Characterization of new recombinant 3-ketosteroid-Δ1-dehydrogenases for the biotransformation of steroids

Abstract

3-Ketosteroid-Δ-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, we cloned 12 putative KstD-encoding (kstd) genes from both fungal and Gram-positive microorganisms and attempted to overproduce the recombinant proteins in E. coli BL21(DE3). Five successful recombinant enzymes catalyzed the Δ-desaturation of a variety of steroidal compounds such as 4-androstene-3,17-dione (AD), 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), hydrocortisone, cortisone, and cortexolone. However, the substrate specificity and catalytic efficiency of the enzymes differ depending on their sources. The purified KstD from Mycobacterium smegmatis mc155 (MsKstD1) displayed high catalytic efficiency toward hydrocortisone, progesterone, and 9-OH-AD, where it had the highest affinity (K 36.9 ± 4.6 μM) toward 9-OH-AD. On the other hand, the KstD from Rhodococcus erythropolis WY 1406 (ReKstD) exhibited high catalytic efficiency toward androst-4,9(11)-diene-3,17-dione (Diene), 21-acetoxy-pregna-4,9(11),16-triene-3,20-dione (Triene), and cortexolone, where in all three cases the K values (12.3 to 17.8 μM) were 2.5-4-fold lower than that toward hydrocortisone (46.3 μM). For both enzymes, AD was a good substrate although ReKstD had a 3-fold higher affinity than MsKstD1. Reaction conditions were optimized for the biotransformation of AD or hydrocortisone in terms of pH, temperature, and effects of hydrogen peroxide, solvent, and electron acceptor. For the biotransformation of hydrocortisone with 20 g/L wet resting E. coli cells harboring MsKstD1 enzyme, the yield of prednisolone was about 90% within 3 h at the substrate concentration of 6 g/L, demonstrating the application potential of the newly cloned KstDs.

Biochemical and structural characterization of 3‐ketosteroid‐Δ1‐dehydrogenase, a candidate enzyme for efficient bioconversion of steroids

“…Six steroid substrates were used to investigate the substrate specificity of KsdD4, including AD, methyltestosterone, testosterone, hydrocortisone, progesterone and cortisone, which could be used for the production of intermediates or steroidal pharmaceuticals. The length of the C17 side chain on the D‐ring of substrates affected the catalytic activity of the KsdD family enzymes . The catalytic activity of KsdD4 toward AD was higher than that of other steroidal substrates (Fig.…”

“…The length of the C17 side chain on the D-ring of substrates affected the catalytic activity of the KsdD family enzymes. 14,47 The catalytic activity of KsdD4 toward AD was higher than that of other steroidal substrates ( Fig. 3(A)).…”

“…Various microorganisms can be exploited for biotransformation and the great diversity of microbial metabolic reactions represents a rich repository of desirable chemical modifications of steroids . Δ 1 ‐dehydrogenation in steroids is one of the crucial modifications owing to the increasing biological activity and economic value of the resulting steroid derivatives, in which a C1‐C2 double bond is introduced into the ring A of 3‐ketosteroid substrates by 3‐ketosteroid‐Δ 1 ‐dehydrogenase …”

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum

“…Phytosterols mainly exist in plants and are abundant in crops, such as beans and cereals, which are starting materials for steroid drugs production through mycobacterium transformation because of their low cost and environment-friendly attributes [4]. The gene-editing of the sterol side chain degradation metabolic pathway has made the industrial production of the steroidal pharmaceutical precursor by phytosterol biotransformation possible [5][6][7][8][9][10][11], but on the premise of improving production efficiency, reducing production costs is still the continuous pursuit of industrial production. Recently, studies on toxic metabolites, such as reactive oxygen species (ROS) in the production process of strains, have provided new ideas for the study of phytosterol conversion [12,13].…”

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates

“…For instance, Arthrobacter simplex has been used for steroid hydroxylation, dehydrogenation and sterol side-chain cleavage because of its high tolerance to organic solvents and remarkable bioconversion [ 14 – 16 ], involving the ∆ 1 -dehydrogenation of steroids. This dehydrogenation is a crucial modification in steroid synthesis because it increases the biological activity and economical value of the original steroidal substrate [ 17 , 18 ]. Both cortisone acetate and prednisone acetate are used as anti-inflammatory and anti-allergy drugs clinically [ 14 , 19 , 20 ].…”