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Nonribosomal Peptide and Polyketide Biosynthesis pp 103–119Cite as

Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1401))

Abstract

The emergence of next-generation sequencing has provided new opportunities in the discovery of new nonribosomal peptides (NRPs) and NRP synthethases (NRPSs). However, there remain challenges for the characterization of these megasynthases. While genetic methods in native hosts are critical in elucidation of the function of fungal NRPS, in vitro assays of intact heterologously expressed proteins provide deeper mechanistic insights in NRPS enzymology. Our previous work in the study of NRPS takes advantage of Saccharomyces cerevisiae strain BJ5464-npgA as a robust and versatile platform for characterization of fungal NRPSs. Here we describe the use of yeast recombination strategies in S. cerevisiae for cloning of the NRPS coding sequence in 2μ-based expression vector; the use of affinity chromatography for purification of NRPS from the total S. cerevisiae soluble protein fraction; and strategies for reconstitution of NRPSs activities in vitro.

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References

  1. Fischbach MA, Walsh CT (2006) Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem Rev 106:3468–3496

    Article  PubMed  CAS  Google Scholar 

  2. Felnagle EA, Jackson EE, Chan YA et al (2008) Nonribosomal peptide synthetases involved in the production of medically relevant natural products. Mol Pharm 5:191–211

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  3. Schwarzer D, Finking R, Marahiel MA (2003) Nonribosomal peptides: from genes to products. Nat Prod Rep 20:275–287

    Article  PubMed  CAS  Google Scholar 

  4. Hur GH, Vickery CR, Burkart MD (2012) Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Nat Prod Rep 29:1074–1098

    Article  PubMed  CAS  Google Scholar 

  5. Koglin A, Walsh CT (2009) Structural insights into nonribosomal peptide enzymatic assembly lines. Nat Prod Rep 26:987–1000

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  6. Balibar CJ, Walsh CT (2006) GliP, a multimodular nonribosomal peptide synthetase in Aspergillus fumigatus, makes the diketopiperazine scaffold of gliotoxin. Biochemistry 45:15029–15038

    Article  PubMed  CAS  Google Scholar 

  7. Balibar CJ, Howard-Jones AR, Walsh CT (2007) Terrequinone A biosynthesis through L-tryptophan oxidation, dimerization and bisprenylation. Nat Chem Biol 3:584–592

    Article  PubMed  CAS  Google Scholar 

  8. Fang F, Salmon K, Shen MWY et al (2011) A vector set for systematic metabolic engineering in Saccharomyces cerevisiae. Yeast 28:123–136

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  9. Kealey JT, Liu L, Santi DV et al (1998) Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci U S A 95:505–509

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  10. Jones EW (1991) Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol 194:428–453

    Article  PubMed  CAS  Google Scholar 

  11. Johnson RE, Washington MT, Prakash S, Prakash L (2000) Fidelity of human DNA polymerase eta. J Biol Chem 275:7447–7450

    Article  PubMed  CAS  Google Scholar 

  12. Shusta EV, Raines RT, Pluckthun A et al (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat Biotechnol 16:773–777

    Article  PubMed  CAS  Google Scholar 

  13. Mootz HD, Schörgendorfer K, Marahiel MA (2002) Functional characterization of 4′-phosphopantetheinyl transferase genes of bacterial and fungal origin by complementation of Saccharomyces cerevisiae lys5. FEMS Microbiol Lett 213:51–57

    PubMed  CAS  Google Scholar 

  14. Gao X, Haynes SW, Ames BD et al (2012) Cyclization of fungal nonribosomal peptides by a terminal condensation-like domain. Nat Chem Biol 8:823–830

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  15. Ishiuchi K, Nakazawa T, Ookuma T et al (2012) Establishing a new methodology for genome mining and biosynthesis of polyketides and peptides through yeast molecular genetics. Chembiochem 13:846–854

    Article  PubMed  CAS  Google Scholar 

  16. Haynes SW, Gao X, Tang Y et al (2013) Complexity generation in fungal peptidyl alkaloid biosynthesis: a two-enzyme pathway to the hexacyclic MDR export pump inhibitor ardeemin. ACS Chem Biol 8:741–748

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  17. Gao X, Jiang W, Jiménez-Osés G et al (2013) An iterative, bimodular nonribosomal peptide synthetase that converts anthranilate and tryptophan into tetracyclic asperlicins. Chem Biol 20:870–878

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  18. Yu D, Xu F, Gage D, Zhan J (2013) Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases. Chem Commun 49:6176–6178

    Article  CAS  Google Scholar 

  19. Zhou H, Qiao K, Gao Z et al (2010) Enzymatic synthesis of resorcylic acid lactones by cooperation of fungal iterative polyketide synthases involved in hypothemycin biosynthesis. J Am Chem Soc 132:4530–4531

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  20. Zhou H, Qiao K, Gao Z et al (2010) Insights into radicicol biosynthesis via heterologous synthesis of intermediates and analogs. J Biol Chem 285:41412–41421

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  21. Cacho RA, Chooi YH, Zhou H et al (2013) Complexity generation in fungal polyketide biosynthesis: a spirocycle-forming P450 in the concise pathway to the antifungal drug griseofulvin. ACS Chem Biol 8:2322–2330

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  22. Xu Y, Zhou T, Zhou Z et al (2013) Rational reprogramming of fungal polyketide first-ring cyclization. Proc Natl Acad Sci U S A 110:5398–5403

    Article  PubMed Central  PubMed  Google Scholar 

  23. Xu Y, Zhou T, Espinosa-Artiles P et al (2014) Insights into the biosynthesis of 12-membered resorcylic acid lactones from heterologous production in Saccharomyces cerevisiae. ACS Chem Biol 9(5):1119–1127

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  24. Ma SM, Li JW, Choi JW et al (2009) Complete reconstitution of a highly reducing iterative polyketide synthase. Science 326:589–592

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  25. Xie X, Meehan MJ, Xu W et al (2009) Acyltransferase mediated polyketide release from a fungal megasynthase. J Am Chem Soc 131:8388–8389

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  26. Zhou H, Gao Z, Qiao K et al (2012) A fungal ketoreductase domain that displays substrate-dependent stereospecificity. Nat Chem Biol 8:331–333

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  27. Lin HC, Chooi YH, Dhingra S et al (2013) The fumagillin biosynthetic gene cluster in Aspergillus fumigatus encodes a cryptic terpene cyclase involved in the formation of beta-trans-bergamotene. J Am Chem Soc 135:4616–4619

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  28. Xu W, Cai X, Jung ME et al (2010) Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae. J Am Chem Soc 132:13604–13607

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  29. Gibson DG (2012) Oligonucleotide assembly in yeast to produce synthetic DNA fragments. Methods Mol Biol 852:11–21

    Article  PubMed  CAS  Google Scholar 

  30. Shao Z, Zhao H (2012) DNA assembler: a synthetic biology tool for characterizing and engineering natural product gene clusters. Methods Enzymol 517:203–224

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  31. Gietz RD, Woods RA (2006) Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol 313:107–120

    PubMed  CAS  Google Scholar 

  32. Lee KM, DaSilva NA (2005) Evaluation of the Saccharomyces cerevisiae ADH2 promoter for protein synthesis. Yeast 22:431–440

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We think Dr. Wei Xu for the construction of pXW55 plasmid. Work from our group is supported by the NIH grants 1R01GM085128 and 1DP1GM106413. R.A.C. is supported by the NRSA grant GM008496 and the UCLA Graduate Division.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA, 90095, USA

    Ralph A. Cacho & Yi Tang

  2. Department of Chemistry and Biochemistry, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA, 90095, USA

    Yi Tang

  3. Department of Bioengineering, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA, 90095, USA

    Yi Tang

Authors
  1. Ralph A. Cacho
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  2. Yi Tang
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Corresponding author

Correspondence to Yi Tang .

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Editors and Affiliations

  1. Donald Danforth Plant Science Center, Saint Louis, Missouri, USA

    Bradley S. Evans

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Cacho, R.A., Tang, Y. (2016). Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro. In: Evans, B. (eds) Nonribosomal Peptide and Polyketide Biosynthesis. Methods in Molecular Biology, vol 1401. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3375-4_7

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  • DOI: https://doi.org/10.1007/978-1-4939-3375-4_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3373-0

  • Online ISBN: 978-1-4939-3375-4

  • eBook Packages: Springer Protocols

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