstract:

Microorganisms have consistently been a crucial source for researchers to explore and develop new natural products. Currently, research methods involving gene editing tools for the discovery, biosynthesis, and metabolic engineering of natural products have garnered broad attention in this field. However, traditional methods for gene editing usually rely on the recombination ability of the host or introduced proteins. It’s difficult to establish a general platform for all bacteria mainly because of their complicated genetic background. This genetic diversity often causes laborious experimental operations with low efficiency. The CRISPR/Cas9 gene editing system, with its unique and flexible targeting advantages, overcomes common limitations such as sequence homology or site constraint in other gene editing methods and thus is more likely to function in diverse bacteria species. This simplifies experimental procedures, enhances work efficiency, and promotes the development of natural product research. This article introduces the applications of the CRISPR/Cas9 system for the discovery, biosynthesis, and metabolic engineering of natural products in microorganisms. It covers the development of the CRISPR/Cas9 system, cloning and genetic editing of natural product biosynthetic gene clusters, structural derivatization and metabolic engineering of natural products, and the activation of silenced natural product biosynthetic gene clusters. These aspects highlight the advantages of the CRISPR/Cas9 system in the research of natural products with microorganisms. Finally, solutions are proposed for addressing challenges that the CRISPR/Cas9 system currently faces in overcoming low recombination efficiency and host adaptability issues. Especially the CRISPR/Cas12a system which has broadened applications of the CRISRP/Cas9 system by preferring different PAM sites. In addition to functions that CRISPR/Cas9 system has realized, its potent multiple targeting ability further enhances the efficiency of target editing. It is believed that with the development of synthetic biology and information technology, an increasing number of genetic manipulation tools and methods related to the CRISPR/Cas9 system will be developed, continually driving progress in the research of natural products.

Key words: CRISPR/Cas9, natural products, microorganisms, synthetic biology, heterologous expression, structure derivative, promoter engineering, metabolism engineering

中图分类号:

Q812

惠真, 唐啸宇. CRISPR/Cas9编辑系统在微生物天然产物研究中的应用[J]. 合成生物学, 2024, 5(3): 658-671.

Zhen HUI, Xiaoyu TANG. Applications of the CRISPR/Cas9 editing system in the study of microbial natural products[J]. Synthetic Biology Journal, 2024, 5(3): 658-671.

图/表 4

参考文献 73

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	策略	生物合成基因簇	功能	生物合成基因簇来源/宿主	参考文献
基因簇克隆	CATCH	Bacillaene	基因簇线性化	Bacillus subtilis str. 168	[29]
		Jadomycin		Streptomyces venezuelae ISP52030
		Chlortetracycline		S. aureofaciens ATCC 10762
		Pentaminomycins A-H		S. cacaoi CA-170360	[30]
		BH-18257 A-C		S. cacaoi CA-170360	[30]
	ICE & λ packaging system	Tu3010		S. thiolactonus NRRL 15439	[31]
	ICE & λ packaging system	Sisomicin		Micromonospora inyonensis DSM 46123	[31]
	CAPTURE	43 uncharacterized BGCs		Streptomyces，Bacillus	[32]
	CAT-FISHING	Marinolactam A		Micromonospora sp. 181	[33]
基因簇遗传编辑	ICE	Tetronate RK-682	遗传突变	Streptomyces sp. Strain 88-682	[34]
	ICE	Holomycin		S. clavuligerus TK24	[34]
	pCRISPomyces	Undecylprodigiosin, Actinorhodin		S. lividans 66	[35]
		Phosphinothricin tripeptide		S. viridochromogenes DSM 40736
		Macrolactam, Lanthipeptide		S. albus J1074
	CRISPR/Cas9	Actinorhodin, Undecylprodigiosin		S. coelicolor M14	[36]
	CRISPR/Cas9-LigD	Actinorhodin		S. coelicolor A3(2)	[37]
	CRISPRi	Actinorhodin		S. coelicolor A3(2)	[37]
	CRISPR/ Cas9-CodA(sm)	Actinorhodin		S. coelicolor M145	[38]
	CRISPR/Cas9	Violacein		E. coli HME68	[39]
	CRISPR/Cas9	Thalassospiramides		Pseudomonas putida EM383	[39]
	CBE/ABE	Undecylprodigiosin, Actinorhodin		S. coelicolor M145	[40]
	CBE/ABE	Avermectin		S. avermitilis MA4680	[40]
产物衍生化	CRISPR/Cas9 & Gibson Assembly	Rapamycin	组装模块编辑	S. avermitilis SUKA	[41]
产物衍生化	CRISPR/Cas9	Enduracidin	组装模块编辑	Streptomyces fungicidicus ATCC 21013	[42]
产物代谢调节	CRISPR/Cas9 & TAR	Actinorhodin	启动子工程	S. albus J1074	[43]
	MSGE	Pristinamyicn Ⅱ, Chloramphenicol, YM-216391	多拷贝	S. pristinaespiralis HCCB10218 S. coelicolor M145	[44]
	CRISPR/Cas9	Amorphadiene	启动子工程,遗传突变, 多拷贝	Bacillus subtilis	[45]
	CCTL	Actinorhodin	启动子工程	Streptomyces sp. 4F	[46]
基因簇激活	CRISPR/Cas9	Alteramide A, Macrolactam 2, FR-900098	启动子工程	S. roseosporus NRRL15998	[47]
	mCRISTAR	Tetarimycin, Lazarimide, AB1210		S. albus	[48]
	mpCRISTAR	Acinorhodin		S. cerevisiae BY4727	[49]
	miCASTAR	Atolypene		Amycolatopsis tolypomycina NRRL B-24205	[50]
	CRISPR/Cas9-LigD	Amicetin	遗传突变	Streptomyces WAC6237	[51]
		Thiolactomycin, 5-chloro-3-formylindole		Streptomyces WAC5374
		Phenanthroviridin aglycone		Streptomyces WAC8241
	CRISPRi/CRISPRa	Jadomycinb	转录调控	Streptomyces venezuelae	[52]

