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.

诸如4-雄甾烯-3,17-二酮（AD）和1,4-雄甾二烯-3,17-二酮（ADD）的中间体在类固醇药物的生产中具有广泛的临床应用。本研究探讨了两个因素在新分枝杆菌中这些中间体的产生中的作用。JC-12：前体，植物固醇和增加AD / ADD溶解度的分子，羟丙基-β-环糊精（HP-β-CD）。使用2D凝胶电泳（2-DE）和基质辅助激光解吸/电离飞行时间/飞行时间串联质谱（MALDI-TOF / TOF-MS / MS）分离并鉴定差异表达的蛋白质。总共鉴定出31种蛋白质，植物甾醇和/或HP-β-CD诱导了10种参与代谢的蛋白质的表达水平提高，从而增强了菌株的抗逆性。在存在植物甾醇和/或HP-β-CD的情况下，五种蛋白质参与AD / ADD，乙酰辅酶A乙酰转移酶（AAT），醇脱氢酶（ADH），烯酰辅酶A水合酶（EH）和短链的合成脱氢酶1和2增加了它们的表达水平。逆转录定量PCR（RT-qPCR）用于验证2-DE结果和这五个蛋白的转录水平。该分析确定了AAT，ADH，EH和电子转移黄素蛋白亚基α/β是AD / ADD合成中可能的瓶颈因此，新孢霉JC-12被建议作为菌株修饰的靶标。