Skip to main content
SpringerLink
Log in

Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product

  • Review
  • Published:
Journal of Biosciences Aims and scope Submit manuscript

Abstract

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  • Anand S, Prasad MVR, Yadav G, Kumar N, Shehara J, Ansari MZ and Mohanty D 2010 SBSPKS: structure based sequence analysis of polyketide synthases. Nucleic Acids Res. 38 W487–W496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ansari MZ, Yadav G, Gokhale RS and Mohanty D 2004 NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases. Nucleic Acids Res. 32 W405–W413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ansari MZ, Sharma J, Gokhale RS and Mohanty D 2008 In silico analysis of methyltransferase domains involved in biosynthesis of secondary metabolites. BMC Bioinf. 9 454

    Article  Google Scholar 

  • Bachmann BO and Ravel J 2009 Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data. In Methods in Enzymology. Elsevier Inc., pp. 181–217.

  • Baltz RH 2011a Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery. J. Ind. Microbiol. Biotechnol. 38 1747–1760

    Article  CAS  PubMed  Google Scholar 

  • Baltz RH 2011b Strain improvement in actinomycetes in the postgenomic era. J. Ind. Microbiol. Biotechnol. 38 657–666

    Article  CAS  PubMed  Google Scholar 

  • Baltz RH 2014 MbtH homology codes to identify gifted microbes for genome mining. J. Ind. Microbiol. Biotechnol. 41 357–369

    Article  CAS  PubMed  Google Scholar 

  • Baranašić D, Zucko J, Diminic J, Gacesa R, Long PF, Cullum J, et al. 2014 Predicting substrate specificity of adenylation domains of nonribosomal peptide synthetases and other protein properties by latent semantic indexing. J. Ind. Microbiol. Biotechnol. 41 461–467

    Article  PubMed  Google Scholar 

  • Blin K, Medema MH, Kazempour D, Fischbach MA, Breitling R, Takano E and Weber T 2013 antiSMASH 2.0 - a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 41 204–212

    Article  Google Scholar 

  • Boll B, Taubitz T and Heide L 2011 Role of MbtH-like proteins in the adenylation of tyrosine during aminocoumarin and vancomycin biosynthesis. J. Biol. Chem. 286 36281–36290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bouslimani A, Sanchez LM, Garg N and Dorrestein PC 2014 Mass spectrometry of natural products: current, emerging and future technologies. Nat. Prod. Rep. 31 718–729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Buchko GW, Kim C-Y, Terwilliger TC and Myler PJ 2010 Solution structure of Rv2377c-founding member of the MbtH-like protein family. Tuberculosis. 90 245–251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Caboche S, Pupin M, Leclère V, Fontaine A, Jacques P and Kucherov G 2008 NORINE: A database of nonribosomal peptides. Nucleic Acids Res. 36 326–331

    Article  Google Scholar 

  • Cane DE and Walsh CT 1999 The parallel and convergent universes of polyketide synthases and nonribosomal peptide synthetases. Chem. Biol. 6 R319–R325

    Article  CAS  PubMed  Google Scholar 

  • Challis GL 2008 Mining microbial genomes for new natural products and biosynthetic pathways. Microbiology. 154 1555–1569

    Article  CAS  PubMed  Google Scholar 

  • Chatfield CH, Mulhern BJ, Viswanathan VK and Cianciotto NP 2012 The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila. Microbiology 158 721–735

  • Drake EJ, Cao J, Qu J, Shah MB, Straubinger RM and Gulick AM 2007 The 1.8 A° crystal structure of PA2412, an MbtH-like protein from the Pyoverdine cluster of Pseudomonas aeruginosa. J. Biol. Chem. 282 20425–20434

    Article  CAS  PubMed  Google Scholar 

  • Felnagle EA, Barkei JJ, Park H, Podevels AM, Matthew D, Drott DW and Thomas MG 2010 MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases. Biochemistry 49 8815–8817

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Forseth RR, Amaike S, Schwenk D, Affeldt KJ, Hoffmeister D, Schroeder FC and Keller NP 2013 Homologous NRPS-like gene clusters mediate redundant small-molecule biosynthesis in Aspergillus flavus. Angew. Chem. Int. Ed. Engl. 18 1199–1216