	策略	生物合成基因簇	功能	生物合成基因簇来源/宿主	参考文献
基因簇克隆	CATCH	Bacillaene	基因簇线性化	Bacillus subtilis str. 168	[29]
		Jadomycin		Streptomyces venezuelae ISP52030
		Chlortetracycline		S. aureofaciens ATCC 10762
		Pentaminomycins A-H		S. cacaoi CA-170360	[30]
		BH-18257 A-C		S. cacaoi CA-170360	[30]
	ICE & λ packaging system	Tu3010		S. thiolactonus NRRL 15439	[31]
	ICE & λ packaging system	Sisomicin		Micromonospora inyonensis DSM 46123	[31]
	CAPTURE	43 uncharacterized BGCs		Streptomyces，Bacillus	[32]
	CAT-FISHING	Marinolactam A		Micromonospora sp. 181	[33]
基因簇遗传编辑	ICE	Tetronate RK-682	遗传突变	Streptomyces sp. Strain 88-682	[34]
	ICE	Holomycin		S. clavuligerus TK24	[34]
	pCRISPomyces	Undecylprodigiosin, Actinorhodin		S. lividans 66	[35]
		Phosphinothricin tripeptide		S. viridochromogenes DSM 40736
		Macrolactam, Lanthipeptide		S. albus J1074
	CRISPR/Cas9	Actinorhodin, Undecylprodigiosin		S. coelicolor M14	[36]
	CRISPR/Cas9-LigD	Actinorhodin		S. coelicolor A3(2)	[37]
	CRISPRi	Actinorhodin		S. coelicolor A3(2)	[37]
	CRISPR/ Cas9-CodA(sm)	Actinorhodin		S. coelicolor M145	[38]
	CRISPR/Cas9	Violacein		E. coli HME68	[39]
	CRISPR/Cas9	Thalassospiramides		Pseudomonas putida EM383	[39]
	CBE/ABE	Undecylprodigiosin, Actinorhodin		S. coelicolor M145	[40]
	CBE/ABE	Avermectin		S. avermitilis MA4680	[40]
产物衍生化	CRISPR/Cas9 & Gibson Assembly	Rapamycin	组装模块编辑	S. avermitilis SUKA	[41]
产物衍生化	CRISPR/Cas9	Enduracidin	组装模块编辑	Streptomyces fungicidicus ATCC 21013	[42]
产物代谢调节	CRISPR/Cas9 & TAR	Actinorhodin	启动子工程	S. albus J1074	[43]
	MSGE	Pristinamyicn Ⅱ, Chloramphenicol, YM-216391	多拷贝	S. pristinaespiralis HCCB10218 S. coelicolor M145	[44]
	CRISPR/Cas9	Amorphadiene	启动子工程,遗传突变, 多拷贝	Bacillus subtilis	[45]
	CCTL	Actinorhodin	启动子工程	Streptomyces sp. 4F	[46]
基因簇激活	CRISPR/Cas9	Alteramide A, Macrolactam 2, FR-900098	启动子工程	S. roseosporus NRRL15998	[47]
	mCRISTAR	Tetarimycin, Lazarimide, AB1210		S. albus	[48]
	mpCRISTAR	Acinorhodin		S. cerevisiae BY4727	[49]
	miCASTAR	Atolypene		Amycolatopsis tolypomycina NRRL B-24205	[50]
	CRISPR/Cas9-LigD	Amicetin	遗传突变	Streptomyces WAC6237	[51]
		Thiolactomycin, 5-chloro-3-formylindole		Streptomyces WAC5374
		Phenanthroviridin aglycone		Streptomyces WAC8241
	CRISPRi/CRISPRa	Jadomycinb	转录调控	Streptomyces venezuelae	[52]

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