Synthetic glucocorticoids are generally preferred over their natural counterparts as these compounds exhibit improved anti-inflammatory potency and glucocorticoid receptor selectivity. However, the biotechnological production of these molecules is often subject to limitations inferred by restricted enzyme stability, selectivity or inhibition thereof. The latter is particularly important during 6α-methylprednisolone production, as the essential C21-hydroxylation of its precursor medrane appears to be hampered by product inhibition of the steroid-21-hydroxylase (CYP21A2). To circumvent this bottleneck, we established a two-step reaction for controlled mixed-culture fermentation, using recombinant E. coli. This process comprises the previously reported C21-hydroxylation of medrane by CYP21A2, followed by an instant derivatization of the hydroxylated product premedrol by chloramphenicol acetyl transferase 1 (CAT1). The CAT1-mediated C21-acetylation prevents the product from regaining access to the enzyme's active site which effectively shifts the chemical equilibrium toward premedrol formation. The successful circumvention of product inhibition at optimized conditions resulted in the formation of more than 1.5 g of product per liter which corresponds to an increase by more than 100 %. Taken together, we demonstrate an efficient system to enhance cytochrome P450-mediated biotransformations, holding great ecologic and economic potential to be applied in industrial processes.

中文翻译：

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混合培养发酵可增强糖皮质激素的C21-羟基化作用。

合成的糖皮质激素通常优于天然的糖皮质激素，因为这些化合物显示出改善的抗炎能力和糖皮质激素受体选择性。但是，这些分子的生物技术生产通常受到限制的酶稳定性，选择性或抑制作用的限制。后者在6α-甲基泼尼松龙的生产过程中尤其重要，因为其前体甲炔的必要C21-羟基化似乎受到类固醇21-羟化酶（CYP21A2）的抑制作用。为了克服这一瓶颈，我们使用重组大肠杆菌建立了两步反应，以进行受控的混合培养发酵。此过程包括先前报道的CYP21A2对甲磺酸的C21-羟基化反应，然后立即通过氯霉素乙酰基转移酶1（CAT1）衍生化羟化前药的羟脯氨酸。CAT1介导的C21乙酰化作用会阻止产物重新获得酶的活性位点，从而有效地将化学平衡转移至前醇形成。在最佳条件下成功地规避了产品抑制作用，每升形成了超过1.5 g的产品，相当于增加了100％以上。综上所述，我们展示了一种有效的系统来增强细胞色素P450介导的生物转化，具有在工业过程中应用的巨大生态和经济潜力。活性位点，有效地将化学平衡转移至前药形成。在最佳条件下成功地规避了产品抑制作用，每升形成了超过1.5 g的产品，相当于增加了100％以上。综上所述，我们展示了一种有效的系统来增强细胞色素P450介导的生物转化，具有在工业过程中应用的巨大生态和经济潜力。活性位点，有效地将化学平衡转移至前药形成。在最佳条件下成功地规避了产品抑制作用，每升形成了超过1.5 g的产品，相当于增加了100％以上。综上所述，我们展示了一种有效的系统来增强细胞色素P450介导的生物转化，具有在工业过程中应用的巨大生态和经济潜力。