A New 3-Ketosteroid-Δ1–Dehydrogenase with High Activity and Broad Substrate Scope for Efficient Transformation of Hydrocortisone at High Substrate Concentration

Abstract

3-Ketosteroid-Δ1-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ1-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, nine putative kstD genes from different origins were selected and overexpressed in Escherichia coli BL21(DE3). These recombinant enzymes catalyzed the Δ1-desaturation of a variety of steroidal compounds. Among them, the KstD from Propionibacterium sp. (PrKstD) displayed the highest specific activity and broad substrate spectrum. The detailed catalytic characterization of PrKstD showed that it can convert a wide range of 3-ketosteroid compounds with diverse substituents, ranging from substituents at the C9, C10, C11 and C17 position through substrates without C4-C5 double bond, to previously inactive C6-substituted ones such as 11β,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione. Reaction conditions were optimized for the biotransformation of hydrocortisone in terms of pH, temperature, co-solvent and electron acceptor. By using 50 g/L wet resting E. coli cells harboring PrKstD enzyme, the conversion of hydrocortisone was about 92.5% within 6 h at the substrate concentration of 80 g/L, much higher than the previously reported results, demonstrating the application potential of this new KstD.

Reconstruction of the Steroid 1(2)-Dehydrogenation System from Nocardioides simplex VKM Ac-2033D in Mycolicibacterium Hosts

“…At the same time, in the most successful studies on the expression of genes of heterologous 3-KSD in microbial hosts that do not possess endogenous sterol catabolism systems, significantly higher activities were demonstrated during the conversion of steroids by washed recombinant cells. Thus, the specific productivity at the conversion of hydrocortisone to prednisolone by E. coli BL21 cells expressing the synthetic prkstD gene [30] and the conversion of AD to ADD by B. subtilis cells expressing the codon-optimized kstD gene from M. neoaurum JC-12 [31] were approximately 30-40 times higher than the activity obtained in this study for M. smegmatis BD/pMhsp_k.…”

“…Noteworthy, the engineering of N. simplex strains is complicated by the current lack of appropriate genetic tools [22,23]. On the contrary, the expression of 3-KSD genes in strains that do not have endogenous steroid catabolism (E. coli [5,[23][24][25][26][27][28][29][30], Bacillus subtilis [5,25,31], Corynebacterium crenatum [32], and Pichia pastoris [33]) made it possible to effectively produce target steroids, but mainly in the presence of exogenous electron acceptors (EEA). For example, the expression of the synthetic PrKstD gene from The problem can be solved either by suppressing the undesirable 20β-reducing activity in N. simplex or by heterologous expression of the 3-KSD genes in strains that do not have such activity.…”

“…For example, the expression of the synthetic PrKstD gene from Propionibacterium sp. in E. coli BL21(DE3) effectively converted 40-70 g/L of hydrocortisone to prednisolone with a 92.5-95.5% yield [30].…”

Recent developments in the enzymatic modifications of steroid scaffolds

“…103 In recent years, various KstDs with high activities have been discovered and applied. [104][105][106][107][108][109][110][111][112] For instance, Guo and coworkers used Nocardioides for the Δ 1,2 -dehydrogenation of 71 to produce 72, which is an important intermediate for the synthesis of fluocinolone acetonide (Fig. 13B).…”

Rational design of a cyclohexanone dehydrogenase for enhanced α,β-desaturation and substrate specificity

“…6 These studies indicate the importance of KSTDs in the synthesis of various steroid drugs. [5][6][7] Therefore, the development of a new class of enzyme which will perform enantio-and regioselective dehydrogenation of cyclic ketones, which are found in the core of many steroid molecules, will be an attractive and greener approach for the pharmaceutical industry.…”