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Biocatalysis of Steroids by Mycobacterium sp. in Aqueous and Organic Media

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Microbial Steroids

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

Abstract

Mycobacterium sp. can convert steroids such as β-sitosterol, campesterol, and cholesterol, by selective side-chain cleavage and oxidation of the C3 hydroxyl group to a ketone, into key intermediates that can be easily functionalized to yield commercially interesting pharmaceutical products. In aqueous systems, the biocatalysis is limited by the low solubility of the steroids in water. Several strategies have been introduced to tackle this limitation, e.g., formation of cyclodextrin–steroid complexes and generation of aqueous microdispersions with steroid particle size in the range of hundreds of nanometers. Still, the introduction of an organic phase acting as a substrate and/or product reservoir is a well-established and relatively easy to implement strategy to overcome the sparing water solubility of steroid molecules. However, the organic phase has to be carefully chosen to prevent tampering with the activity/viability of microbial cells.

In this chapter, we describe the methodology for the biocatalysis of β-sitosterol to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD), both in aqueous and organic:aqueous systems. In the latter case, both traditional organic solvents and green solvents are proposed.

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References

  1. Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B (2001) Industrial biocatalysis today and tomorrow. Nature 409:258–268

    Article  CAS  PubMed  Google Scholar 

  2. de Carvalho CCCR, Cruz A, Angelova B, Fernandes P, Pons MN, Pinheiro HM, Cabral JMS, da Fonseca MMR (2004) Behaviour of Mycobacterium sp. NRRL B-3805 whole cells in aqueous, organic-aqueous and organic media studied by fluorescence microscopy. Appl Microbiol Biotechnol 64:696–701

    Article  Google Scholar 

  3. Batth R, Nicolle C, Cuciurean IS, Simonsen HT (2020) Biosynthesis and industrial production of androsteroids. Plants 9:1144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. van Schie MMCH, Spöring J-D, Bocola M, Domínguez de María P, Rother D (2021) Applied biocatalysis beyond just buffers – from aqueous to unconventional media. Options and guidelines. Green Chem 23:3191–3206

    Article  PubMed  PubMed Central  Google Scholar 

  5. de Carvalho CCCR (2017) Whole cell biocatalysts: essential workers from Nature to the industry. Microb Biotechnol 10:250–263

    Article  PubMed  Google Scholar 

  6. Grundtvig IPR, Heintz S, Krühne U, Gernaey KV, Adlercreutz P, Hayler JD, Wells AS, Woodley JM (2018) Screening of organic solvents for bioprocesses using aqueous-organic two-phase systems. Biotechnol Adv 36:1801–1814

    Article  Google Scholar 

  7. Rokade R, Ravindran S, Singh P, Suthar J (2018) Microbial biotransformation for the production of steroid medicament. In: Vijayakumar R, Raja SS (eds) Secondary metabolites – sources and applications. IntechOpen, London, p 115

    Google Scholar 

  8. Heipieper HJ, Neumann G, Cornelissen S, Meinhardt F (2007) Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Appl Microbiol Biotechnol 74:961–973

    Article  CAS  PubMed  Google Scholar 

  9. de Carvalho CCCR (2010) Adaptation of Rhodococcus to organic solvents. In: Alvarez MH (ed) Biology of Rhodococcus. Springer, Berlin, Heidelberg, p 368

    Google Scholar 

  10. Little AD (2001) Making EHS an integral part of process design. American Institute of Chemical Engineers, CWRT, CCPS, New York

    Google Scholar 

  11. Tao J, Kazlauskas RJ (2011) Biocatalysis for green chemistry and chemical process development. John Wiley & Sons, Inc., Hoboken

    Book  Google Scholar 

  12. Sheldon RA, Woodley JM (2018) Role of biocatalysis in sustainable chemistry. Chem Rev 118:801–838

    Article  CAS  PubMed  Google Scholar 

  13. 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 

  14. Nunes VO, Vanzellotti ND, Fraga JL, Pessoa FL, Ferreira TF, Amaral PF (2022) Biotransformation of phytosterols into androstenedione – a technological prospecting study. Molecules 27:3164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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 

