title: [Overexpressing 3-ketosteroid-Δ1-dehydrogenase for degrading phytosterols into androst-1,4-diene-3,17-dione].
abstract: We constructed plasmid pMTac to overexpress 3-ketosteroid-Δ1-dehydrogenase (KSDD) in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione (ADD) production. To construct pMTac, pACE promoter on pMF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein (GFP) was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2% compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD (4-androstene-3,17-dione) production of JC-12/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.
authors.forename: Lele
authors.initials: L
authors.lastname: Zhang
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.forename: Minglong
authors.initials: M
authors.lastname: Shao
authors.forename: Rongrong
authors.initials: R
authors.lastname: Chen
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.forename: Hu
authors.initials: H
authors.lastname: Li
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Xu
mesh_terms: D000730:Androstadienes / Q000378:metabolism*; D005285:Fermentation; D007218:Industrial Microbiology; D009161:Mycobacterium; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010840:Phytosterols / Q000378:metabolism*; D010957:Plasmids
chemical_list: D000730:Androstadienes; D010840:Phytosterols; C006573:1,4-androstadiene-3,17-dione; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase