The chemical and biotechnological modification of steroids is of utmost importance for the development of new functionalities of steroid drugs. One of such important modification is the introduction of a double bond between the atoms C1 and C2 of ring A, so-called Δ dehydrogenation, which increases the potency and selectivity of steroid drugs. For example, the oxidation of hydrocortisone to prednisolone results in increased anti-inflammatory activity while reducing salt-retaining activity . Δ 1 -Dehydrogenation, which starts the central degradation pathway in bacteria (the opening of ring A leading to the complete mineralization of the steroids), is catalysed by a microbial flavin enzyme, 3-ketosteroid Δ -dehydrogenase (KstD, EC 1.3.99.4). The genes encoding KstDs, characterized by a rather high sequence diversity, are abundant among various bacteria and fungi . As a result, microbial systems are routinely used by the pharmaceutical industry in the biotransformation of various sterols and steroids . Steroid-transforming bacteria frequently have several isoenzymes at their disposal. These isoenzymes usually differ in their substrate preference and are up-regulated in the presence of different steroids . Multiple isoenzymes in a genome may help microorganisms degrade steroids with different structural characteristics . Recently, several reports confirmed that KstDs are capable of dehydrogenating steroids with undegraded isooctyl C17 substituents .