    Google Scholar 

  • Geladopoulos TP, Sotiroudis TG and Evangelopoulos A 1991 A malachite green colorimetric assay for protein phosphatase activity. Anal. Biochem. 192 112–116

    Article  CAS  PubMed  Google Scholar 

  • Gocht M and Marahiel MA 1994 Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis. J. Bacteriol. 176 2654–2662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gulick AM 2009 Conformational dynamics in the acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem. Biol. 4 811–827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Haynes SW and Challis GL 2007 Non-linear enzymatic logic in natural product modular mega-synthases and -synthetases. Curr. Opin. Drug Discov. Dev. 10 203–218

    CAS  Google Scholar 

  • Helfrich EJN and Piel J 2016 Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 33 231–316

    Article  CAS  PubMed  Google Scholar 

  • Herbst DA, Boll B, Zocher G, Stehle T and Heide L 2013 Structural basis of the interaction of MbtH-like proteins, putative regulators of nonribosomal peptide biosynthesis, with adenylating enzymes. J. Biol. Chem. 288 1991–2003

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Imker HJ, Krahn D, Clerc J, Kaiser M and Walsh CT 2010 N-acylation during glidobactin biosynthesis by the tridomain nonribosomal peptide synthetase module GlbF. Chem. Biol. 17 1077–1083

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Keller U, Lang M, Crnovcic I, Pfennig F and Schauwecker F 2010 The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus: a genetic hall of mirrors for synthesis of a molecule with mirror symmetry. J. Bacteriol. 192 2583–2595

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khayatt BI, Overmars L, Siezen RJ and Francke C 2013 Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific Hidden Markov Models. PLoS ONE 8 e62136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kleinkauf H and Von Döhren H 1996 A nonribosomal system of peptide biosynthesis. Eur. J. Biochem. 236 335–351

    Article  CAS  PubMed  Google Scholar 

  • Kries H, Wachtel R, Pabst A, Wanner B, Niquille D and Hilvert D 2014 Reprogramming nonribosomal peptide synthetases for “clickable” amino acids. Angew. Chem. Int. Ed. Engl. 53 1–5

    Article  Google Scholar 

  • Krug D and Müller R 2014 Secondary metabolomics: the impact of mass spectrometry-based approaches on the discovery and characterization of microbial natural products. Nat. Prod. Rep. 31 768–783

    Article  CAS  PubMed  Google Scholar 

  • Lambalot RH, Gehring AM, Flugel RS, Zuber P, LaCelle M, Marahiel MA, et al. 1996 A new enzyme superfamily - the phosphopantetheinyl transferases. Chem. Biol. 3 923–936

    Article  CAS  PubMed  Google Scholar 

  • Lautru S, Oves-Costales D, Pernodet J-L and Challis GL 2007 MbtH-like protein-mediated cross-talk between non-ribosomal peptide antibiotic and siderophore biosynthetic pathways in Streptomyces coelicolor M145. Microbiology. 153 1405–1412

    Article  CAS  PubMed  Google Scholar 

  • Li MH, Ung PM, Zajkowski J, Garneau-Tsodikova S and Sherman DH 2009 Automated genome mining for natural products. BMC Bioinf. 10 185

    Article  Google Scholar 

  • Lombó F, Velasco A, Castro A, de la Calle F, Braña AF, Sánchez-Puelles JM, et al. 2006 Deciphering the biosynthesis pathway of the antitumor thiocoraline from a marine actinomycete and its expression in two Streptomyces species. ChemBioChem. 7 366–376

    Article  PubMed  Google Scholar 

  • McMahon MD, Rush JS and Thomas MG 2012 Analysis of MbtB, MbtE, and MbtF suggest revisions to the mycobactin biosynthesis pathway in Mycobacterium tuberculosis. J. Bacteriol. 194 2809–2818

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mcquade TJ, Shallop AD, Sheoran A, Delproposto JE and Tsodikov OV 2009 A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis. Anal. Biochem. 386 244–250

