Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor

  • Park, Ji-Hee (Department of Biological Sciences and Bioengineering, Inha University) ;
  • Park, Heung-Soon (Department of Biological Sciences and Bioengineering, Inha University) ;
  • Nah, Hee-Ju (Department of Biological Sciences and Bioengineering, Inha University) ;
  • Kang, Seung-Hoon (Department of Biological Sciences and Bioengineering, Inha University) ;
  • Choi, Si-Sun (Department of Biological Sciences and Bioengineering, Inha University) ;
  • Kim, Eung-Soo (Department of Biological Sciences and Bioengineering, Inha University)
  • Received : 2022.05.22
  • Accepted : 2022.06.06
  • Published : 2022.07.28
Download PDF
⟨ Previous Next ⟩

Abstract

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

Keywords

Acknowledgement

The authors appreciate the BAC-associated technical support provided by Bio S&T Inc. (Quebec, Canada). This work was funded by the National Research Foundation of Korea (Project No. NRF-2021R1A2C2012203).

References

  1. Genilloud O. 2017. Actinomycetes: still a source of novel antibiotics. Nat. Prod. Rep. 34: 1203-1232. https://doi.org/10.1039/C7NP00026J
  2. Xu M, Wright GD. 2019. Heterologous expression-facilitated natural products' discovery in actinomycetes. J. Ind. Microbiol. Biotechnol. 46: 415-431. https://doi.org/10.1007/s10295-018-2097-2
  3. Martinet L, Naome A, Deflandre B, Maciejewska M, Tellatin D, Tenconi E, et al. 2019. A single biosynthetic gene cluster is responsible for the production of bagremycin antibiotics and Ferroverdin Iron Chelators. mBio 10: e01230-19.
  4. Dhakal D, Sohng JK, Pandey RP. 2019. Engineering actinomycetes for biosynthesis of macrolactone polyketides. Microb. Cell Fact. 18: 137.
  5. Kim HJ, Kim MK, Lee MJ, Won HJ, Choi SS, Kim ES. 2015. Post-PKS tailoring steps of a disaccharide-containing polyene NPP in Pseudonocardia autotrophica. PLoS One 10: e0123270.
  6. Park HS, Kim HJ, Han CY, Nah HJ, Choi SS, Kim ES. 2020. Stimulated biosynthesis of an C10-Deoxy Heptaene NPP B2 via regulatory genes overexpression in Pseudonocardia autotrophica. Front. Microbiol. 11: 19.
  7. Fjaervik E, Zotchev SB. 2005. Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei. Appl. Microbiol. Biotechnol. 67: 436-443. https://doi.org/10.1007/s00253-004-1802-4
  8. Caffrey P, De Poire E, Sheehan J, Sweeney P. 2016. Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies. Appl. Microbiol. Biotech. 100: 3893-3908. https://doi.org/10.1007/s00253-016-7474-z
  9. Lee MJ, Kong D, Han K, Sherman DH, Bai L, Deng Z, et al. 2012. Structural analysis and biosynthetic engineering of a solubilityimproved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica. Appl. Microbiol. Biotechnol. 95: 157-168. https://doi.org/10.1007/s00253-012-3955-x
  10. Kim HJ, Han CY, Park JS, Oh SH, Kang SH, Choi SS, et al. 2018. Nystatin-like Pseudonocardia polyene B1, a novel disaccharidecontaining antifungal heptaene antibiotic. Sci. Rep. 8: 13584.
  11. Han CY, Jang JY, Kim HJ, Choi SS, Kim ES. 2019. Pseudonocardia strain improvement for stimulation of the di-sugar heptaene Nystatin-like Pseudonocardia polyene B1 biosynthesis. J. Ind. Microbiol. Biotechnol. 46: 649-655. https://doi.org/10.1007/s10295-019-02149-7
  12. Caffrey P, Hogan M, Song Y. 2022. New glycosylated polyene macrolides: refining the ore from genome mining. Antibiotics 11: 334.
