Skip to main content
SpringerLink
Log in

Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Pancreatic lipase inhibitors, such as tetrahydrolipstatin (orlistat), are used in anti-obesity treatments. Orlistat is the only anti-obesity drug approved by the European Medicines Agency (EMA). The drug is synthesized by saturation of lipstatin, a β-lactone compound, isolated from Streptomyces toxytricini and S. virginiae. To identify producers of novel pancreatic lipase inhibitors or microbial strains with improved lipstatin production and higher chemical purity remains still a priority. In this study, a high-throughput screening method to identify Streptomyces strains producing potent pancreatic lipase inhibitors was established. The assay was optimized and validated using S. toxytricini NRRL 15443 and its mutants. Strains grew in 24-well titer plates. Lipstatin levels were assessed directly in culture medium at the end of cultivation by monitoring lipolytic activity in the presence of a chromogenic substrate, 1,2-Di-O-lauryl-rac-glycero-3-glutaric acid 6-methylresorufin ester (DGGR). The lipase activity decreased in response to lipstatin production, and this was demonstrated by accumulation of red-purple methylresorufin, a product of DGGR digestion. The sensitivity of the assay was achieved by adding a lipase of high lipolytic activity and sensitivity to lipstatin to the reaction mixture. In the assay, the fungal lipase from Mucor javanicus was used as an alternative to the human pancreatic lipase. Many fungal lipases preserve high lipolytic activity in extreme conditions and are not colipase dependent. The assay proved to be reliable in differentiation of strains with high and low lipstatin productivity.

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

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Hochuli E, Kupfer E, Maurer R et al (1987) Lipstatin, an inhibitor of pancreatic lipase, produced by Streptomyces toxytricini. II. Chemistry and structure elucidation. J Antibiot (Tokyo) 8:1086–1091. https://doi.org/10.7164/antibiotics.40.1086

    Article  Google Scholar 

  2. Weibel EK, Hadvàry P, Kupfer E et al (1987) Lipstatin, an inhibitor of pancreatic lipase, produced by Streptomyces toxytricini. I. Producing organism, fermentation, isolation and biological activity. J Antiobiot 40:1081–1085. https://doi.org/10.7164/antibiotics.40.1081

    Article  CAS  Google Scholar 

  3. Mulzer M, Tiegs BJ, Wang Y et al (2014) Total synthesis of tetrahydrolipstatin and stereoisomers via a highly regio- and diastereoselective carbonylation of epoxyhomoallylic alcohols. J Am Chem Soc 136:10814–10820. https://doi.org/10.1021/ja505639u

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Eisenreich W, Kupfer E, Stohler P et al (2003) Biosynthetic origin of a branched chain analogue of the lipase inhibitor, lipstatin. J Med Chem 46:4209–4212. https://doi.org/10.1021/jm030780x

    Article  CAS  PubMed  Google Scholar 

  5. Eisenreich W, Kupfer E, Weber W, Bacher A (1997) Tracer studies with crude U-13C-lipid mixtures. Biosynthesis of the lipase inhibitor lipstatin. J Biol Chem 272:867–874. https://doi.org/10.1074/jbc.272.2.867

    Article  CAS  PubMed  Google Scholar 

  6. Goese M, Eisenreich W, Kupfer E et al (2000) Biosynthetic origin of hydrogen atoms in the lipase inhibitor lipstatin. J Biol Chem 275:21192–21196. https://doi.org/10.1074/jbc.M003094200

    Article  CAS  PubMed  Google Scholar 

  7. Bai T, Zhang D, Lin S et al (2014) Operon for biosynthesis of lipstatin, the beta-lactone inhibitor of human pancreatic lipase. Appl Environ Microbiol 80:7473–7483. https://doi.org/10.1128/AEM.01765-14

    Article  PubMed  PubMed Central  Google Scholar 

  8. Demirev AV, Khanal A, Sedai BR et al (2010) The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini. Appl Microbiol Biotechnol 87:1129–1139. https://doi.org/10.1007/s00253-010-2587-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Demirev AV, Lee JS, Sedai BR et al (2009) Identification and characterization of acetyl-CoA carboxylase gene cluster in Streptomyces toxytricini. J Microbiol 47:473–478. https://doi.org/10.1007/s12275-009-0135-5

