In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
11 08 2020
11 08 2020
Historique:
received:
07
11
2019
accepted:
10
07
2020
entrez:
13
8
2020
pubmed:
13
8
2020
medline:
12
9
2020
Statut:
epublish
Résumé
One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.
Identifiants
pubmed: 32782248
doi: 10.1038/s41467-020-17769-2
pii: 10.1038/s41467-020-17769-2
pmc: PMC7419507
doi:
Substances chimiques
Biological Products
0
Polyketide Synthases
79956-01-7
Sirolimus
W36ZG6FT64
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
4022Références
Giordanetto, F. & Kihlberg, J. Macrocyclic drugs and clinical candidates: what can medicinal chemists learn from their properties? J. Med. Chem.57, 278–295 (2014).
pubmed: 24044773
Bogdan, A. R., Davies, N. L. & James, K. Comparison of diffusion coefficients for matched pairs of macrocyclic and linear molecules over a drug-like molecular weight range. Org. Biomol. Chem.9, 7727–7733 (2011).
pubmed: 21979439
Mallinson, J. & Collins, I. Macrocycles in new drug discovery. Future Med. Chem.4, 1409–1438 (2012).
pubmed: 22857532
Driggers, E. M., Hale, S. P., Lee, J. & Terrett, N. K. The exploration of macrocycles for drug discovery–an underexploited structural class. Nat. Rev. Drug Discov.7, 608–624 (2008).
pubmed: 18591981
Hann, M. M. & Keseru, G. M. Finding the sweet spot: the role of nature and nurture in medicinal chemistry. Nat. Rev. Drug Discov.11, 355–365 (2012).
pubmed: 22543468
Bauer, A. & Bronstrup, M. Industrial natural product chemistry for drug discovery and development. Nat. Prod. Rep.31, 35–60 (2014).
pubmed: 24142193
Medema, M. H., Cimermancic, P., Sali, A., Takano, E. & Fischbach, M. A. A systematic computational analysis of biosynthetic gene cluster evolution: lessons for engineering biosynthesis. PLoS Comput. Biol.10, e1004016 (2014).
pubmed: 25474254
pmcid: 4256081
Zhang, L. et al. Characterization of giant modular PKSs provides insight into genetic mechanism for structural diversification of aminopolyol polyketides. Angew. Chem. Int. Ed. Engl.56, 1740–1745 (2017).
pubmed: 28133950
Jenke-Kodama, H. & Dittmann, E. Evolution of metabolic diversity: insights from microbial polyketide synthases. Phytochemistry70, 1858–1866 (2009).
pubmed: 19619887
Wlodek, A. et al. Diversity oriented biosynthesis via accelerated evolution of modular gene clusters. Nat. Commun.8, 1206 (2017).
pubmed: 29089518
pmcid: 5663706
McDaniel, R. et al. Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products. Proc. Natl Acad. Sci. USA96, 1846–1851 (1999).
pubmed: 10051557
Sugimoto, Y., Ding, L., Ishida, K. & Hertweck, C. Rational design of modular polyketide synthases: morphing the aureothin pathway into a luteoreticulin assembly line. Angew. Chem. Int. Ed. Engl.53, 1560–1564 (2014).
pubmed: 24402879
Kushnir, S. et al. Minimally invasive mutagenesis gives rise to a biosynthetic polyketide library. Angew. Chem. Int. Ed. Engl.51, 10664–10669 (2012).
pubmed: 22996590
Hashimoto, T. et al. Biosynthesis of quinolidomicin, the largest known macrolide of terrestrial origin: identification and heterologous expression of a biosynthetic gene cluster over 200 kb. Org. Lett.20, 7996–7999 (2018).
pubmed: 30543302
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337, 816–821 (2012).
pubmed: 22745249
pmcid: 6286148
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods6, 343–345 (2009).
pubmed: 19363495
Komatsu, M. et al. Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites. ACS Synth. Biol.2, 384–396 (2013).
pubmed: 23654282
pmcid: 3932656
Guduru, S. K. R. & Arya, P. Synthesis and biological evaluation of rapamycin-derived, next generation small molecules. Medchemcomm9, 27–43 (2018).
pubmed: 30108899
Schwecke, T. et al. The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proc. Natl Acad. Sci. USA92, 7839–7843 (1995).
pubmed: 7644502
Kuscer, E. et al. Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus. J. Bacteriol.189, 4756–4763 (2007).
pubmed: 17468238
pmcid: 1913445
Choi, J., Chen, J., Schreiber, S. L. & Clardy, J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science273, 239–242 (1996).
pubmed: 8662507
Caufield, C. E., Musser, J. H. & Rinker, J. M. Hydrogenated rapamycin derivatives. United States patent US5023262 (1990).
