DOI QR코드

DOI QR Code

A CRISPR/Cas9 Cleavage System for Capturing Fungal Secondary Metabolite Gene Clusters

  • Xu, Xinran (State Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences) ;
  • Feng, Jin (State Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences) ;
  • Zhang, Peng (State Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences) ;
  • Fan, Jie (State Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences) ;
  • Yin, Wen-Bing (State Key Laboratory of Mycology and CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences)
  • 투고 : 2020.08.20
  • 심사 : 2020.10.26
  • 발행 : 2021.01.28

초록

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

키워드

참고문헌

  1. Romsdahl J, Wang CCC. 2019. Recent advances in the genome mining of Aspergillus secondary metabolites (covering 2012-2018). Medchemcomm. 10: 840-866. https://doi.org/10.1039/C9MD00054B
  2. Araujo R, Sampaio-Maia B. 2018. Fungal genomes and genotyping. Adv. Appl. Microbiol. 102: 37-81. https://doi.org/10.1016/bs.aambs.2017.10.003
  3. Sharma KK. 2016. Fungal genome sequencing: basic biology to biotechnology. Crit. Rev. Biotechnol. 36: 743-759. https://doi.org/10.3109/07388551.2015.1015959
  4. Li W, Yin WB. 2019. Genetic mining of the "dark matter" in fungal natural products. Sci. China Life Sci. 62: 1250-1252. https://doi.org/10.1007/s11427-019-9818-3
  5. Wang B, Guo F, Dong SH, Zhao H. 2019. Activation of silent biosynthetic gene clusters using transcription factor decoys. Nat. Chem. Biol. 15: 111-114. https://doi.org/10.1038/s41589-018-0187-0
  6. Keller NP. 2019. Fungal secondary metabolism: regulation, function and drug discovery. Nat. Rev. Microbiol. 17: 167-180. https://doi.org/10.1038/s41579-018-0121-1
  7. Chiang YM, Oakley CE, Ahuja M, Entwistle R, Schultz A, Chang S-L, et al. 2013. An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans. J. Am. Chem. Soc. 135: 7720-7731. https://doi.org/10.1021/ja401945a
  8. Harvey CJB, Tang M, Schlecht U, Horecka J, Fisher C, Lin H-C, et al. 2018. Hex: a heterologous expression platform for the discovery of fungal natural products. Sci. Adv. 4: eaar5459. https://doi.org/10.1126/sciadv.aar5459
  9. Chiang YM, Szewczyk E, Davidson AD, Keller N, Oakley BR, Wang CC, et al. 2009. A gene cluster containing two fungal polyketide synthases encodes the biosynthetic pathway for a polyketide, asperfuranone, in Aspergillus nidulans. J. Am. Chem. Soc. 131: 2965- 2970. https://doi.org/10.1021/ja8088185
  10. Nielsen MT, Nielsen JB, Anyaogu DC, Holm DK, Nielsen KF, Larsen TO, et al. 2013. Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach. PLoS One 8: e72871. https://doi.org/10.1371/journal.pone.0072871
  11. WB Yin, YH Chooi, Smith AR, Cacho RA, Hu Y, White TC, et al. 2013. Discovery of cryptic polyketide metabolites from dermatophytes using heterologous expression in Aspergillus nidulans. ACS Synth. Biol. 2: 629-634. https://doi.org/10.1021/sb400048b
  12. Lin TS, Chiang YM, Wang CC. 2016. Biosynthetic pathway of the reduced polyketide product citreoviridin in Aspergillus terreus var. Aureus revealed by heterologous expression in Aspergillus nidulans. Org. Lett. 18: 1366-1369. https://doi.org/10.1021/acs.orglett.6b00299
  13. Chiang YM, Ahuja M, Oakley CE, Entwistle R, Asokan A, Zutz C, et al. 2016. Development of genetic dereplication strains in Aspergillus nidulans results in the discovery of aspercryptin. Angew. Chem. Int. Ed. Engl. 55: 1662-1665. https://doi.org/10.1002/anie.201507097
  14. Bok JW, Ye R, Clevenger KD, Mead D, Wagner M, Krerowica A, et al. 2015. Fungal artificial chromosomes for mining of the fungal secondary metabolome. BMC Genomics 16: 343. https://doi.org/10.1186/s12864-015-1561-x
  15. Kouprina N, Larionov V. 2006. Tar cloning: insights into gene function, long-range haplotypes and genome structure and evolution. Nat. Rev. Genet. 7: 805-812. https://doi.org/10.1038/nrg1943
  16. Lee NC, Larionov V, Kouprina N. 2015. Highly efficient CRISPR/Cas9-mediated tar cloning of genes and chromosomal loci from complex genomes in yeast. Nucleic Acids Res. 43: e55. https://doi.org/10.1093/nar/gkv112
  17. Clevenger KD, JW Bok, Ye R, Miley GP, Verdan MH, Velk T, et al. 2017. A scalable platform to identify fungal secondary metabolites and their gene clusters. Nat. Chem. Biol. 13: 895-901. https://doi.org/10.1038/nchembio.2408
  18. Doudna JA, Charpentier E. 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346: 1258096. https://doi.org/10.1126/science.1258096