  13. Nah HJ, Pyeon HR, Kang SH, Choi SS, Kim ES. 2017. Cloning and heterologous expression of a large-sized natural product biosynthetic gene cluster in Streptomyes Species. Front. Microbiol 8: 394.
  14. Choi SS, Katsuyama Y, Bai L, Deng Z, Ohnishi Y, Kim ES. 2018. Genome engineering for microbial natural product discovery. Curr. Opin. Microbiol. 45: 55-60.
  15. Zhang JJ, Yamanaka K, Tang X, Moore BS. 2019. Direct cloning and heterologous expression of natural product biosynthetic gene clusters by transformation-associated recombination. Methods Enzymol. 621: 87-110. https://doi.org/10.1016/bs.mie.2019.02.026
  16. Greunke C, Duell ER, D'Agostino PM, Glockle A, Lamm K, Gulder T. 2018. Direct pathway cloning (DiPaC) to unlock natural product biosynthetic potential. Metab. Eng. 47: 334-345. https://doi.org/10.1016/j.ymben.2018.03.010
  17. Larson CB, Crusemann M, Moore BS. 2017. PCR-independent method of transformation-associated recombination reveals the cosmomycin biosynthetic gene cluster in an ocean Streptomycete. J. Nat. Prod. 80: 1200-1204. https://doi.org/10.1021/acs.jnatprod.6b01121
  18. Kang HS, Kim ES. 2021. Recent advances in heterologous expression of natural product biosynthetic gene clusters in Streptomyces hosts. Curr. Opin. Biotechnol. 69: 118-127. https://doi.org/10.1016/j.copbio.2020.12.016
  19. Hun K, Kim SJ, Park SN, Oh JW. 2004. Antiviral effect of amphotericin B on Japanese Encephalitis virus replication. J. Microbiol. Biotechnol. 14: 121-127.
  20. Al-Khikani FHO. 2020. Amphotericin B as antiviral drug: possible efficacy against COVID-19. Ann. Thorac. Med. 15: 118-124. https://doi.org/10.4103/atm.ATM_147_20
  21. Khare P, Sahu U, Pandey SC, Samant M. 2020. Current approaches for target-specific drug discovery using natural compounds against SARS-CoV-2 infection. Virus Res. 290: 198169.
  22. Xu M, Wang Y, Zhao Z, Gao G, Huang SX, Kang Q, et al. 2016. Functional genome mining for metabolites encoded by large gene clusters through heterologous expression of a whole-genome bacterial artificial chromosome library in Streptomyces spp. Appl. Environ. Microbiol. 82: 5795-5805. https://doi.org/10.1128/AEM.01383-16
  23. Won HJ, Kim HJ, Jang JY, Kang SH, Choi SS, Kim ES. 2017. Improved recovery and biological activities of an engineered polyene NPP analogue in Pseudonocardia autotrophica. J. Ind. Microbiol. Biotechnol. 44: 1293-1299. https://doi.org/10.1007/s10295-017-1954-8
  24. Saez-Alvarez Y, Arias A, Del Aguila C, Agudo R. 2019. Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs. Sci. Rep. 9: 5397.
  25. Nah HJ. Woo MW, Choi SS, Kim ES. 2015. Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system. Microb. Cell Fact. 14: 140.
  26. Jeon HG, Seo J, Lee MJ, Han K, Kim ES. 2011. Analysis and functional expression of NPP pathway-specific regulatory genes in Pseudonocardia autotrophica. J. Ind. Microbiol. Biotechnol. 38: 573-579. https://doi.org/10.1007/s10295-011-0939-2
  27. Zhang B, Wang KB, Wang W, Bi SF, Mei YN, Deng XZ, et al. 2018. Discovery, biosynthesis, and heterologous production of Streptoseomycin, an anti-microaerophilic bacteria macrodilactone. Org. Lett. 20: 2967-2971. https://doi.org/10.1021/acs.orglett.8b01006
  28. Lu J, Long Q, Zhao Z, Chen L, He W, Hong J, et al. 2020. Engineering the erythromycin-producing strain Saccharopolyspora erythraea HOE107 for the heterologous production of polyketide antibiotics. Front. Microbiol. 11: 593217.