Skip to main content
SpringerLink
Log in

Bioconversion of Phytosterols into Androstenedione by Mycolicibacterium

  • Protocol
  • First Online:
Microbial Steroids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2704))

  • 252 Accesses

Abstract

The chapter describes the bioconversion of phytosterols into androstenedione (AD) by Mycolicibacterium spp. in shake flasks and fermenters, as well as LC–MS-based methods for analysis of phytosterols and steroid products. Phytosterols are derived as by-products of vegetable oil refining and manufacture of wood pulp. They contain the same four-ring nucleus as steroids and may be converted to high-value steroids by removing the sidechain at C17 and minor changes at other sites in the ring structure. Many bacteria, including Mycolicibacterium spp., can degrade phytosterols. Mutants of Mycolicibacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and androstadienedione (ADD). The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic for the cells. Two main strategies have been employed to overcome this challenge: the use of two-phase systems and the addition of chemically modified cyclodextrins. The latter method is used here. Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as hints how to minimize the practical problems due to the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC–MS analysis are described.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Moreau RA, Whitaker BD, Hicks KB (2002) Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Prog Lipid Res 41:457–500

    Article  CAS  PubMed  Google Scholar 

  2. Cantrill R, Kawamura Y (2008) Phytosterols, phytostanols and their esters, chemical and technical assessments. Proceedings of 69th JEFCA, 1–13. Available at http://www.fao.org/fileadmin/templates/agns/pdf/jecfa/cta/69/Phytosterols.pdf

  3. Al Jasem Y, Khan M, Taha A, Thiemann T (2014) Preparation of steroidal hormones with an emphasis on transformations of phytosterols and cholesterol - a review. Mediterranean J Chem 3:796–830

    Article  Google Scholar 

  4. Szentirmai A (1990) Microbial physiology of sidechain degradation of sterols. J Ind Microbiol 6:101–116

    Article  CAS  Google Scholar 

  5. Malaviya A, Gomes J (2008) Androstenedione production by biotransformation of phytosterols. Bioresour Technol 99:6725–6737

    Article  CAS  PubMed  Google Scholar 

  6. The Human metabolome database, http://www.hmdb.ca/metabolites/hmdb00852

  7. Saad HY, Higuchi WI (1965) Water solubility of cholesterol. J Pharm Sci 54:1205–1206

    Article  CAS  PubMed  Google Scholar 

  8. Yuan JJ, Guan YX, Wang YT, Wang HQ, Yao SJ (2016) Side-chain cleavage of phytosterols by Mycolicibacterium sp. MB 3683 in a biphasic ionic liquid/aqueous system. J Chem Technol Biotechnol. Published online in Wiley Online Library: (wileyonlinelibrary.com). https://doi.org/10.1002/jctb.4865

  9. Pendharkar GB, Anjum SD, Patil S (2014) Enhanced biotransformation of phytosterols, a byproduct of soybean refineries, to a key intermediate used for synthesis of steroidal drugs. Asian J Pharm Clin Res 7:178–180

    Google Scholar 

  10. Yalkowski SH, He Y (2003) Handbook of Aqueous Solubility Data. CRC Press, Boca Raton

    Book  Google Scholar 

  11. NIH PubChem open chemistry database, https://pubchem.ncbi.nlm.nih.gov/

  12. Khomutov SM, Dovbnya DV, Donova MV (2001) Dissolution of a mixture of steroids in cyclodextrin solutions: A model description. Pharmaceut Chem J 55:627–629

    Article  Google Scholar 

  13. Lee CY, Liu WH (1992) Production of androsta-l,4-diene-3,17-dione from cholesterol using immobilized growing cells of Mycolicibacterium sp. NRRL B-3683 adsorbed on solid carriers. Appl Microbiol Biotechnol 36:598–603

    Article  CAS  PubMed  Google Scholar 

  14. Smith M, Zahnley J, Pfeifer D, Goff D (1993) Growth and cholesterol oxidation by Mycolicibacterium species in Tween 80 medium. Appl Environ Microbiol 59:1425–1429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pérez C, Falero A, Llanes N, Hung BR, Herve ME, Palmero A, Marti E (2003) Resistance to androstanes as an approach for androstandienedione yield enhancement in industrial mycobacteria. J Ind Microbiol Biotechnol 30:623–626

    Article  PubMed  Google Scholar 

  16. Donova MV (2007) Transformation of steroids by actinobacteria: a review. Appl Biochem Microbiol 43:1–14

    Article  CAS  Google Scholar 

  17. Xu YG, Guan YX, Wang HQ, Yao SJ (2014) Microbial side-chain cleavage of phytosterols by mycobacteria in vegetable oil/aqueous two-phase system. Appl Biochem Biotechnol 174:522–533

    Article  CAS  PubMed  Google Scholar 

  18. Stefanov S, Yankov D, Beschkov V (2006) Biotransformation of phytosterols to androstenedione in two phase water-oil system. Chem Biochem Eng Q 20:421–427

    CAS  Google Scholar 

  19. Carvalho F, Marques MPC, de Carvalho CCCR, Cabral JMS, Fernandes P (2009) Sitosterol bioconversion with resting cells in liquid polymer based systems. Bioresour Technol 100:4050–4053

    Article  CAS  PubMed  Google Scholar 

  20. Marques MPC, Carvalho F, de Carvalho CCCR, Cabral JMS, Fernandes P (2010) Steroid bioconversion: towards green processes. Food Bioprod Process 88:12–20

    Article  CAS  Google Scholar 

  21. Cruz A, Fernandes P, Cabral JMS, Pinheiro HM (2002) Effect of phase composition on the whole-cell bioconversion of β-sitosterol in biphasic media. J Mol Catal B Enzym 19-20:371–375

