Skip to main content
Log in

Redesign of antifungal polyene glycosylation: engineered biosynthesis of disaccharide-modified NPP

  • Methods and protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Polyene macrolides such as nystatin A1 and amphotericin B have been known to be potent antifungal antibiotics for several decades. Because the therapeutic application of polyenes is restricted by severe side effects such as nephrotoxicity, various chemical and biological studies to modify the polyene structure have been conducted to develop less-toxic polyene antifungals. A newly discovered nystatin-like polyene compound NPP was shown to contain an aglycone that was identical to nystatin but harbored a unique di-sugar moiety, mycosaminyl-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic toxicity. Additionally, a NPP-specific second sugar extending gene, nppY, was recently identified to be responsible for the transfer of a second sugar, N-acetyl-glucosamine, in NPP biosynthesis. In this study, we investigated biosynthesis of the glycoengineered NPP analog through genetic manipulation of the NPP A1 producer, Pseudonocardia autotrophica KCTC9441. NypY is another second sugar glycosyltransferase produced by Pseudonocardia sp. P1 that is responsible for the transfer of a mannose to the mycosaminyl sugar residue of nystatin. We blocked the transfer of a second sugar through nppY disruption, then expressed nypY in P. autotrophica △nppY mutant strain. When compared with nystain A1 and NPP A1, the newly engineered mannosylated NPP analog showed reduced in vitro antifungal activity, while exhibiting higher nephrotoxical activities against human hepatocytes. These results suggest for the first time that not only the number of sugar residues but also the type of extended second sugar moiety could affect biological activities of polyene macrolides.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Barke J, Seipke RF, Grüschow S, Heavens D, Drou N, Bibb MJ, Goss RJM, Yu DW, Hutchings MI (2010) A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus BMC Biol:8

  • Boros-Majewska J, Salewska N, Borowski E, Milewski S, Malic S, Wei XQ, Hayes AJ, Wilson MJ, Williams DW (2014) Novel nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis. Med Microbiol Immunol 203:341–355

    Article  CAS  PubMed  Google Scholar 

  • Brautaset T, Sletta H, Degnes KF, Sekurova ON, Bakke I, Volokhan O, Andreassen T, Ellingsen TE, Zotchev SB (2011) New nystatin-related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces noursei. Appl Environ Microbiol 77:6636–6643

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 Biotechnol 100:3893–3908

    Article  CAS  PubMed  Google Scholar 

  • Cereghetti DM, Carreira EM (2006) Amphotericin B: 50 years of chemistry and biochemistry. Synthesis:914–942

  • CPfaller MA, Chaturvedi V, Espinel-Ingroff A, Ghannoum MA, Gosey LL, Odds FC, Rex JH, Rinaldi MG, Sheehan DJ, Walsh TJ, Warnock DW (2002) Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard-second edition. NCCLS document M27-A2 [ISBN 1-56238-469-4]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA

  • De Poire E, Stephens N, Rawlings B, Caffrey P (2013) Engineered biosynthesis of disaccharide-modified polyene macrolides. Appl Environ Microbiol 79:6156–6159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ehrenfreund-Kleinman T, Azzam T, Falk R, Polacheck I, Golenser J, Domb AJ (2002) Synthesis and characterization of novel water soluble amphotericin B-arabinogalactan conjugates. Biomaterials 23:1327–1335

    Article  CAS  PubMed  Google Scholar 

  • Gregory MA, Till R, Smith MCM (2003) Integration site for Streptomyces phage fBT1 and development of site-specific integrating vectors. J Bacteriol 185:5320–5323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hong HJ, Hutchings MI, Hill LM, Buttner MJ (2005) The role of the novel fem protein Van K in vancomycin resistance in Streptomyces coelicolor. J Biol Chem 280:13055–13061

    Article  CAS  PubMed  Google Scholar 

  • Hutchinson E, Murphy B, Dunne T, Breen C, Rawlings B, Caffrey P (2010) Redesign of polyene macrolide glycosylation: engineered biosynthesis of 19-(O)-perosaminyl-amphoteronolide B. Chem Biol 17:174–182

