Centro de Investigaciones Biológicas Margarita Salas, Agencia del Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Microb Biotechnol. 2021 Nov;14(6):2514-2524. doi: 10.1111/1751-7915.13735. Epub 2021 Mar 4.
11α-hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α-hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α-hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH-AD and 11αOH-ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α-hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end-products.
11α-羟化甾体前体是用于生产避孕药和糖皮质激素的最重要的商业医药中间体之一。这些化合物目前通过真菌菌株的生物转化，在两步连续发酵步骤中生产。在这项工作中，我们通过合理设计，开发了一种新的重组细菌，能够使用胆固醇（CHO）或植物固醇（PHYTO）作为原料，在一步发酵中生产 11α-羟化前体。我们设计了一个表达来自真菌米根霉的 11α-羟化酶的合成操纵子，该操纵子被克隆到先前为了从甾体中生产 4-雄烯-3,17-二酮（AD）和 1,4-雄二烯-3,17-二酮（ADD）而构建的分枝杆菌突变菌株中。在这些经过修饰的细菌底盘中引入真菌合成操纵子，首次允许在一步发酵中直接从甾体中高产量生产 11αOH-AD 和 11αOH-ADD。值得注意的是，分枝杆菌甾醇分解代谢途径的酶将 11α-羟化中间产物识别为替代底物，并能够有效地将甾醇转化为所需的羟化终产物。