    Article  CAS  PubMed  Google Scholar 

  • Medema MH and Fischbach MA 2015 Computational approaches to natural product discovery. Nat. Chem. Biol. 11 639–648

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Medema MH, Blin K, Cimermancic P, de Jager V, Zakrzewski P, Fischbach MA, et al. 2011 antiSMASH: Rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 39 W339–W346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Minowa Y, Araki M and Kanehisa M 2007 Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial genomes. J. Mol. Biol. 368 1500–1517

    Article  CAS  PubMed  Google Scholar 

  • Miyanaga A, Hayakawa Y, Numakura M, Hashimoto J, Teruya K, Hirano T, et al. 2016 Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the β-Amino Acid Selective Adenylating Enzyme FlvN. Biosci. Biotechnol. Biochem. 8451 1–7

    Google Scholar 

  • Mootz HD and Marahiel MA 1997 The tyrocidine biosynthesis operon of Bacillus brevis: Complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J. Bacteriol. 179 6843–6850

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Munk C, Lapidus A, Copeland A, Jando M, Mayilraj S, Glavina Del Rio T, et al. 2009 Complete genome sequence of Stackebrandtia nassauensis type strain (LLR-40K-21). Stand. Genomic Sci. 1 234–241

    Article  PubMed  PubMed Central  Google Scholar 

  • O’Brien RV, Davis RW, Khosla C and Hillenmeyer ME 2014 Computational identification and analysis of orphan assembly-line polyketide synthases. J. Antibiot. 67 89–97

    Article  PubMed  Google Scholar 

  • Ogasawara Y, Yackley BJ, Greenberg JA, Rogelj S and Melançon CE 2015 Expanding our understanding of sequence-function relationships of Type II polyketide biosynthetic gene clusters: Bioinformatics-guided identification of Frankiamicin A from Frankia sp. EAN1pec. PLoS ONE 10 1–25

    Google Scholar 

  • Prieto C, García-estrada C, Lorenzana D and Martín JF 2012 NRPSsp: non-ribosomal peptide synthase substrate predictor. Bioinformatics 28 426–427

    Article  CAS  PubMed  Google Scholar 

  • Quadri LE, Sello J, Keating TA, Weinreb PH and Walsh CT 1998 Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin. Chem. Biol. 5 631–645

    Article  CAS  PubMed  Google Scholar 

  • Rausch C, Weber T, Kohlbacher O, Wohlleben W and Huson DH 2005 Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs). Nucleic Acids Res. 33 5799–5808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rausch C, Hoof I, Weber T, Wohlleben W and Huson DH 2007 Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution. BMC Evol. Biol. 7 78

    Article  PubMed  PubMed Central  Google Scholar 

  • Reger AS, Wu R, Dunaway-mariano D and Gulick AM 2008 Structural characterization of a 140° domain movement in the two-step reaction catalyzed by 4-chlorobenzoate : CoA Ligase. Biochemistry 47 8016–8025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Röttig M, Medema MH, Blin K, Weber T, Rausch C and Kohlbacher O 2011 NRPSpredictor2 - A web server for predicting NRPS adenylation domain specificity. Nucleic Acids Res. 39 362–367

    Article  Google Scholar 

  • Singh M and Sareen D 2014 Novel LanT associated lantibiotic clusters identified by genome database mining. PLoS ONE 9 e91352

    Article  PubMed  PubMed Central  Google Scholar 

  • Skinnider MA, Dejong CA, Rees PN, Johnston CW, Li H, Webster ALH, et al. 2015 Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM). Nucleic Acids Res. 43 9645–9662

    CAS  PubMed  PubMed Central  Google Scholar 

  • Stachelhaus T and Marahiel MA 1995 Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis. FEMS Microbiol. Lett. 125 3–14

    Article  CAS  PubMed  Google Scholar 

  • Stachelhaus T, Mootz H and Marahiel M 1999 The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem. Biol. 6 493–505

