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Obtaining of 24-Norchol-4-ene-3,22-dione from Phytosterol with Mutants of Mycolicibacterium neoaurum

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Microbial Steroids

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

Abstract

Engineered mutants of Mycolicibacterium spp. are known producers of valuable steroid synthons with C19 or C22 skeleton. Here we describe a method for site-directed mutagenesis of Mycolicibacterium neoaurum strains, bioconversion from phytosterol, and selective purification of C23 steroid 24-norchol-4-ene-3,22-dione (24-NCED) and C22 steroid 20-hydroxymethylpregn-4-ene-3-one (20-HMP). The yields of crystalline products with 95% purity by the method here described are 2.74 ± 0.085 g for 24-NCED and 1.42 ± 0.085 g for 20-HMP from 10 g/L phytosterol. 20-HMP is recognized as the key precursor in chemical syntheses of pharmaceutical corticosteroids and 24-NCED is a promising synthon for the synthesis of valuable steroids and own potent biological activity.

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References

  1. Donova MV (2007) Transformation of steroids by actinobacteria: a review. Appl Biochem Microbiol 43:1–14. https://doi.org/10.1134/S0003683807010012

    Article  CAS  Google Scholar 

  2. Fernández-Cabezón L, Galán B, García JL (2018) New insights on steroid biotechnology. Front Microbiol 9:958. https://doi.org/10.3389/fmicb.2018.00958

    Article  PubMed  PubMed Central  Google Scholar 

  3. Marsheck WJ, Kraychy S, Muir RD (1972) Microbial degradation of sterols. Appl Microbiol 23:72–77. https://doi.org/10.1128/am.23.1.72-77.1972

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Andor A, Jekkel A, Hopwood DA, Jeanplong F, Ilkőy É, Kónya A, Kurucz I, Ambrus G (2006) Generation of useful insertionally blocked sterol degradation pathway mutants of fast-growing Mycobacteria and cloning, characterization, and expression of the terminal oxygenase of the 3-ketosteroid 9α-hydroxylase in Mycobacterium smegmatis mc 2 155. Appl Environ Microbiol 72:6554–6559. https://doi.org/10.1128/AEM.00941-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Shtratnikova VY, Bragin EY, Dovbnya DV, Pekov YA, Schelkunov MI, Strizhov N, Ivashina TV, Ashapkin VV, Donova MV (2014) Complete genome sequence of sterol-transforming Mycobacterium neoaurum strain VKM Ac-1815D. Genome Announc 2:e01177–e01113. https://doi.org/10.1128/genomeA.01177-13

    Article  PubMed  PubMed Central  Google Scholar 

  6. Rodríguez-García A, Fernández-Alegre E, Morales A, Sola-Landa A, Lorraine J, Macdonald S, Dovbnya D, Smith MCM, Donova M, Barreiro C (2016) Complete genome sequence of ‘Mycobacterium neoaurum’ NRRL B-3805, an androstenedione (AD) producer for industrial biotransformation of sterols. J Biotechnol 224:64–65. https://doi.org/10.1016/j.jbiotec.2016.03.021

    Article  CAS  PubMed  Google Scholar 

  7. Xu L-Q, Liu Y-J, Yao K, Liu H-H, Tao X-Y, Wang F-Q, Wei D-Z (2016) Unraveling and engineering the production of 23,24-bisnorcholenic steroids in sterol metabolism. Sci Rep 6:21928. https://doi.org/10.1038/srep21928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Peng H, Wang Y, Jiang K, Chen X, Zhang W, Zhang Y, Deng Z, Qu X (2021) A dual role reductase from phytosterols catabolism enables the efficient production of valuable steroid precursors. Angew Chem Int Ed 60:5414–5420. https://doi.org/10.1002/anie.202015462

    Article  CAS  Google Scholar 

  9. Imada Y, Takahashi K (1978) A process for producing steroidal alcohols. Patent Eur 0001622

    Google Scholar 

  10. Wovcha MG, Antosz FJ, Beaton JM, Garcia AB, Kominek LA (1980) Process for preparing 9α-OH BN acid methyl ester. US Patent 4214051

    Google Scholar 

  11. Wang Y, Li X, Feng J, Wu Q, Zhu D, Ma Y. (2017) The mycobacteria of one plant of ketone of 4 alkene of biosynthesis 20 hydroxyl, 23,24 dinorchola 3 and synthetic method. Patent CN 106854630

    Google Scholar 

  12. 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. https://doi.org/10.1007/s10529-020-02991-1

    Article  CAS  PubMed  Google Scholar 

  13. Jiu J, Marsheck WJ (1976) 3-Oxo-pregna-4,17(20)-dien-20-carboxylic acid and esters. US Patent 3994933

    Google Scholar 

  14. Toró A, Ambrus G (1990) Oxidative decarboxylation of 17(20)-dehydro-23,24-dinorcholanoic acids. Tetrahedron Lett 31:3475–3476. https://doi.org/10.1016/S0040-4039(00)97426-4

    Article  Google Scholar 

  15. Toró A, Ambrus G (1992) Synthesis of 17α-hydroxy-20-oxo-pregnanes from 17(20)-dehydro-23,24-dinorcholan-22-oic acids. Tetrahedron Lett 33:5265–5266. https://doi.org/10.1016/S0040-4039(00)79150-7

    Article  Google Scholar 

  16. Gavagnin M, Ungur N, Mollo E, Templado J, Cimino G (2002) Structure and synthesis of a progesterone homologue from the skin of the dorid nudibranch Aldisa smaragdina. Eur J Org Chem 2002:1500–1504. https://doi.org/10.1002/1099-0690(200205)2002:9<1500::AID-EJOC1500>3.0.CO;2-D

