Identification of bottlenecks in 4-androstene-3,17-dione/1,4-androstadiene-3,17-dione synthesis by Mycobacterium neoaurum JC-12 through comparative proteomics

Intermediates such as 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) have extensive clinical applications in the production of steroid pharmaceuticals. The present study explores the effect of two factors in the production of these intermediates in Mycobacterium neoaurum JC-12: the precursor, phytosterol and a molecule that increases AD/ADD solubility, hydroxypropyl-β-cyclodextrin (HP-β-CD). Differentially expressed proteins were separated and identified using 2D gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS). In total, 31 proteins were identified, and improved expression levels of ten proteins involved in metabolism was induced by phytosterol and/or HP-β-CD, which strengthened the stress resistance of the strain. In the presence of phytosterol and/or HP-β-CD, five proteins involved in the synthesis of AD/ADD, acetyl-CoA acetyltransferase (AAT), alcohol dehydrogenase (ADH), enoyl-CoA hydratase (EH) and short-chain dehydrogenase 1 and 2, increased their expression levels. Reverse transcription-quantitative PCR (RT-qPCR) was used to verify the 2-DE results and the transcriptional level of these five proteins. This analysis identified AAT, ADH, EH, and electron transfer flavoprotein subunit α/β as the possible bottlenecks for AD/ADD synthesis in M. neoaurum JC-12, which therefore are suggested as targets for strain modification. •A comprehensive proteomic analysis of AD/ADD producing M. neoaurum JC-12 at different fermentation conditions is performed.•Herein, the analysis is performed by combing 2DE analysis and mass spectrometry.•This study facilitates an understanding of global regulation mechanisms of M. neoaurum JC-12 under various conditions.•The possible bottlenecks (AAT, ADH, EH, ETF subunit α/β) for AD/ADD synthesis pathway in M. neoaurum JC-12 are identified.