The application of the CRISPR/Cas9 editing system in the study of microbial natural products

doi:10.12211/2096-8280.2023-110

Abstract

Abstract:

Microorganisms, as an immense treasure trove of natural products, have consistently been a crucial source for researchers to explore and develop new natural products. Currently, the research methods involving gene editing tools for the discovery, biosynthesis, and metabolic engineering of natural products have garnered broad attention in this field. However, the traditional methods of genetic editing usually rely on the recombination ability from the host or introduced proteins. It's difficult to establish a general platform for all the bacteria mainly because of the complicated genetic background. This genetic diversity often causes laborious experimental operations in low efficiency. The CRISPR/Cas9 genetic editing system, with its unique and flexible targeting advantages, overcomes common limitations such as sequence homology or site constraints in other genetic editing methods and more likely to function in diverse bacteria species. This simplifies experimental procedures, enhances experimental efficiency, and promotes the development of the field of natural product research. This article primarily introduces the application of the CRISPR/Cas9 system in 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 elucidate the advantages of the CRISPR/Cas9 system in the research of natural products in the field of microorganisms. Finally, solutions are proposed for the challenges that the CRISPR/Cas9 system currently faces in overcoming recombination efficiency and host adaptability issues. Especially the CRISPR/Cas12a system which has broadened the application scope of CRISRP/Cas9 system by preferring different PAM site. In addition to the 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 field of natural products.

Key words: CRISPR/Cas9, natural products, microorganism, synthetic biology

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

CLC Number:

Q812

Zhen HUI, Xiaoyu TANG. The application of the CRISPR/Cas9 editing system in the study of microbial natural products[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2023-110.

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, DOI: 10.12211/2096-8280.2023-110.