    Article  CAS  PubMed  Google Scholar 

  10. Sladič G, Urukalo M, Kirn M et al (2014) Identification of lipstatin-producing ability in Streptomyces virginiae CBS 314.55 using dereplication approach. Food Technol Biotechnol 52:276–284

    Google Scholar 

  11. Gong G, Sun X, Liu X et al (2007) Mutation and a high-throughput screening method for improving the production of Epothilones of Sorangium. J Ind Microbiol Biotechnol 34:615–623. https://doi.org/10.1007/s10295-007-0236-2

    Article  CAS  PubMed  Google Scholar 

  12. Xu W, Huang J, Lin R et al (2010) Regulation of morphological differentiation in S. coelicolor by RNase III (AbsB) cleavage of mRNA encoding the AdpA transcription factor. Mol Microbiol 75:781–791. https://doi.org/10.1111/j.1365-2958.2009.07023.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zeng W, Du G, Chen J et al (2015) A high-throughput screening procedure for enhancing α-ketoglutaric acid production in Yarrowia lipolytica by random mutagenesis. Process Biochem 50:1516–1522. https://doi.org/10.1016/j.procbio.2015.06.011

    Article  CAS  Google Scholar 

  14. Zamzuri NA, Abd-Aziz S, Rahim RA et al (2014) A rapid colorimetric screening method for vanillic acid and vanillin-producing bacterial strains. J Appl Microbiol 116:903–910. https://doi.org/10.1111/jam.12410

    Article  CAS  PubMed  Google Scholar 

  15. Lewis DR, Liu DJ (2012) Direct measurement of lipase inhibition by orlistat using a dissolution linked in vitro assay. Clin Pharmacol Biopharm 1:1–11. https://doi.org/10.4172/2167-065X.1000103

    Article  Google Scholar 

  16. Beisson F, Rivière C (2000) Methods for lipase detection and assay: a critical review. Eur J Lipid Sci Technol 9312:133–153

    Article  Google Scholar 

  17. Gupta N, Rathi P, Gupta R (2002) Simplified para-nitrophenyl palmitate assay for lipases and esterases. Anal Biochem 311:98–99. https://doi.org/10.1016/S0003-2697(02)00379-2

    Article  CAS  PubMed  Google Scholar 

  18. Gupta R, Rathi P, Gupta N, Bradoo S (2003) Lipase assays for conventional and molecular screening: an overview. Biotechnol Appl Biochem 37:63–71. https://doi.org/10.1042/BA20020059

    Article  CAS  PubMed  Google Scholar 

  19. Mateos-Díaz E, Rodríguez JA, de los Ángeles Camacho-Ruiz M, Mateos-Díaz JC (2012) High-throughput screening method for Lipases/Esterases. In: Sandoval G (ed) Methods in molecular biology Humana Press, New York, pp 89–100

    Google Scholar 

  20. Panteghini M, Bonora R, Pagani F (2001) Measurement of pancreatic lipase activity in serum by a kinetic colorimetric assay using a new chromogenic substrate. Ann Clin Biochem 38:365–370. https://doi.org/10.1258/0004563011900876

    Article  CAS  PubMed  Google Scholar 

  21. Souri E, Jalalizadeh H, Kebriaee-Zadeh A, Zadehvakili B (2007) HPLC analysis of orlistat and its application to drug quality control studies. Chem Pharm Bull (Tokyo) 55:251–254. https://doi.org/10.1248/cpb.55.251

    Article  CAS  Google Scholar 

  22. Brabcová J, Zarevúcka M, Macková M (2010) Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013. Yeast 27:1029–1038. https://doi.org/10.1002/yea.1812

    Article  PubMed  Google Scholar 

  23. Kumar MS, Verma V, Soni S, Rao ASP (2011) Identification and process development for enhanced production of lipstatin from Streptomyces toxytricini (NRRL 15443) and further downstream processing. Adv Biol Tech 11:6–11

    Google Scholar 

  24. Zhu T, Wang L, Wang W et al (2014) Enhanced production of lipstatin from Streptomyces toxytricini by optimizing fermentation conditions and medium. J Gen Appl Microbiol 60:106–111. https://doi.org/10.2323/jgam.60.106

    Article  CAS  PubMed  Google Scholar 

  25. Zulauf M, Fürstenberger U, Grabo M et al (1989) Critical micellar concentrations of detergents. Methods Enzymol 172:528–538. https://doi.org/10.1016/S0076-6879(89)72032-2

    Article  CAS  PubMed  Google Scholar 

  26. Wilcox MD, Brownlee IA, Richardson JC et al (2014) The modulation of pancreatic lipase activity by alginates. Food Chem 146:479–484. https://doi.org/10.1016/j.foodchem.2013.09.075

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mayr LM, Bojanic D (2009) Novel trends in high-throughput screening. Curr Opin Pharmacol 9:580–588. https://doi.org/10.1016/j.coph.2009.08.004

    Article  CAS  PubMed  Google Scholar 

  28. Lookene A, Skottova N, Olivecrona G (1994) Interactions of lipoprotein lipase with the active-site inhibitor tetrahydrolipstatin (Orlistat). Eur J Biochem 222:395–403. https://doi.org/10.1111/j.1432-1033.1994.tb18878.x

    Article  CAS  PubMed  Google Scholar 

  29. Aloulou A, Rodriguez JA, Puccinelli D et al (2007) Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica. Biochim Biophys Acta 1771:228–237. https://doi.org/10.1016/j.bbalip.2006.12.006

    Article  CAS  PubMed  Google Scholar 

  30. Bénarouche A, Point V, Carrière F, Cavalier J-FF (2014) An interfacial and comparative in vitro study of gastrointestinal lipases and Yarrowia lipolytica LIP2 lipase, a candidate for enzyme replacement therapy. Biochimie 102:145–153. https://doi.org/10.1016/j.biochi.2014.03.004

    Article  PubMed  Google Scholar 

  31. Frikha F, Miled N, Bacha AB et al (2010) Structural homologies, importance for catalysis and lipid binding of the N-terminal peptide of a fungal and a pancreatic lipase. Protein Pept Lett 17:254–259. https://doi.org/10.2174/092986610790226049

    Article  CAS  PubMed  Google Scholar 

  32. Stoytcheva M, Stoytcheva M, Montero G et al (2012) Analytical methods for lipases activity determination: a review analytical methods for lipases activity determination: a review. Curr Anal Chem 8:400–407. https://doi.org/10.2174/157341112801264879

    Article  CAS  Google Scholar 

  33. Kumar P, Dubey KK (2017) Mycelium transformation of Streptomyces toxytricini into pellet: role of culture conditions and kinetics. Bioresour Technol 228:339–347. https://doi.org/10.1016/j.biortech.2017.01.002

    Article  CAS  PubMed  Google Scholar 

  34. Minas W, Bailey JE, Duetz W (2000) Streptomycetes in micro-cultures: growth, production of secondary metabolites, and storage and retrieval in the 96-well format. Antonie Van Leeuwenhoek 78:297–305. https://doi.org/10.1023/A:1010254013352

    Article  CAS  PubMed  Google Scholar 

  35. van Wezel GP, McDowall KJ (2011) The regulation of the secondary metabolism of Streptomyces: new links and experimental advances. Nat Prod Rep 28:1311–1333. https://doi.org/10.1039/c1np00003a

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Veronika Palušková for help with isolation of strains by UV mutagenesis.

Funding

This study was funded by ITMS 26220220093, Grant of Science and Technology Assistance Agency (Grant No. APVV-0719-12) and KEGA (Grant No. 047STU-4/2016).

Author information

Authors and Affiliations

  1. Institute of Biochemistry and Microbiology, Slovak University of Technology, Radlinského 9, 81 237, Bratislava, Slovakia

    Michal Híreš, Nora Rapavá, Martin Šimkovič, Ľudovít Varečka & Svetlana Kryštofová

  2. Department of Organic Chemistry, Slovak University of Technology, Radlinského 9, 81 237, Bratislava, Slovakia

    Dušan Berkeš

Authors
  1. Michal Híreš
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nora Rapavá
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Šimkovič
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ľudovít Varečka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dušan Berkeš
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Svetlana Kryštofová
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michal Híreš.

Ethics declarations

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Híreš, M., Rapavá, N., Šimkovič, M. et al. Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains. Curr Microbiol 75, 580–587 (2018). https://doi.org/10.1007/s00284-017-1420-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-017-1420-x

Search

Navigation