Kim, J. H., Komatsu, M., Shin-Ya, K., Omura, S. & Ikeda, H. Distribution and functional analysis of the phosphopantetheinyl transferase superfamily in Actinomycetales microorganisms. Proc. Natl Acad. Sci. USA115, 6828–6833 (2018).
pubmed: 29903901
Kellenberger, L. et al. A polylinker approach to reductive loop swaps in modular polyketide synthases. ChemBioChem9, 2740–2749 (2008).
pubmed: 18937219
Donadio, S., McAlpine, J. B., Sheldon, P. J., Jackson, M. & Katz, L. An erythromycin analog produced by reprogramming of polyketide synthesis. Proc. Natl Acad. Sci. USA90, 7119–7123 (1993).
pubmed: 8346223
Zheng, J., Gay, D. C., Demeler, B., White, M. A. & Keatinge-Clay, A. T. Divergence of multimodular polyketide synthases revealed by a didomain structure. Nat. Chem. Biol.8, 615–621 (2012).
pubmed: 22634636
pmcid: 3477503
Strom, T. et al. Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns. J. Mass Spectrom.46, 615–624 (2011).
pubmed: 21671437
Artzi, N. et al. Sustained efficacy and arterial drug retention by a fast drug eluting cross-linked fatty acid coronary stent coating. Ann. Biomed. Eng.44, 276–286 (2016).
pubmed: 26314990
Keatinge-Clay, A. T. Stereocontrol within polyketide assembly lines. Nat. Prod. Rep.33, 141–149 (2016).
pubmed: 26584443
pmcid: 4742408
Luengo, J. I., Konialian-Beck, A., Rozamus, L. W. & Holt, D. A. Manipulation of the rapamycin effector domain. selective nucleophilic substitution of the C7 methoxy group. J. Org. Chem.59, 6512–6513 (1994).
Law, B. J. C., Struck, A. W., Bennett, M. R., Wilkinson, B. & Micklefield, J. Site-specific bioalkylation of rapamycin by the RapM 16-O-methyltransferase. Chem. Sci.6, 2885–2892 (2015).
pubmed: 29403635
pmcid: 5729408
Watanabe, T. et al. Genetic visualization of protein interactions harnessing liquid phase transitions. Sci. Rep.7, 46380 (2017).
pubmed: 28406179
pmcid: 5390312
Li, J., Kim, S. G. & Blenis, J. Rapamycin: one drug, many effects. Cell Metab.19, 373–379 (2014).
pubmed: 24508508
pmcid: 3972801
Cottens, S. & Sedrani, R. Rapamycin derivatives. WO 96/41807 (1996).
Liu, Y. et al. In vitro CRISPR/Cas9 system for efficient targeted DNA editing. mBio6, e01714–e01715 (2015).
pubmed: 26556277
pmcid: 4659471
Jiang, W. et al. Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters. Nat. Commun.6, 8101 (2015).
pubmed: 26323354
pmcid: 4569707
Alberti, F. & Corre, C. Editing streptomycete genomes in the CRISPR/Cas9 age. Nat. Prod. Rep.36, 1237–1248 (2019).
pubmed: 30680376
Zhao, Y., Li, G., Chen, Y. & Lu, Y. Challenges and advances in genome editing technologies in Streptomyces. Biomolecules10, 734 (2020).
Kapur, S., Chen, A. Y., Cane, D. E. & Khosla, C. Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase. Proc. Natl Acad. Sci. USA107, 22066–22071 (2010).
pubmed: 21127271
Swinney, D. C. & Anthony, J. How were new medicines discovered? Nat. Rev. Drug Discov.10, 507–519 (2011).
pubmed: 21701501
Schreiber, K. H. et al. A novel rapamycin analog is highly selective for mTORC1 in vivo. Nat. Commun.10, 3194 (2019).
pubmed: 31324799
pmcid: 6642166
Komatsu, M., Uchiyama, T., Omura, S., Cane, D. E. & Ikeda, H. Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proc. Natl Acad. Sci. USA107, 2646–2651 (2010).
pubmed: 20133795
Chu, G., Vollrath, D. & Davis, R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science234, 1582–1585 (1986).
pubmed: 3538420
Ikeda, H., Kotaki, H. & Omura, S. Genetic studies of avermectin biosynthesis in Streptomyces avermitilis. J. Bacteriol.169, 5615–5621 (1987).
pubmed: 3680172
pmcid: 214006
Gregory, M. A. et al. Rapamycin biosynthesis: elucidation of gene product function. Org. Biomol. Chem.4, 3565–3568 (2006).
pubmed: 16990929