Figures/Tables 4

References 73

1	BAKER D D, CHU M, OZA U, et al. The value of natural products to future pharmaceutical discovery[J]. Natural Product Reports, 2007, 24(6): 1225-1244.
2	NEWMAN D J, CRAGG G M. Natural products as sources of new drugs from 1981 to 2014[J]. Journal of Natural Products, 2016, 79(3): 629-661.
3	MOLONEY M G. Natural products as a source for novel antibiotics[J]. Trends in Pharmacological Sciences, 2016, 37(8): 689-701.
4	KIM H U, BLIN K, LEE S Y, et al. Recent development of computational resources for new antibiotics discovery[J]. Current Opinion in Microbiology, 2017, 39: 113-120.
5	TRACANNA V, JONG A D, MEDEMA M H, et al. Mining prokaryotes for antimicrobial compounds: from diversity to function[J]. FEMS Microbiology Reviews, 2017, 41(3): 417-429.
6	JIANG F G, DOUDNA J A. CRISPR-Cas9 structures and mechanisms[J]. Annual Review of Biophysics, 2017, 46: 505-529.
7	BARRANGOU R, FREMAUX C, DEVEAU H, et al. CRISPR provides acquired resistance against viruses in prokaryotes[J]. Science, 2007, 315(5819): 1709-1712.
8	MOJICA F J M, DÍEZ-VILLASEÑOR C, GARCÍA-MARTÍNEZ J, et al. Short motif sequences determine the targets of the prokaryotic CRISPR defence system[J]. Microbiology, 2009, 155(Pt 3): 733-740.
9	SAPRANAUSKAS R, GASIUNAS G, FREMAUX C, et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli [J]. Nucleic Acids Research, 2011, 39(21): 9275-9282.
10	DELTCHEVA E, CHYLINSKI K, SHARMA C M, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III[J]. Nature, 2011, 471(7340): 602-607.
11	STERNBERG S H, REDDING S, JINEK M, et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9[J]. Nature, 2014, 507(7490): 62-67.
12	JINEK M, CHYLINSKI K, FONFARA I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096): 816-821.
13	GASIUNAS G, BARRANGOU R, HORVATH P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(39): E2579-E2586.
14	JIANG W Y, BIKARD D, COX D, et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature Biotechnology, 2013, 31(3): 233-239.
15	THOMASON L C, COSTANTINO N, LI X T, et al. Recombineering: genetic engineering in Escherichia coli using homologous recombination[J]. Current Protocols, 2023, 3(2): e656.
16	QI L S, LARSON M H, GILBERT L A, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression[J]. Cell, 2013(5): 1173-1183.
17	BIKARD D, JIANG W Y, SAMAI P, et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system[J]. Nucleic Acids Research, 2013, 41(15): 7429-7437.
18	DOVE S L, HOCHSCHILD A. Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target[J]. Genes & Development, 1998, 12(5): 745-754.
19	HILTON I B, GERSBACH C A. Enabling functional genomics with genome engineering[J]. Genome Research, 2015, 25(10): 1442-1455.
20	KOMOR A C, KIM Y B, PACKER M S, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603): 420-424.
21	GAUDELLI N M, KOMOR A C, REES H A, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage[J]. Nature, 2017, 551(7681): 464-471.
22	LIU H B, JIANG H, HALTLI B, et al. Rapid cloning and heterologous expression of the meridamycin biosynthetic gene cluster using a versatile Escherichia coli-streptomyces artificial chromosome vector, pSBAC[J]. Journal of Natural Products, 2009, 72(3): 389-395.
23	NAH H J, WOO M W, CHOI S S, et al. Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system[J]. Microbial Cell Factories, 2015, 14: 140.
24	KIM J H, FENG Z Y, BAUER J D, et al. Cloning large natural product gene clusters from the environment: piecing environmental DNA gene clusters back together with TAR[J]. Biopolymers, 2010, 93(9): 833-844.
25	KOUPRINA N, LARIONOV V. Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology[J]. Chromosoma, 2016, 125(4): 621-632.
26	BIAN X Y, HUANG F, STEWART F A, et al. Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering[J]. Chembiochem, 2012, 13(13): 1946-1952.
