The activity of Aspergillus niger ATCC1015 to biotransform 16α,17α-epoxy progesterone was investigated for rational engineering ofAspergillus niger.The transformation product was determined as 11α-hydroxy-16α,17α-epoxy pro-gesterone based on analyses of TLC,HPLC and NMR spectroscopy.In addition,the 11α-hydroxylation activities ofA.niger ATCC1015 on 16α,17α-epoxy progesterone were shown to be induced by substrate 16α,17α-epoxy progesterone.Given that fungal steroid hydroxylases are members of the cytochrome P450 family,57 CYP genes were selected as potential can-didates of the target hydroxylase genes.Quantitative Real-time PCR showed that expression of 2 of 57 CYP genes named AnA100 and AnA154 were highly induced by 16α,17α-epoxy progesterone treatment.RecombinantS.cerevisiae stains pYES2-AnA100 and pYES2-AnA154 were constructed with genes AnA100 and AnA154 respectively.The results of the steroidal transformation showed that recombinantS.cerevisiaepYES2-AnA100 can transform 16α,17α-epoxy progesterone to 11α-hydroxylation of 16α,17α-epoxyprogesterone.%为了定向遗传改造黑曲霉菌种,研究了黑曲霉(Aspergillus niger)ATCC1015转化甾体16α,17α–环氧黄体酮活性.转化产物经过薄层层析(TLC)、高效液相色谱(HPLC)以及氢谱、碳谱分析最终确定为11α–羟基–16α,17α–环氧黄体酮.确定了黑曲霉ATCC101511α–羟基化活性受底物16α,17α–环氧黄体酮的诱导.鉴于真菌的甾体羟化酶属于细胞色素P450酶,从黑曲霉ATCC1015的P450(CYP)基因数据库中筛选出57个具有编码甾体羟化酶潜力的CYP基因;利用实时荧光定量PCR确定了2个受甾体底物高度诱导的候选目标甾体羟化酶基因AnA100和AnA154.分别构建了AnA100基因和AnA154基因的重组酿酒酵母菌株pYES2-AnA100和pYES2-AnA154,甾体转化结果显示重组酵母菌pYES2-An100能够转化16α,17α–环氧黄体酮生成11α–羟基–16α,17α–环氧黄体酮.
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