Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain.
Environ Microbiol. 2018 May;20(5):1815-1827. doi: 10.1111/1462-2920.14114. Epub 2018 Apr 24.
In this work, we have characterized the C-19+ gene cluster (MSMEG_2851 to MSMEG_2901) of Mycobacterium smegmatis. By in silico analysis, we have identified the genes encoding enzymes involved in the modification of the A/B steroid rings during the catabolism of C-19 steroids in certain M. smegmatis mutants mapped in the PadR-like regulator (MSMEG_2868), that constitutively express the C-19+ gene cluster. By using gene complementation assays, resting-cell biotransformations and deletion mutants, we have characterized the most critical genes of the cluster, that is, kstD2, kstD3, kshA2, kshB2, hsaA2, hsaC2 and hsaD2. These results have allowed us to propose a new catabolic route named C-19+ pathway for the mineralization of C-19 steroids in M. smegmatis. Our data suggest that the deletion of the C-19+ gene cluster may be useful to engineer more robust and efficient M. smegmatis strains to produce C-19 steroids from sterols. Moreover, the new KshA2, KshB2, KstD2 and KstD3 isoenzymes may be useful to design new microbial cell factories for the 9α-hydroxylation and/or Δ1-dehydrogenation of 3-ketosteroids.
在这项工作中，我们对分枝杆菌的 C-19+基因簇（MSMEG_2851 至 MSMEG_2901）进行了特征描述。通过计算机分析，我们鉴定了编码参与某些在 PadR 样调控因子（MSMEG_2868）中定位的分枝杆菌突变体中 C-19 甾体代谢过程中 A/B 甾体环修饰的酶的基因，该调控因子使 C-19+基因簇持续表达。通过使用基因互补测定、休止细胞生物转化和缺失突变体，我们对该簇的最关键基因进行了表征，即 kstD2、kstD3、kshA2、kshB2、hsaA2、hsaC2 和 hsaD2。这些结果使我们能够提出一种新的命名为 C-19+途径的矿化途径，用于分枝杆菌中 C-19 甾体的矿化。我们的数据表明，C-19+基因簇的缺失可能有助于构建更健壮和高效的分枝杆菌菌株，用于从甾醇生产 C-19 甾体。此外，新的 KshA2、KshB2、KstD2 和 KstD3 同工酶可能有助于设计用于 3-酮甾体的 9α-羟化和/或Δ1-脱氢的新微生物细胞工厂。
