Colletotrichum lini is used as an industrial stain for the dihydroxylation of steroid compound dehydroepiandrosterone (DHEA) to biosynthesize 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA), a key intermediate of the most popular oral contraceptive “Yasmin”. This work aimed to enhance 7α,15α-diOH-DHEA production in C. lini CGMCC 6051 through ethanol induction. With 0.6% (v/v) ethanol induction and 10 g/L DHEA concentration, the 7α,15α-diOH-DHEA molar yield reached 58.8%, which was increased by 67.5% than that of the control. iTRAQ-based quantitative proteomic analysis was applied to explore the probable molecular mechanism of C. lini response to ethanol induction. A total of 50 differential expressed proteins was affected by ethanol induction, and could be related to multiple metabolic pathways. Most of differently expressed proteins were functionally mapped into pathways of transport, steroids metabolism, or redox reaction. Other proteins for energy, transcription and translation, and carbohydrate metabolism might have important roles in the cellular response to ethanol induction. In addition, the levels of cytochrome P450 and NAD(P)H-cytochrome P450 reductase were remarkably higher under ethanol induction, and their functions on DHEA dihydroxylation were first proposed in C. lini. Our results provide critical clues in revealing the dihydroxylation mechanism and are important for efficient microbiological hydroxylation of steroidal compounds in the future.

Biological significance

iTRAQ strategy was first used to compare the proteomes of ethanol induction during the dihydroxylation reaction by Colletotrichum lini CGMCC 6051. The changes in protein provided a comprehensive overview of DHEA dihydroxylation in C. lini, including the proteins for steroids metabolism, redox reaction, transport, transcription and translation, energy and carbohydrate metabolism. Cytochrome P450, NADPH-cytochrome P450 reductase, and NADH-cytochrome b5 reductase were highlighted due to their outstanding contribution to DHEA dihydroxylation. The results help us understand the molecular mechanism underlying ethanol induction in C. lini and would guide strain engineering to further improve dihydroxylation efficiency.

中文翻译：

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基于iTRAQ的Colletotrichum lini定量蛋白质组学分析揭示了乙醇诱导的增强DHEA二羟基化效率的机制。

Colletotrichum lini被用作工业污渍，用于甾类化合物脱氢表雄酮（DHEA）的二羟基化反应，以生物合成3β，7α，15α-三羟基-5-雄烯酮-17-一（7α，15α-diOH-DHEA）的关键中间体。最受欢迎的口服避孕药“ Yasmin”。这项工作旨在通过乙醇诱导提高C. lini CGMCC 6051中7α，15α-diOH-DHEA的产量。以0.6％（v / v）乙醇诱导和10 g / L DHEA浓度，7α，15α-diOH-DHEA摩尔产率达到58.8％，比对照提高了67.5％。基于iTRAQ的定量蛋白质组学分析被用于探索C. lini的可能的分子机制对乙醇诱导的反应。共有50种差异表达蛋白受到乙醇诱导的影响，并且可能与多种代谢途径有关。大多数差异表达的蛋白质在功能上定位于运输，类固醇代谢或氧化还原反应的途径。其他用于能量，转录和翻译以及碳水化合物代谢的蛋白质可能在细胞对乙醇诱导的反应中起重要作用。此外，在乙醇诱导下，细胞色素P450和NAD（P）H-细胞色素P450还原酶的水平显着升高，并且它们首次在C. lini中提出了其对DHEA二羟基化的作用。。我们的结果为揭示二羟基化机理提供了关键线索，对于将来甾类化合物的有效微生物羟基化具有重要意义。

生物学意义

的iTRAQ策略最早是由二羟基化反应过程中的乙醇诱导的蛋白质组比较炭疽病利尼CGMCC 6051中蛋白质的变化在提供DHEA二羟基化的全面概述C.利尼，包括蛋白质为类固醇代谢，氧化还原反应，运输，转录和翻译，能量和碳水化合物代谢。细胞色素P450，NADPH-细胞色素P450还原酶和NADH-细胞色素b 5还原酶因其对DHEA二羟基化的杰出贡献而得到了强调。这些结果有助于我们了解C. lini中乙醇诱导的分子机制，并指导菌株工程技术进一步提高二羟基化效率。