  16. Cruz A, Fernandes P, Cabral JMS, Pinheiro HM (2001) Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems. J Mol Catal B Enzym 11:579–585

    Article  CAS  Google Scholar 

  17. Donova MV, Egorova OV (2012) Microbial steroid transformations: current state and prospects. Appl Microbiol Biotechnol 94:1423–1447

    Article  CAS  PubMed  Google Scholar 

  18. Hu Y, Wang D, Wang X, Wei D (2020) A recycled batch biotransformation strategy for 22-hydroxy-23,24-bisnorchol-4-ene-3-one production from high concentration of phytosterols by mycobacterial resting cells. Biotechnol Lett 42:2589–2594

    Article  CAS  PubMed  Google Scholar 

  19. Wang D, Zhang J, Cao D-D, Wang X, Wei D (2021) Identification and in situ removal of an inhibitory intermediate to develop an efficient phytosterol bioconversion process using a cyclodextrin-resting cell system. RSC Adv 11:24787–24793

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Angelova B, Fernandes P, Spasova D, Mutafov S, Pinheiro HM, Cabral JMS (2006) Scanning electron microscopy investigations on bis(2-ethylhexyl)phthalate treated Mycobacterium cells. Microsc Res Tech 69:613–617

    Article  CAS  PubMed  Google Scholar 

  21. Gulla V, Banerjee T, Patil S (2010) Bioconversion of soysterols to androstenedione by Mycobacterium fortuitum subsp. fortuitum NCIM 5239, a mutant derived from total sterol degrader strain. J Chem Technol Biotechnol 85:1135–1141

    Article  CAS  Google Scholar 

  22. Kutney JP, Milanova RK, Vassilev CD, Stefanov SS, Nedelcheva NV, Kutney P, Milanova K (1999) High yield microbial conversion of phytosterol to androstadienedione and androstenedione, in presence of solubilizer, e.g. silicone. WO9949075-A; EP1066399-A; WO9949075-A1. (Forbes Medi-Tech Inc; Akzo Nobel Nv; Organon Nv)

    Google Scholar 

  23. Rodina NV, Molchanova MA, Voishvillo NE, Andryushina VA, Stytsenko TS (2008) Conversion of phytosterols into androstenedione by Mycobacterium neoaurum. Appl Biochem Microbiol 44:48–54

    Article  CAS  Google Scholar 

  24. Li X, Chen T, Peng F, Song S, Yu J, Sidoine DN, Cheng X, Huang Y, He Y, Su Z (2021) Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria. Microb Cell Factories 20:158

    Article  CAS  Google Scholar 

  25. Zhao W, Xie H, Zhang X, Wang Z (2022) Crystal substrate inhibition during microbial transformation of phytosterols in Pickering emulsions. Appl Microbiol Biotechnol 106:2403–2414

    Article  CAS  PubMed  Google Scholar 

  26. Andriushina VA, Rodina NV, Stytsenko TC, Luu DH, Druzhinina AV, Iaderets VV, Voishvillo NE (2011) Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis. Prikl Biokhim Mikrobiol 47:297–301

    CAS  PubMed  Google Scholar 

  27. Liu Y-J, W-t J, Song L, Tao X-Y, Zhao M, Gao B, Meng H, Wang F-Q, Wei D-Z (2022) Transformation of phytosterols into pregnatetraenedione by a combined microbial and chemical process. Green Chem 24:3759–3771

    Article  CAS  Google Scholar 

  28. Zhang Y, Zhou X, Wang X, Wang L, Xia M, Luo J, Shen Y, Wang M (2020) Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum. Microb Cell Factories 19:13

