The use of Aspergillus ochraceus TCCC41060 for synthesis of 11α-OH-ethylgonendione, an important intermediate for synthesis of desogestrel-a major ingredient of the “third-generation” oral contraceptives, is hampered by its low regioselectivity of hydroxylation. In the present study, we sought to characterize gene(s) involved in steroid hydroxylation specificity in strain TCCC41060. Taking advantage of the fact that expression of the 11α-hydroxylase, a member of the cytochrome P450 family, is highly induced by steroid substrates, we combined RNA-seq, qRT-PCR, and yeast functional expression to search for responsible steroid hydroxylase gene(s). Two highly inducible P450 genes (CYP68L8 and CYP68J5) were isolated and recombinant yeast cells expressing CYP68J5 were capable of 11α-hydroxylating both 16,17α-epoxyprogesterone and D-ethylgonendione. Disruption of CYP68J5 in strain TCCC41060 resulted in complete loss of hydroxylation activities towards D-ethylgonendione, indicating that CYP68J5 was solely responsible for hydroxylation activity on D-ethylgonendione in TCCC41060. The above results demonstrated that low hydroxylation specificity of CYP68J5 on D-ethylgonendione fully accounted for high by-product contents in TCCC41060, thus pointing to a strategy to engineer 11α-hydroxylase variants with higher hydroxylation specificity.

中文翻译：

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用细胞色素P450基因CYP68J5测定工业菌株曲霉TCCC41060的甾体羟基化特异性

曲霉TCCC41060用于合成11α-OH-乙基gonendione（去甲孕烯醇（“第三代”口服避孕药的主要成分）的重要中间体）的合成因其低羟基选择性而受阻。在本研究中，我们试图表征TCCC41060菌株中涉及甾体羟基化特异性的基因。利用类固醇底物高度诱导细胞色素P450家族成员11α-羟化酶的表达这一事实，我们结合RNA-seq，qRT-PCR和酵母功能性表达来寻找负责任的类固醇羟化酶基因（ s）。分离出两个高诱导性的P450基因（CYP68L8和CYP68J5），表达CYP68J5的重组酵母细胞能够11α-羟基化16,17α-环氧孕酮和D-乙基gonendione。菌株TCCC41060中CYP68J5的破坏导致对D-乙基gonendione的羟基化活性完全丧失，表明CYP68J5完全负责TCCC41060中D-乙基gonendione的羟基化活性。以上结果表明，CYP68J5在D-乙基gonendione上的低羟基化特异性完全解释了TCCC41060中高副产物的含量，因此提出了设计具有更高羟基化特异性的11α-羟化酶变异体的策略。