  19. Hsu PD, Lander ES, Zhang F. 2014. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157: 1262-1278. https://doi.org/10.1016/j.cell.2014.05.010
  20. Liu Y, Tao W, Wen S, Li Z, Yang A, Deng Z, et al. 2015. In vitro CRISPR/Cas9 system for efficient targeted DNA editing. mBio 6: e01714-01715.
  21. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, Zhu TF. 2015. Cas9-assisted targeting of chromosome segments catch enables onestep targeted cloning of large gene clusters. Nat. Commun. 6: 8101. https://doi.org/10.1038/ncomms9101
  22. Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, et al. 2015. CRISPR/Cas9 nuclease cleavage combined with gibson assembly for seamless cloning. Biotechniques 58: 161-170. https://doi.org/10.2144/000114261
  23. Zhang C, Lu L. 2017. Precise and efficient in-frame integration of an exogenous gfp tag in Aspergillus fumigatus by a CRISPR system. Methods Mol. Biol. 1625: 249-258. https://doi.org/10.1007/978-1-4939-7104-6_17
  24. Zhang C, Meng X, Wei X, Lu L. 2016. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet. Biol. 86: 47-57. https://doi.org/10.1016/j.fgb.2015.12.007
  25. Zhang P, Wang X, Fan A, Zheng Y, Liu X, Wang S, et al. 2017. A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in pestalotiopsis fici. Mol. Microbiol. 105: 469-483. https://doi.org/10.1111/mmi.13711
  26. Cacho RA, Jiang W, Chooi YH Walsh CT, Tang Y. 2012. Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440. J. Am. Chem. Soc. 134: 16781-16790. https://doi.org/10.1021/ja307220z
  27. Jiang W, Zhu TF. 2016. Targeted isolation and cloning of 100-kb microbial genomic sequences by cas9-assisted targeting of chromosome segments. Nat. Protoc. 11: 960-975. https://doi.org/10.1038/nprot.2016.055
  28. Xu X, Liu L, Zhang F, Wang W, Ki J, Guo L, et al. 2014. Identification of the first diphenyl ether gene cluster for pestheic acid biosynthesis in plant endophyte pestalotiopsis fici. Chembiochem 15: 284-292. https://doi.org/10.1002/cbic.201300626
  29. Wu G, Zhou H, Zhang P, Wang W, Li J, Gou L, et al. 2016. Polyketide production of pestaloficiols and macrodiolide ficiolides revealed by manipulations of epigenetic regulators in an endophytic fungus. Org. Lett. 18: 1832-1835. https://doi.org/10.1021/acs.orglett.6b00562
  30. Anders C, Jinek M. 2014. In vitro enzymology of cas9. Methods Enzymol. 546: 1-20. https://doi.org/10.1016/B978-0-12-801185-0.00001-5
  31. Alberti F, Foster GD, Bailey AM. 2017. Natural products from filamentous fungi and production by heterologous expression. Appl. Microbiol. Biotechnol. 101: 493-500. https://doi.org/10.1007/s00253-016-8034-2
  32. Qiao Y-M, Yu R-L, Zhu P. 2019. Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites. RSC Adv. 9: 35124-35134. https://doi.org/10.1039/C9RA06908A
  33. Cobb RE, Zhao H. 2012. Direct cloning of large genomic sequences. Nat. Biotechnol. 30: 405-406. https://doi.org/10.1038/nbt.2207
  34. Kouprina N, Noskov VN, Larionov V. 2020. Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast. Nat. Protoc. 15: 734-749. https://doi.org/10.1038/s41596-019-0280-1
  35. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas9 systems. Nat. Biotechnol. 31: 233-239. https://doi.org/10.1038/nbt.2508
  36. Karvelis T, Gasiunas G, Siksnys V. 2013. Programmable DNA cleavage in vitro by cas9. Biochem. Soc. Trans. 41: 1401-1406. https://doi.org/10.1042/BST20130164
  37. Huang MY, Mitchell AP. 2017. Marker recycling in Candida albicans through CRISPR-Cas9-induced marker excision. mSphere 2: 2.
  38. Krappmann S. 2017. CRISPR-Cas9, the new kid on the block of fungal molecular biology. Med. Mycol. 55: 16-23. https://doi.org/10.1093/mmy/myw097
  39. Sarkari P, Marx H, Blumhoff ML, Mattanovich D, Sauer M, Steiger MG. 2017. An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger. Bioresour. Technol. 245: 1327-1333. https://doi.org/10.1016/j.biortech.2017.05.004
  40. Muller H, Annaluru N, Schwerzmann JW, Rhichardson SM, Dymond J, Cooper EM, et al. 2012. Assembling large DNA segments in yeast. Methods Mol. Biol. 852: 133-150. https://doi.org/10.1007/978-1-61779-564-0_11
  41. Wortman JR, Fedorova N, Crabtree J, Jordar V, Maiti BJ, Amedeo P, et al. 2006. Whole genome comparison of the A. fumigatus family. Med. Mycol. 44: S3-S7.
  42. Wang X, Wu F, Liu L, Liu X, Che Y, Keller NP, et al. 2015. The bzip transcription factor pfzipa regulates secondary metabolism and oxidative stress response in the plant endophytic fungus pestalotiopsis fici. Fungal Genet. Biol. 81: 221-228. https://doi.org/10.1016/j.fgb.2015.03.010