27	FU J, BIAN X Y, HU S B, et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting[J]. Nature Biotechnology, 2012, 30(5): 440-446.
28	MURPHY K C. Phage recombinases and their applications[J]. Advances in Virus Research, 2012, 83: 367-414.
29	JIANG W J, ZHAO X J, GABRIELI T, et al. Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters[J]. Nature Communications, 2015, 6: 8101.
30	ROMÁN-HURTADO F, SÁNCHEZ-HIDALGO M, MARTÍN J, et al. One pathway, two cyclic non-ribosomal pentapeptides: heterologous expression of BE-18257 antibiotics and pentaminomycins from Streptomyces cacaoi CA-170360[J]. Microorganisms, 2021, 9(1): 135.
31	TAO W X, CHEN L, ZHAO C H, et al. In vitro packaging mediated one-step targeted cloning of natural product pathway[J]. ACS Synthetic Biology, 2019, 8(9): 1991-1997.
32	ENGHIAD B, HUANG C S, GUO F, et al. Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination[J]. Nature Communications, 2021, 12(1): 1171.
33	LIANG M D, LIU L S, XU F, et al. Activating cryptic biosynthetic gene cluster through a CRISPR-Cas12a-mediated direct cloning approach[J]. Nucleic Acids Research, 2022, 50(6): 3581-3592.
34	LIU Y K, TAO W X, WEN S S, et al. In vitro CRISPR/Cas9 system for efficient targeted DNA editing[J]. mBio, 2015, 6(6): e01714-15.
35	COBB R E, WANG Y J, ZHAO H M. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system[J]. ACS Synthetic Biology, 2015, 4(6): 723-728.
36	HUANG H, ZHENG G S, JIANG W H, et al. One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces [J]. Acta Biochimica et Biophysica Sinica, 2015, 47(4): 231-243.
37	TONG Y J, CHARUSANTI P, ZHANG L X, et al. CRISPR-Cas9 based engineering of actinomycetal genomes[J]. ACS Synthetic Biology, 2015, 4(9): 1020-1029.
38	ZENG H, WEN S S, XU W, et al. Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system[J]. Applied Microbiology and Biotechnology, 2015, 99(24): 10575-10585.
39	ZHANG J J, MOORE B S. Site-directed mutagenesis of large biosynthetic gene clusters via oligonucleotide recombineering and CRISPR/Cas9 targeting[J]. ACS Synthetic Biology, 2020, 9(7): 1917-1922.
40	ZHONG Z Y, GUO J H, DENG L, et al. Base editing in Streptomyces with Cas9-deaminase fusions[EB/OL]. bioRxiv. (20190507)[2023-11-01]. .
41	KUDO K, HASHIMOTO T, HASHIMOTO J, et al. In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives[J]. Nature Communications, 2020, 11(1): 4022.
42	THONG W L, ZHANG Y X, ZHUO Y, et al. Gene editing enables rapid engineering of complex antibiotic assembly lines[J]. Nature Communications, 2021, 12(1): 6872.
43	JI C H, KIM H, KANG H S. Synthetic inducible regulatory systems optimized for the modulation of secondary metabolite production in Streptomyces [J]. ACS Synthetic Biology, 2019, 8(3): 577-586.
44	LI L, ZHENG G S, CHEN J, et al. Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes[J]. Metabolic Engineering, 2017, 40: 80-92.
45	SONG Y F, HE S Q, ABDALLAH I I, et al. Engineering of multiple modules to improve amorphadiene production in Bacillus subtilis using CRISPR-Cas9[J]. Journal of Agricultural and Food Chemistry, 2021, 69(16): 4785-4794.
46	LEI C, LI S Y, LIU J K, et al. The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro [J]. Nucleic Acids Research, 2017, 45(9): e74.
47	ZHANG M M, WONG F T, WANG Y J, et al. CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters[J]. Nature Chemical Biology, 2017, 13: 607–609.
48	KANG H S, CHARLOP-POWERS Z, BRADY S F. Multiplexed CRISPR/Cas9- and TAR-mediated promoter engineering of natural product biosynthetic gene clusters in yeast[J]. ACS Synthetic Biology, 2016, 5(9): 1002-1010.
49	KIM H Y, JI C H, JE H W, et al. mpCRISTAR: multiple plasmid approach for CRISPR/Cas9 and TAR-mediated multiplexed refactoring of natural product biosynthetic gene clusters[J]. ACS Synthetic Biology, 2020, 9(1): 175-180.
