Engineering phytosterol transport system in Mycobacterium sp. strain MS136 enhances production of 9α-hydroxy-4-androstene-3,17-dione
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.
Keywords9α-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.
Supplementary Table 1—Strains, primers and plasmids used in this study.
Supplementary Fig. 1—HPLC analysis of 9-OHAD produced by engineered strains.
- 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
- 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