Abstract
3-Ketosteroid-∆1-dehydrogenase (KstD), a key enzyme in microbial steroid catabolism, catalyzes the trans-axial elimination of the C1 and C2 hydrogen atoms of the A-ring from the polycyclic ring structure of 3-ketosteroids, and it was usually used to transform androst-4-ene-3,17-dione (AD) to produce androsta-1,4-diene-3,17-dione. Here, the KstD from Gordonia neofelifaecis was expressed efficiently in Escherichia coli. E. coli cells expressing KstD3gor were subjected to the investigation of dehydrogenation activity for different steroids. The results showed that KstD3gor has a clear preference for steroid substrates with 3-keto-4-ene configuration, and it exhibits higher activity towards steroid substrates carrying a small or no aliphatic side chain than towards substrates having a bulky side chain at the C-17 atom. The recombinant strain could efficiently convert androst-4,9(11)-dien-3,17-dione into androst-1,4,9(11)-trien-3,17-dione (with conversion rate of 96%). 1(2)-Dehydrogenation of androst-4,9(11)-dien-3,17-dione is one of the key steps in glucocorticoid production. To the best of our knowledge, this is the first study reporting on the conversion of androst-4,9(11)-dien-3,17-dione catalyzed by recombinant KstD; the expression system of KstD3gor reported here would have an impact in the industrial production of glucocorticoid in the future.
Highlights
Significant progress has been made over the last ten years in the use of enzymes and microorganisms for the manufacturing of complex chemical compounds and replacing multi-steps chemical syntheses
To expand our knowledge about KstD3gor from G. neofelifaecis, the gene of this enzyme was sub-cloned into a commercial E. coli vector pET28a(?) and recombinant KstD3gor protein was expressed in BL21(DE3) cells
The electron transfer from AD via phenazine methosulphate to nitroblue tetrazolium (NBT) catalyzed by KstD3gor resulted in the formation of a purplecolored product of the reaction, formazan (Fig. 2b, lanes 2, 3)
Summary
Significant progress has been made over the last ten years in the use of enzymes and microorganisms for the manufacturing of complex chemical compounds and replacing multi-steps chemical syntheses. Microbial transformation could be carried out under mild reaction conditions with excellent yields of products and remarkable regio- and stereo-selectivity, which is hardly available for chemical synthesis. For producing novel steroidal drugs and generating active pharmaceutical ingredients, microbial transformation is employed as a novel, efficient and economical tool (Donova 2007; Garcıa et al 2012; Yang et al 2015). The efficiency of enzymatic processes and purity of their products have obvious advantages in comparison with multi-steps chemical syntheses of hormonal drugs. The development of steroid biotechnology requires further studies of microorganisms able to degrade/modify steroids as well as enzymes catalyzing these reactions on the molecular level (Yang et al 2015)
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