50	KIM S H, LU W L, AHMADI M K, et al. Atolypenes, tricyclic bacterial sesterterpenes discovered using a multiplexed in vitro Cas9-TAR gene cluster refactoring approach[J]. ACS Synthetic Biology, 2019, 8(1): 109-118.
51	CULP E J, YIM G, WAGLECHNER N, et al. Hidden antibiotics in actinomycetes can be identified by inactivation of gene clusters for common antibiotics[J]. Nature Biotechnology, 2019, 37(10): 1149-1154.
52	AMERUOSO A, VILLEGAS KCAM M C, COHEN K P, et al. Activating natural product synthesis using CRISPR interference and activation systems in Streptomyces [J]. Nucleic Acids Research, 2022, 50(13): 7751-7760.
53	WANG J W, WANG A, LI K Y, et al. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning[J]. BioTechniques, 2015, 58(4): 161-170.
54	LEE N C O, LARIONOV V, KOUPRINA N. Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast[J]. Nucleic Acids Research, 2015, 43(8): e55.
55	ZHOU J T, WU R H, XUE X L, et al. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA[J]. Nucleic Acids Research, 2016, 44(14): e124.
56	HOHN B, WURTZ M, KLEIN B, et al. Phage lambda DNA packaging, in vitro [J]. Journal of Supramolecular Structure, 1974, 2(2-4): 302-317.
57	DATSENKO K A, WANNER B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(12): 6640-6645.
58	TISCHER B K, VON EINEM J, KAUFER B, et al. Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli [J]. BioTechniques, 2006, 40(2): 191-197.
59	SIEGL T, PETZKE L, WELLE E, et al. I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in Streptomyces [J]. Applied Microbiology & Biotechnology, 2010, 87(4): 1525-1532.
60	HERRMANN S, SIEGL T, LUZHETSKA M, et al. Site-specific recombination strategies for engineering actinomycete genomes[J]. Applied & Environmental Microbiology, 2012, 78(6): 1804-1812.
61	WOLF T, GREN T, THIEME E, et al. Targeted genome editing in the rare actinomycete Actinoplanes sp. SE50/110 by using the CRISPR/Cas9 System[J]. Journal of Biotechnology, 2016, 231: 122-128.
62	ZHANG Y, YUN K Y, HUANG H M, et al. Antisense RNA interference-enhanced CRISPR/Cas9 base editing method for improving base editing efficiency in Streptomyces lividans 66[J]. ACS Synthetic Biology, 2021, 10(5): 1053-1063.
63	SHAFEE T, LOWE R. Eukaryotic and prokaryotic gene structure[J]. WikiJournal of Medicine, 2017, 4(1): 2.
64	JIN L Q, JIN W R, MA Z C, et al. Promoter engineering strategies for the overproduction of valuable metabolites in microbes[J]. Applied Microbiology and Biotechnology, 2019, 103(21-22): 8725-8736.
65	WANG Y, CHENG H J, LIU Y, et al. In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing[J]. Nature Communications, 2021, 12(1): 678.
66	RUTLEDGE P J, CHALLIS G L. Discovery of microbial natural products by activation of silent biosynthetic gene clusters[J]. Nature Reviews Microbiology, 2015, 13(8): 509-523.
67	ZETSCHE B, GOOTENBERG J S, ABUDAYYEH O O, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 2015, 163(3): 759-771.
68	FONFARA I, RICHTER H, BRATOVIČ M, et al. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA[J]. Nature, 2016, 532(7600): 517-521.
69	ZETSCHE B, HEIDENREICH M, MOHANRAJU P, et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array[J]. Nature Biotechnology, 2017, 35: 31-34.
70	LI L, WEI K K, ZHENG G S, et al. CRISPR-Cpf1-assisted multiplex genome editing and transcriptional repression in Streptomyces [J]. Applied and Environmental Microbiology, 2018, 84(18): e00827-18.
71	SCHILLING C, KOFFAS M A G, SIEBER V, et al. Novel prokaryotic CRISPR-Cas12a-based tool for programmable transcriptional activation and repression[J]. ACS Synthetic Biology, 2020, 9(12): 3353-3363.
72	LI S Y, ZHAO G P, WANG J. C-brick: a new standard for assembly of biological parts using Cpf1[J]. ACS Synthetic Biology, 2016, 5(12): 1383-1388.
73	BAUMAN K D, BUTLER K S, MOORE B S, et al. Genome mining methods to discover bioactive natural products[J]. Natural Product Reports, 2021, 38(11): 2100-2129.