    Article  CAS  PubMed  Google Scholar 

  • Starcevic A, Zucko J, Simunkovic J, Long PF, Cullum J and Hranueli D 2008 ClustScan: an integrated program package for the semi-automatic annotation of modular biosynthetic gene clusters and in silico prediction of novel chemical structures. Nucleic Acids Res. 36 6882–6892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Staunton J and Wilkinson B 1997 Biosynthesis of Erythromycin and Rapamycin. Chem. Rev. 97 2611–2630

    Article  CAS  PubMed  Google Scholar 

  • Stegmann E, Rausch C, Stockert S, Burkert D and Wohlleben W 2006 The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production. FEMS Microbiol. Lett. 262 85–92

    Article  CAS  PubMed  Google Scholar 

  • Taboada B, Ciria R, Martinez-Guerrero CE and Merino E 2012 ProOpDB: Prokaryotic operon database. Nucleic Acids Res. 40 627–631

    Article  Google Scholar 

  • Wang Y, Zhi X, Zhang Y, Cui X, Xu L and Li W 2009 Stackebrandtia albiflava sp. nov. and emended description of the genus Stackebrandtia. Int. J. Syst. Evol. Microbiol. 59 574–577

    Article  CAS  PubMed  Google Scholar 

  • Wang M, Beissner M and Zhao H 2014 Aryl-aldehyde formation in fungal polyketides: Discovery and characterization of a distinct biosynthetic mechanism. Chem. Biol. 21 257–263

    Article  PubMed  PubMed Central  Google Scholar 

  • Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, et al. 2015 antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 39 W339–W346

    Google Scholar 

  • Weissman KJ and Müller R 2008 Protein-protein interacions in multienzyme megasynthetases. Chem. Bio. Chem. 9 826–848

    Article  CAS  PubMed  Google Scholar 

  • Wolpert M, Gust B, Kammerer B and Heide L 2007 Effects of deletions of MbtH-like genes on clorobiocin biosynthesis in Streptomyces coelicolor. Microbiology 153 1413–1423

    Article  CAS  PubMed  Google Scholar 

  • Xia S, Ma Y, Zhang W, Yang Y, Wu S, Zhu M, et al. 2012 Identification of Sare0718 as an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205. PLoS ONE. 7 e37487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yeh E, Kohli RM, Bruner SD and Walsh CT 2004 Type II thioesterase restores activity of a NRPS module stalled with an aminoacyl-S-enzyme that cannot be elongated. Chembiochem. 5 1290–1293

    Article  CAS  PubMed  Google Scholar 

  • Yu Y, Zhang Q and van der donk WA 2013 Insights into the evolution of lanthipeptide biosynthesis. Protein Sci. 22 1478–1489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang W, Heemstra JR Jr, Walsh CT and Imker HJ 2010 Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins. Biochemistry 49 9946–9947

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ziemert N, Podell S, Penn K, Badger JH, Allen E and Jensen PR 2012 The natural product domain seeker NaPDoS: a phylogeny based bioinformatic tool to classify secondary metabolite gene diversity. PLoS ONE 7 e34064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zolova OE and Garneau-Tsodikova S 2012 Importance of the MbtH-like protein TioT for production and activation of the thiocoraline adenylation domain of TioK. Med. Chem. Commun. 3 950

    Article  CAS  Google Scholar 

Download references

Acknowledgements

MS acknowledges the independent Senior Research Fellowship (SRF) No. 3/1/3/JRF-2011/HRD-99(11005), awarded by the Indian Council of Medical Research, New Delhi. The financial assistance received from DST-PURSE grant, Department of Science and Technology, and UGC-SAP, New Delhi, are also acknowledged.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Panjab University, Chandigarh, India

    Mangal Singh, Sandeep Chaudhary & Dipti Sareen

Authors
  1. Mangal Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sandeep Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dipti Sareen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dipti Sareen.

Additional information

[Singh M, Chaudhary S and Sareen D 2017 Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product. J. Biosci.]

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Singh, M., Chaudhary, S. & Sareen, D. Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product. J Biosci 42, 175–187 (2017). https://doi.org/10.1007/s12038-017-9663-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12038-017-9663-z

Keywords

Search

Navigation