    Article  CAS  PubMed  Google Scholar 

  • Jeon HG, Seo JY, Lee MJ, Han KB, Kim ES (2011) Analysis and functional expression of NPP pathway-specific regulatory genes in Pseudonocardia autotrophica. J Ind Microbiol Biotechnol 38:573–579

    Article  CAS  PubMed  Google Scholar 

  • Kim BK, Lee MJ, Seo J, Hwang YB, Lee MY, Han KB, Sherman DH, Kim ES (2009) Identification of functionally clustered nystatin-like biosynthetic genes in a rare actinomycetes, Pseudonocardia autotrophica. J Ind Microbiol Biotechnol 36:1425–1434

    Article  CAS  PubMed  Google Scholar 

  • Kim HJ, Kim MK, Jin YY, Kim ES (2014) Effect of antibiotic down-regulatory gene wblA ortholog on antifungal polyene production in rare actinomycetes Pseudonocardia autotrophica. J Microbiol Biotechnol 24:1226–1231

    Article  CAS  PubMed  Google Scholar 

  • 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

  • Lee MJ, Kong D, Han K, Sherman DH, Bai L, Deng Z, Lin S, Kim ES (2012) Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica. Appl Microbiol Biotechnol 95:157–168

    Article  CAS  PubMed  Google Scholar 

  • Paquet V, Carreira EM (2006) Significant improvement of antifungal activity of polyene macrolides by bisalkylation of the mycosamine. Org Lett 8:1807–1809

    Article  CAS  PubMed  Google Scholar 

  • Seipke RF, Grüschow S, Goss RJM, Hutchings MI (2012) Isolating antifungals from fungus-growing ant symbionts using a genome-guided chemistry approach. Methods Enzymol 517:47–70

    Article  CAS  PubMed  Google Scholar 

  • Stephens N, Rawlings B, Caffrey P (2013) Versatility of enzymes catalyzing late steps in polyene 67-121C biosynthesis. Biosci Biotechnol Biochem 77:880–883

    Article  CAS  PubMed  Google Scholar 

  • Szlinder-Richert J, Mazerski J, Cybulska B, Grzybowska J, Borowski E (2001) MFAME, N-methyl-N-D-fructosyl amphotericin B methyl ester, a new amphotericin B derivative of low toxicity: relationship between self-association and effects on red blood cells. Biochim Biophys Acta Gen Subj 1528:15–24

    Article  CAS  Google Scholar 

  • Walmsley S, De Poire E, Rawlings B, Caffrey P (2016) Engineered biosynthesis and characterization of disaccharide-modified 8-deoxyamphoteronolides. Appl Microbiol Biotechnol: 1-7

  • Wilcock BC, Endo MM, Uno BE, Burke MD (2013) C2'-OH of amphotericin B plays an important role in binding the primary sterol of human cells but not yeast cells. J Am Chem Soc 135:8488–8491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wright JJ, Greeves D, Mallams AK, Picker DH (1977) Structural elucidation of heptaene macrolide antibiotics 67-121-A and 67-121-C. Journal of the Chemical Society, Chemical Communications, pp 710–712

    Google Scholar 

  • Zhang C, Moretti R, Jiang J, Thorson JS (2008) The in vitro characterization of polyene glycosyltransferases AmphDI and NysDI. Chem Bio Chem 9:2506–2514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The plasmid pIJ10257-nypY was a kind gift from Ryan Seipke and Matt Hutchings of the University of East Anglia, UK.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Eung-Soo Kim.

Ethics declarations

Funding

This work was conducted with the support of the “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01129601)” Rural Development Administration, Republic of Korea. This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No. NRF-2014R1A2A1A11052236).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Electronic supplementary material

ESM 1

(PDF 663 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, HJ., Kang, SH., Choi, SS. et al. Redesign of antifungal polyene glycosylation: engineered biosynthesis of disaccharide-modified NPP. Appl Microbiol Biotechnol 101, 5131–5137 (2017). https://doi.org/10.1007/s00253-017-8303-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-017-8303-8

Keywords

Navigation