title: Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione.
abstract: The gene encoding the putative reductase component (KshB) of 3-ketosteroid 9α-hydroxylase was cloned from Rhodococcus equi USA-18, a cholesterol oxidase-producing strain formerly named Arthrobacter simplex USA-18, by PCR according to consensus amino acid motifs of several bacterial KshB subunits. Deletion of the gene in R. equi USA-18 by a PCR-targeted gene disruption method resulted in a mutant strain that could accumulate up to 0.58 mg/ml 1,4-androstadiene-3,17-dione (ADD) in the culture medium when 0.2% cholesterol was used as the carbon source, indicating the involvement of the deleted enzyme in 9α-hydroxylation of steroids. In addition, this mutant also accumulated 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ1,4-BNC). Because both ADD and Δ1,4-BNC are important intermediates for the synthesis of steroid drugs, this mutant derived from R. equi USA-18 may deserve further investigation for its application potential.
authors.forename: Chin-Hsing
authors.initials: CH
authors.lastname: Yeh
authors.forename: Yung-Shun
authors.initials: YS
authors.lastname: Kuo
authors.forename: Che-Ming
authors.initials: CM
authors.lastname: Chang
authors.forename: Wen-Hsiung
authors.initials: WH
authors.lastname: Liu
authors.forename: Meei-Ling
authors.initials: ML
authors.lastname: Sheu
authors.forename: Menghsiao
authors.initials: M
authors.lastname: Meng
mesh_terms: D000730:Androstadienes / Q000737:chemistry / Q000378:metabolism*; D001711:Biotransformation; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D002876:Chromosomes, Bacterial / Q000235:genetics; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D015203:Reproducibility of Results; D016929:Rhodococcus equi / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D013256:Steroids / Q000737:chemistry* / Q000378:metabolism; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: C077549:3-oxo-bisnorchola-1,4-dien-22-oic acid; D000730:Androstadienes; D013256:Steroids; D013261:Sterols; C006573:1,4-androstadiene-3,17-dione; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases