Kollerov Vyacheslav V, Timakova Tatiana A, Shutov Andrei A, Donova Marina V
Federal Research Center, Pushchino Center for Biological Research of Russian Academy of Sciences, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Prospekt Nauki, 5, 142290 Pushchino, Moscow Region, Russia.
J Fungi (Basel). 2024 Nov 28;10(12):830. doi: 10.3390/jof10120830.
Testosterone (TS) and its 1(2)-dehydrogenated derivative boldenone (BD) are widely used in medicine, veterinary science and as precursors in organic synthesis of many therapeutic steroids. Green production of these compounds is possible from androstenedione (AD) enzymatically, or from phytosterol (PS) using fermentation stages. In this study, the ascomycete sp. VKM F-3040 was shown to convert androstadienedione (ADD, 4 and 10 g/L) to yield 97% and 78% (mol/mol) of BD, respectively. Based on its high 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, a novel cascade biotransformation of PS was developed for production of TS and BD. At the first stage, the strains of VKM Ac-1815D or VKM Ac-1816D converted PS (5 or 10 g/L) into AD or ADD (each in a concentration of 2.5 or 5 g/L), respectively. At the second stage, mycelium of the fungus under the revealed optimal conditions reduced AD or ADD with more than 90% efficiency to form TS or BD, respectively. Based on transcriptome analysis, six candidate genes that might encode 17β-HSDs in the sp. genome were revealed. Along with 17β-HSDs, the fungus possessed inducible P450 7-monooxygenase, which led to the accumulation of 7α-hydroxytestosterone (7α-OH-TS) as a major product from AD (up to 83% within 24 h after mycelium addition at the second stage of cascade biotransformation). The presence of protein synthesis inhibitor cycloheximide (CHX) prevented 7α/β-hydroxylation due to inhibition of de novo synthesis of the enzyme in the fungal cells. The results demonstrate the high biotechnological potential of the sp. strain and open up prospects for the synthesis of valuable 17β-reduced and 7-hydroxylated steroids by cascade biotransformations.
睾酮（TS）及其1(2)-脱氢衍生物勃地龙（BD）在医学、兽医学中被广泛使用，并且作为许多治疗性甾体有机合成的前体。这些化合物可以通过酶法由雄烯二酮（AD）绿色生产，或者通过发酵阶段由植物甾醇（PS）生产。在本研究中，子囊菌属的某种菌株VKM F-3040被证明可将雄二烯二酮（ADD，4 g/L和10 g/L）分别转化为产率为97%和78%（摩尔/摩尔）的BD。基于其高17β-羟基甾体脱氢酶（17β-HSD）活性，开发了一种用于生产TS和BD的新型PS级联生物转化方法。在第一阶段，VKM Ac-1815D或VKM Ac-1816D菌株分别将PS（5 g/L或10 g/L）转化为AD或ADD（各自浓度为2.5 g/L或5 g/L）。在第二阶段，在揭示的最佳条件下，真菌菌丝体将AD或ADD分别以超过90%的效率还原形成TS或BD。基于转录组分析，在该菌株基因组中揭示了六个可能编码17β-HSD的候选基因。除了17β-HSD之外，该真菌还具有诱导型P450 7-单加氧酶，这导致在级联生物转化的第二阶段添加菌丝体后24小时内，作为AD的主要产物7α-羟基睾酮（7α-OH-TS）积累（高达83%）。蛋白质合成抑制剂环己酰亚胺（CHX）的存在由于抑制真菌细胞中该酶的从头合成而阻止了7α/β-羟基化。结果证明了该菌株的高生物技术潜力，并为通过级联生物转化合成有价值的17β-还原和7-羟基化甾体开辟了前景。