    Article  Google Scholar 

  17. Schwarz V, Pihera P, Protiva J, Mickova R (1984) Biodegradation of cholesterol by a mutant of the Mycobacterium species. Collect Czechoslov Chem Commun 49:2713–2719

    Article  CAS  Google Scholar 

  18. Imada Y, Takahashi K (1981) Steroid and its preparation. Patent Japan JPS56120698

    Google Scholar 

  19. Shtratnikova VY, Schelkunov MI, Fokina VV, Pekov YA, Ivashina T, Donova MV (2016) Genome-wide bioinformatics analysis of steroid metabolism-associated genes in Nocardioides simplex VKM Ac-2033D. Curr Genet 62:643–656. https://doi.org/10.1007/s00294-016-0568-4

    Article  CAS  PubMed  Google Scholar 

  20. Nesbitt NM, Yang X, Fontán P, Kolesnikova I, Smith I, Sampson NS, Dubnau E (2010) A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol. Infect Immun 78:275–282. https://doi.org/10.1128/IAI.00893-09

    Article  CAS  PubMed  Google Scholar 

  21. Xiong L-B, Liu H-H, Xu L-Q, Sun W-J, Wang F-Q, Wei D-Z (2017) Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes. Microb Cell Factories 16:89. https://doi.org/10.1186/s12934-017-0705-x

    Article  CAS  Google Scholar 

  22. Dovbnya DV, Ivashina TV, Khomutov SM, Shutov AA, Donova MV (2022) Recombinant mycobacterial producer strains and their use to produce 22-functionalised steroids. Patent RU 2767610

    Google Scholar 

  23. Loraine JK, Smith MCM (2017) Genetic techniques for manipulation of the phytosterol biotransformation strain Mycobacterium neoaurum NRRL B-3805. In: Barredo J-L, Herráiz I (eds) Microbial Steroids. Springer New York, New York, pp 93–108. https://doi.org/10.1007/978-1-4939-7183-1_7

    Chapter  Google Scholar 

  24. Ivashina TV, Nikolayeva VM, Dovbnya DV, Donova MV (2012) Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D. J Steroid Biochem Mol Biol 129:47–53. https://doi.org/10.1016/j.jsbmb.2011.09.008

    Article  CAS  PubMed  Google Scholar 

  25. Hesselink PGM, van Vliet S, de Vries H, Witholt B (1989) Optimization of steroid side chain cleavage by Mycobacterium sp. in the presence of cyclodextrins. Enz Microb Technol 11:398–404. https://doi.org/10.1016/0141-0229(89)90133-6

    Article  CAS  Google Scholar 

  26. Zhang L, Wang M, Shen Y, Ma Y, Luo J (2009) Improvement of steroid biotransformation with hydroxypropyl-β-cyclodextrin induced complexation. Appl Biochem Biotechnol 159:642–654. https://doi.org/10.1007/s12010-008-8499-2

    Article  CAS  PubMed  Google Scholar 

  27. Zhao A, Zhang X, Li Y, Wang Z, Lv Y, Liu J, Alam MA, Xiong W, Xu J (2021) Mycolicibacterium cell factory for the production of steroid-based drug intermediates. Biotechnol Adv 53:107860. https://doi.org/10.1016/j.biotechadv.2021.107860

    Article  CAS  PubMed  Google Scholar 

  28. Weber A, Muller R, Kurzidim J (1976) Method for isolation of 3-hydroxy steroids and 3-keto steroids. US Patent 4057541

    Google Scholar 

  29. Donova MV, Dovbnya DV, Kalinichenko AN, Arinbasarova AYu, Vagabova LM, Morozova ZV, Koshcheyenko KA (1997) Method of androst-4-ene-3,17-dione preparing. Patent RU 2079258

    Google Scholar 

  30. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  31. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86. https://doi.org/10.1016/S0378-1119(98)00130-9

    Article  CAS  PubMed  Google Scholar 

  32. Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268. https://doi.org/10.1016/0378-1119(82)90015-4

    Article  CAS  PubMed  Google Scholar 

  33. Oka A, Sugisaki H, Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147:217–226. https://doi.org/10.1016/0022-2836(81)90438-1

    Article  CAS  PubMed  Google Scholar 

  34. Bragin EY, Shtratnikova VY, Dovbnya DV, Schelkunov MI, Pekov YA, Malakho SG, Egorova OV, Ivashina TV, Sokolov SL, Ashapkin VV, Donova MV (2013) Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp. strains. J Steroid Biochem Mol Biol 138:41–53. https://doi.org/10.1016/j.jsbmb.2013.02.016

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by Russian Science Foundation under Grant No. 21-64-00024.

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Authors and Affiliations

  1. Institute of Biochemistry & Physiology of Microorganisms, Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences, Federal Research Center, Pushchino, Russia

    Dmitry V. Dovbnya, Tanya V. Ivashina, Sergey M. Khomutov, Andrei A. Shutov, Natalia O. Deshcherevskaya & Marina V. Donova

  2. Pharmins LTD, Pushchino, Russia

    Marina V. Donova

Authors
  1. Dmitry V. Dovbnya
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  2. Tanya V. Ivashina
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  3. Sergey M. Khomutov
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  4. Andrei A. Shutov
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  5. Natalia O. Deshcherevskaya
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  6. Marina V. Donova
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Corresponding author

Correspondence to Dmitry V. Dovbnya .

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

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Dovbnya, D.V., Ivashina, T.V., Khomutov, S.M., Shutov, A.A., Deshcherevskaya, N.O., Donova, M.V. (2023). Obtaining of 24-Norchol-4-ene-3,22-dione from Phytosterol with Mutants of Mycolicibacterium neoaurum. 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_18

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  • DOI: https://doi.org/10.1007/978-1-0716-3385-4_18

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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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