Biotechnology Letters

, Volume 40, Issue 4, pp 673–678 | Cite as

Engineering phytosterol transport system in Mycobacterium sp. strain MS136 enhances production of 9α-hydroxy-4-androstene-3,17-dione

  • Kun He
  • Hong Sun
  • Hao Song
Original Research Paper



To enhance the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from phytosterols, a phytosterol transport system was constructed in Mycobacterium sp. strain MS136.


9-OHAD can be produced via the controlled degradation of phytosterols by mycobacteria. This involves an active transport process that requires trans-membrane proteins and ATP. A phytosterol transport system from Mycobacterium tuberculosis H37Rv was constructed in Mycobacterium sp. strain MS136 by co-expression of an energy-related gene, mceG, and two integrated membrane protein genes, yrbE4A and yrbE4B. The resultant of the Mycobacterium sp. strain MS136-GAB gave 5.7 g 9-OHAD l−1, which was a 20% increase over 4.7 g l−1 by the wild-type strain. The yield of 9-OHAD was increased to 6.0 g l−1 by optimization of fermentation conditions, when 13 g phytosterols l−1 were fermented for 84 h in 30 ml biotransformation medium in shake flasks.


Phytosterol transport system plays an active role in the uptake and transport of sterols, cloning of the system improved the mass transfer of phytosterols and increased the production of 9-OHAD.


9α-Hydroxy-4-androstene-3,17-dione Mycobacteria Optimization Phytosterols Steroids Transport system 



The authors acknowledge the financial support by a startup funding from Tianjin University.

Supporting information

Supplementary Table 1—Strains, primers and plasmids used in this study.

Supplementary Fig. 1—HPLC analysis of 9-OHAD produced by engineered strains.

Supplementary material

10529_2018_2520_MOESM1_ESM.docx (44 kb)
Supplementary material 1 (DOCX 44 kb)


  1. Donova MV, Egorova OV (2012) Microbial steroid transformations: current state and prospects. Appl Microbiol Biotechnol 94:1423–1447CrossRefPubMedGoogle Scholar
  2. Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS (2003) Microbial conversion of steroid compounds: recent developments. Enzym Microbial Technol 32:688–705CrossRefGoogle Scholar
  3. Gao XQ (2016) Enhanced steroid metabolites production by resting cell phytosterol bioconversion. Chem Biochem Eng Q 29:567–573CrossRefGoogle Scholar
  4. Geize RVD, Yam K, Heuser T, Wilbrink MH, Hara H et al (2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci USA 104:1947–1952CrossRefPubMedPubMedCentralGoogle Scholar
  5. Joshi SM, Pandey AK, Capite N, Fortune SM, Rubin EJ, Sassetti CM (2006) Characterization of mycobacterial virulence genes through genetic interaction mapping. Proc Natl Acad Sci USA 103:11760–11765CrossRefPubMedPubMedCentralGoogle Scholar
  6. Klepp LI, Forrellad MA, Osella AV, Blanco FC et al (2012) Impact of the deletion of the six mce operons in Mycobacterium smegmatis. Microbes Infect 14:590–599CrossRefPubMedPubMedCentralGoogle Scholar
  7. Knol J, Bodewits K, Hessels GI, Dijkhuizen L, Geize RVD (2008) 3-Keto-5alpha-steroid delta(1)-dehydrogenase from Rhodococcus erythropolis SQ1 and its orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that function in cholesterol catabolism. Biochem J 410:339–346CrossRefPubMedGoogle Scholar
  8. Mohn WW, Geize RVD, Stewart GR, Okamoto S, Liu J, Dijkhuizen L, Eltis LD (2008) The actinobacterial mce4 locus encodes a steroid transporter. J Biol Chem 283:35368–35374CrossRefPubMedPubMedCentralGoogle Scholar
  9. Pandey AK, Sassetti CM (2008) Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci USA 105:4376–4380CrossRefPubMedPubMedCentralGoogle Scholar
  10. Wang Z, Zhao F, Chen D, Li D (2006) Biotransformation of phytosterol to produce androsta-diene-dione by resting cells of Mycobacterium in cloud point system. Process Biochem 41:557–561CrossRefGoogle Scholar
  11. Wei W, Wang FQ, Fan SY, Wei DZ (2010) Inactivation and augmentation of the primary 3-ketosteroid-Δ1-dehydrogenase in Mycobacterium neoaurum NwIB-01: biotransformation of soybean phytosterols to 4-androstene-3,17-dione or 1,4-androstadiene-3,17-dione. Appl Environ Microbiol 76:4578–4582CrossRefPubMedPubMedCentralGoogle Scholar
  12. Wilbrink MH, Petrsma M, Dijkhuizen L, Geize RVD (2011) FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme a ligase essential for degradation of C-24 branched sterol side chains. Appl Environ Microbiol 77:4455–4464CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Systems Bioengineering, Ministry of Education, School of Chemical Engineering and Technology, and SynBio Research Platform, Collaborative Innovation Centre of Chemical Science and EngineeringTianjin UniversityTianjinChina

Personalised recommendations