	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	B. subtilisstr. 168	[29]
		Jadomycin		S. venezuelae ISP52030
		Chlortetracycline		S. aureofaciens ATCC 10762
		Pentaminomycins A-H		Streptomyces cacaoi CA-170360	[30]
		BH-18257 A-C		Streptomyces cacaoi CA-170360	[30]
	ICE & λ packaging system	Tu3010		S. thiolactonus NRRL 15439	[31]
	ICE & λ packaging system	Sisomicin		M. inyoensis 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		P. 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		A. 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	B. subtilisstr. 168	[29]
		Jadomycin		S. venezuelae ISP52030
		Chlortetracycline		S. aureofaciens ATCC 10762
		Pentaminomycins A-H		Streptomyces cacaoi CA-170360	[30]
		BH-18257 A-C		Streptomyces cacaoi CA-170360	[30]
	ICE & λ packaging system	Tu3010		S. thiolactonus NRRL 15439	[31]
	ICE & λ packaging system	Sisomicin		M. inyoensis 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		P. 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		A. 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]

[1]	Yanyan YUAN, Huifang CHEN, Sihui YANG, Honghui WANG, Zhou NIE. Engineering artificial receptor cluster: chemical synthetic biology strategies and emerging applications [J]. Synthetic Biology Journal, 2024, 5(1): 53-76.
[2]	Jingyu ZHAO, Jian ZHANG, Qingsheng QI, Qian WANG. Research progress in biosensors based on bacterial two-component systems [J]. Synthetic Biology Journal, 2024, 5(1): 38-52.
[3]	Qian MENG, Cong YIN, Weiren HUANG. Tumor organoids and their research progress in synthetic biology [J]. Synthetic Biology Journal, 2024, 5(1): 191-201.
[4]	Xiaojie GUO, Xingjin JIAN, Liyan WANG, Chong ZHANG, Xinhui XING. Progress in bioreactors and instruments for phenotype testing with synthetic biology research [J]. Synthetic Biology Journal, 2024, 5(1): 16-37.
[5]	Qiang ZHOU, Dawei ZHOU, Jingxiang SUN, Jingnan WANG, Wankui JIANG, Wenming ZHANG, Yujia JIANG, Fengxue XIN, Min JIANG. Research progress in synthesis of astaxanthin by microbial fermentation [J]. Synthetic Biology Journal, 2024, 5(1): 126-143.
[6]	Duo LIU, Peiyuan LIU, Lianyue LI, Yaxin WANG, Yuhui CUI, Huimin XUE, Hanjie WANG. Design and synthesis of engineered extracellular vesicles and their biomedical applications [J]. Synthetic Biology Journal, 2024, 5(1): 154-173.
[7]	Han SUN, Jin LIU. Research progress and prospects in lipid metabolic engineering of eukaryotic microalgae [J]. Synthetic Biology Journal, 2023, 4(6): 1140-1160.
[8]	Huili SUN, Jinyu CUI, Guodong LUAN, Xuefeng LYU. Progress of cyanobacterial synthetic biotechnology for efficient light-driven carbon fixation and ethanol production [J]. Synthetic Biology Journal, 2023, 4(6): 1161-1177.
[9]	Xiongying YAN, Zhen WANG, Jiyun LOU, Haoyu ZHANG, Xingyu HUANG, Xia WANG, Shihui YANG. Progress in the construction of microbial cell factories for efficient biofuel production [J]. Synthetic Biology Journal, 2023, 4(6): 1082-1121.
[10]	Chenyue ZHANG, Yingqun MA, Xing WANG, Rongzhan FU, Jiwei HUANG, Xiufu HUA, Daidi FAN, Qiang FEI. Progress in the bioconversion of biogas into sustainable aviation fuel [J]. Synthetic Biology Journal, 2023, 4(6): 1246-1258.
[11]	Yaru CHEN, Yingxiu CAO, Hao SONG. Advances and applications of gene editing and transcriptional regulation in electroactive microorganisms [J]. Synthetic Biology Journal, 2023, 4(6): 1281-1299.
[12]	Guomiao ZHAO, Xin YANG, Yuan ZHANG, Jing WANG, Jian TAN, Chao WEI, Nana ZHOU, Fan LI, Xiaoyan WANG. Biofoundry and its industrial application [J]. Synthetic Biology Journal, 2023, 4(5): 892-903.
[13]	Zhidian DIAO, Xixian WANG, Qing SUN, Jian XU, Bo MA. Advances and applications of single-cell Raman spectroscopy testing and sorting equipment [J]. Synthetic Biology Journal, 2023, 4(5): 1020-1035.
[14]	Hui LU, Fangli ZHANG, Lei HUANG. Establishment of iBioFoundry for synthetic biology applications [J]. Synthetic Biology Journal, 2023, 4(5): 877-891.
[15]	Zhonghu BAI, He REN, Jianqi NIE, Yang SUN. The recent progresses and applications of in-parallel fermentation technology [J]. Synthetic Biology Journal, 2023, 4(5): 904-915.