    Article  CAS  Google Scholar 

  29. Chang H, Zhang H, Zhu L, Zhang W, You S, Qi W, Qian J, Su R, He Z (2020) A combined strategy of metabolic pathway regulation and two-step bioprocess for improved 4-androstene-3,17-dione production with an engineered Mycobacterium neoaurum. Biochem Eng J 164:107789

    Article  CAS  Google Scholar 

  30. Fernández-Cabezón L, Galán B, García JL (2018) New insights on steroid biotechnology. Front Microbiol 9:958

    Article  PubMed  PubMed Central  Google Scholar 

  31. Zhao Y-Q, Liu Y-J, Ji W-T, Liu K, Gao B, Tao X-Y, Zhao M, Wang F-Q, Wei D-Z (2022) One-pot biosynthesis of 7β-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered Mycolicibacterium neoaurum. Microb Cell Factories 21:59

    Article  CAS  Google Scholar 

  32. 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 

  33. Sedlaczek L, Gorminski BM, Lisowska K (1994) Effect of inhibitors of cell-envelope synthesis on beta-sitosterol side-chain degradation by Mycobacterium sp. NRRL-MB-3683. J Basic Microbiol 34:387–399

    Article  CAS  PubMed  Google Scholar 

  34. Llanes N, Hung B, Falero A, Perez C, Aguila B (1995) Glucose and lactose effect on AD and ADD bioconversion by Mycobacterium sp. Biotechnol Lett 17:1237–1240

    Article  CAS  Google Scholar 

  35. Cruz A, Fernandes P, Cabral JMS, Pinheiro HM (2004) Solvent partitioning and whole-cell sitosterol bioconversion activity in aqueous-organic two-phase systems. Enzym Microb Technol 34:342–353

    Article  CAS  Google Scholar 

  36. 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 

  37. Sih CJ (1988) Process for preparing steroids. US 4755463 A

    Google Scholar 

  38. Marsheck WJ, Kraychy S, Muir RD (1972) Microbial degradation of sterols. Appl Microbiol 23:72–77

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu C, Zhang X, Rao Z-M, Shao M-L, Zhang L-L, Wu D, Xu Z-H, Li H (2015) Mutation breeding of high 4-androstene-3,17-dione-producing Mycobacterium neoaurum ZADF-4 by atmospheric and room temperature plasma treatment. J Zhejiang Univ Sci B 16:286–295

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Huang C-L, Chen Y-R, Liu W-H (2006) Production of androstenones from phytosterol by mutants of Mycobacterium sp. Enzym Microb Technol 39:296–300

    Article  CAS  Google Scholar 

  41. Marques MPC, de Carvalho CCCR, Claudino MJC, Cabral JMS, Fernandes P (2007) On the feasibility of the microscale approach for a multistep biotransformation: sitosterol side chain cleavage. J Chem Technol Biotechnol 82:856–863

    Article  CAS  Google Scholar 

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Acknowledgments

The work was partially funded by national funds from FCT in the scope of the project UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit iBB-Institute for Bioengineering and Biosciences and of the project LA/P/0140/2020 of the i4HN-Associate Laboratory Institute for Health and Bioeconomy.

Author information

Authors and Affiliations

  1. iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Carla C. C. R. de Carvalho

  2. Associate Laboratory i4HB – Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Carla C. C. R. de Carvalho & Pedro Fernandes

  3. iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal

    Pedro Fernandes

  4. DREAMS and Faculty of Engineering, Universidade Lusófona de Humanidades e Tecnologias, Lisbon, Portugal

    Pedro Fernandes

Authors
  1. Carla C. C. R. de Carvalho
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  2. Pedro Fernandes
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Corresponding author

Correspondence to Carla C. C. R. de Carvalho .

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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

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de Carvalho, C.C.C.R., Fernandes, P. (2023). Biocatalysis of Steroids by Mycobacterium sp. in Aqueous and Organic Media. 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_13

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  • DOI: https://doi.org/10.1007/978-1-0716-3385-4_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3384-7

  • Online ISBN: 978-1-0716-3385-4

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