    Article  CAS  Google Scholar 

  22. Cruz A, Angelova B, Fernandes P, Cabral JMS, Pinheiro HM (2004) Study of key operational parameters for the side-chain cleavage of sitosterol by free mycobacterial cells in bis-(2-ethylhexyl) phthalate. Biocatal Biotransformation 22:189–194

    Article  CAS  Google Scholar 

  23. Del Valle EM (2004) Cyclodextrins and their uses: a review. Process Biochem 39:1033–1046

    Article  Google Scholar 

  24. Brewster ME, Loftsson T (2007) Cyclodextrins as pharmaceutical solubilizers. Adv Drug Deliv Rev 59:645–666

    Article  CAS  PubMed  Google Scholar 

  25. Gao XQ, Feng JX, Wang XD, Hua Q, Wei DZ (2015) Enhanced steroid metabolites production by resting cell phytosterol bioconversion. Chem Biochem Eng Q 29:567–573

    Article  CAS  Google Scholar 

  26. Donova MV, Nikolayeva VM, Dovbnya DV, Gulevskaya SA, Suzina NE (2007) Methyl-β-cyclodextrin alters growth, activity and cell envelope features of sterol-transforming mycobacteria. Microbiology 153:1981–1992

    Article  CAS  PubMed  Google Scholar 

  27. Shen Y, Wang M, Zhang L, Ma Y, Ma B, Zheng Y et al (2011) Effects of hydroxypropyl-β-cyclodextrin on cell growth, activity, and integrity of steroid-transforming Arthrobacter simplex and Mycolicibacterium sp. Appl Microbiol Biotechnol 90:1995–2003

    Article  CAS  PubMed  Google Scholar 

  28. Gulla V, Banerjee T, Patil S (2008) Quantitative TLC analysis of steroid drug intermediates formed during bioconversion of soysterols. Chromatographia 68:663–667

    Article  CAS  Google Scholar 

  29. Kollerov VV, Shutov AA, Fokina VV, Sukhodolskaya GV, Donova MV (2008) Biotransformation of 3-keto-androstanes by Gongronella butleri VKM F-1033. J Mol Catal B Enzym 55:61–68

    Article  CAS  Google Scholar 

  30. Gomez C, Fabregat A, Pozo OJ, Marcos J, Segura J, Ventura R (2014) Analytical strategies based on mass spectrometric techniques for the study of steroid metabolism. Trends Anal Chem 53:106–116

    Article  CAS  Google Scholar 

  31. Marwah A, Marwah P, Lardy H (2001) High-performance liquid chromatographic analysis of dehydroepiandrosterone. J Chromatogr A 935:279–296

    Article  CAS  PubMed  Google Scholar 

  32. Sánchez-Machado DI, López-Hernández J, Paseiro-Losada P, López-Cervantes J (2004) An HPLC method for the quantification of sterols in edible seaweeds. Biomed Chromatogr 18:183–190

    Article  PubMed  Google Scholar 

  33. Rocco A, Fanali S (2009) Analysis of phytosterols in extra-virgin olive oil by nano-liquid chromatography. J Chromatogr A 1216:7173–7178

    Article  CAS  PubMed  Google Scholar 

  34. Ahonen L, Keski-Rahkonen P, Saarelainen T, Paviala J, Ketola RA, Auriola S et al (2012) Comparison of liquid chromatography-microchip/mass spectrometry to conventional liquid chromatography–mass spectrometry for the analysis of steroids. Anal Chim Acta 721:115–121

    Article  CAS  PubMed  Google Scholar 

  35. Mo S, Dong L, Hurst WJ, van Breemen RB (2013) Quantitative analysis of phytosterols in edible oils using APCI liquid chromatography–tandem mass spectrometry. Lipids 48:949–956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Palmgren JJ, Töyräs A, Mauriala T, Mönkkönen J, Auriola S (2005) Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry. J Chromatogr B 821:144–152

    Article  CAS  Google Scholar 

  37. theLabRat.com - Biotech Jobs and Research Resources http://www.thelabrat.com/protocols/WolfesVitaminSolution.shtmL

Download references

Acknowledgments

This work was supported by grants from the European Union program ERA IB for the MySteri project and ERA CoBioTech for the project Syntheroids. MySteri funding was received from the European Union’s 7th Research Framework Programme (EIB.12.010) and financed through the Research Council of Norway (project number 230037/O30). Syntheroids funding was received from the European Union’s Horizon 2020 research and innovation programme under grant agreement No [722361] and financed through the Research Council of Norway (project number 285849). The authors thank Gunn Broli, Therese Myhre Harbak, and Kai Vernstad for their technical support. They are grateful to have had the MySteri and Syntheroids consortium members as valuable discussions partners during the work.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway

    Kjell Domaas Josefsen, Anna Nordborg, Simone Balzer Le, Silje Malene Olsen & Håvard Sletta

Authors
  1. Kjell Domaas Josefsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anna Nordborg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Simone Balzer Le
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Silje Malene Olsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Håvard Sletta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Simone Balzer Le .

Editor information

Editors and Affiliations

  1. Área de Bioquímica y Biología Molecular, Departamento de Biología Molecular, Facultad de Veterinaria, Universidad de León, León, Spain

    Carlos Barreiro

  2. Department of Biotechnology, Curia Spain, León, Spain

    José-Luis Barredo

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Josefsen, K.D., Nordborg, A., Le, S.B., Olsen, S.M., Sletta, H. (2023). Bioconversion of Phytosterols into Androstenedione by Mycolicibacterium. In: Barreiro, C., Barredo, JL. (eds) Microbial Steroids. Methods in Molecular Biology, vol 2704. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3385-4_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3385-4_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3384-7

  • Online ISBN: 978-1-0716-3385-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation