Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs

Abstract

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1_N and KshA2_N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1_N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1_N led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2_N to be a redundant form of kshA1_N Through a combined modification of kshA1_N, kshA2_N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter^-1 ADD from the transformation of 20 g liter^-1 phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1_N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter^-1 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g liter^-1 phytosterol in 120 h.IMPORTANCE Steroidal drugs are widely used for anti-inflammation, anti-tumor action, endocrine regulation, and fertility management, among other uses. The two main starting materials for the industrial synthesis of steroid drugs are phytosterol and diosgenin. The phytosterol processing is carried out by microbial transformation, which is thought to be superior to the diosgenin processing by chemical conversions, given its simple and environmentally friendly process. However, diosgenin has long been used as the primary starting material instead of phytosterol. This is in response to challenges in developing efficient microbial strains for industrial phytosterol transformation, which stem from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9α-hydroxylase, KshA1_N and KshA2_N, in M. neoaurum and demonstrated their crucial role in determining the yield and variety of products from phytosterol transformation. This work has practical value in developing industrial strains for phytosterol biotransformation.

Keywords: 3-ketosteroid-9α-hydroxylase; 9α-hydroxy-4-androstene-3,17-dione; 9α-hydroxy-4-androstene-3,17-dione (9-OHAD); Mn25795; androst-1,4-diene-3,17-dione; androst-1,4-diene-3,17-dione (ADD); mycobacterium; sterol; sterols.

FIG 1

Role of Ksh in the sterol catabolism pathway under aerobic conditions. (a) Metabolism of the steroid nucleus. (b) Proposed catabolic pathway of sterols (e.g., β-sitosterol). C₁₉ metabolites are shown in the blue box, and C₂₂ metabolites are in the green box. The conversion from 22HOBNC-CoA to AD was designated the AD pathway (blue arrows), and the conversion from 22HOBNC-CoA to 4-HBC was designated the HBC pathway (green arrows). Abbreviations: 22OBNC-CoA, 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA; 22HOBNC-CoA, 22-hydroxy-3-oxo-25,26-bisnorchol-4-en-24-oyl CoA; 4-BNC, 3-oxo-23,24-bisnorchol-4-en-22-oic acid; 4-BNC-CoA, 3-oxo-23,24-bisnorchol-4-en-22-oyl-coenzyme A thioester; AD, 4-androstene-3,17-dione; ADD, 1,4-androstadiene-3,17-dione; T, testosterone; DHT, boldenone; 9-OHAD, 9α-hydroxy-4-androstene-3,17-dione; 9-OHADD, 9α-hydroxy-1,4-androstadiene-3,17-dione; 4-HBC, 22-hydroxy-23,24-bisnorchol-ene-3-one; 1,4-HBC, 22-hydroxy-23,24-bisnorchol-1,4-dien-3-one; 9-OHHBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one.

FIG 2

Molecular characterization of KshA1_N and KshA2_N. (a) Schematic description of the locations of kshA1_N and kshA2_N in the Mn25795 chromosome. Arrow directions indicate the transcriptional direction of genes. Orthologous genes are indicated by gray-shaded regions, kshA1_N and kshA2_N are shown in green, and kstD genes are shown in blue. Intergenic areas containing the conserved palindromic motif for KstR1 binding in Mn25795 are labeled with asterisks. The genes from M. tuberculosis H37Rv that we predicted to be essential for survival in the host are indicated by triangles. (b) ClustalW alignment of the partial sequence from the putative β-sheet region located at the entrance of the KshA active site from M. tuberculosis H37Rv and Mn25795 and R. rhodochrous DSM 43269. Highly conserved residues and the four mutation points in the chimeric KshA1_N are indicated by filled and open rhombuses, respectively. (c) Three-dimensional model structure of KshA_H37Rv and the loop regions of KshA_H37Rv, KshA1_N, KshA2_N, KshA1_DSM43269, and KshA5_DSM43269. Green indicates the putative β-sheet domain; red and blue indicate the first and second α-helix domains, respectively; the orange sphere shows the nonheme ferrous iron; and the orange and yellow cluster spheres indicate the Rieske [2Fe-2S] cluster. The loop region of KshA_H37Rv is indicated with a box. The loop regions from KshA1_N, KshA2_N, KshA1_DSM43269, and KshA5_DSM43269 were homologously modeled with KshA1_H37Rv as a template and are shown as enlarged stick representations.

FIG 3

Determination of the role of kshA1_N and kshA2_N in Mn25795. (a) Growth curves of Mn25795 and its derivative strains using cholesterol (a) or cholesterol-glycerol (b) as carbon sources, respectively. Symbols: Mn25795, ■; Mut_kshA1N, △; Mut_kshA2N, ▽; Mut_kshA1&2N, ♢; Com_kshA1N, ▲; Com_kshA1&2N, ◆; Com-blank, •. The concentration of cholesterol was 2 g liter⁻¹. The concentration of glycerol was the same as that for MYC/01. (c) High-performance liquid chromatography (HPLC) analyses of the metabolites resultant from the metabolism of 2 g liter⁻¹ cholesterol by Mut_kshA1N in cholesterol-glycerol medium at 120 h. (d) Transcriptional changes in kshA1_N and kshA2_N in glycerol medium with the addition of 2.0 mM cholesterol and 1.5 mM AD, respectively. Data are shown as averages from triplicate experiments ± standard errors.

FIG 4

SDS-PAGE analysis of E. coli expression and copurification of KshA_N and KshB_N. (a) KshA1_N and KshB_N. (b) KshA2_N and KshB_N. Lane 1, standard marker protein; 2, cell extracts from BL21-kshA_N; 3, cell extracts from BL21-kshB_N; 4, mixture of cell extracts of BL21-kshA_N and BL21-kshB_N in a KshA_N:kshB_N molar ratio of approximately 1:2; 5, the copurified fraction of KshA_N and KshB_N with 150 mM imidazole.

FIG 5

ADD synthesis metabolic pathway and metabolite analysis of constructed ADD-producing strains. (a) The ADD synthesis metabolic pathway from β-sitosterol in Mn25795. Red arrows indicate strengthened steps, and the crossed arrows represent blocked steps. The structure of the desired product ADD is also indicated in red. (b) HPLC comparison of the products derived from transformation of 2 g liter⁻¹ phytosterol by strains I and II in the growing cell system at the shake flask level. (c) HPLC comparison of the products derived from transformation of 20 g liter⁻¹ phytosterols by strains II and III in the resting cell system at the shake flask level.

FIG 6

Comparative analyses of 9-OHAD-producing strains. (a) The role of Ksh in the production of 9-OHAD. (b) Transcription profiles of kshA1_N in strains IV and V at 72 h (gray bars) and 120 h (dark bars) during the metabolism of 2.0 mM cholesterol. The fold change values indicate mRNA levels in strains IV and V relative to that of Mut_MN-kstD1&2&3. The data represent averages from triplicate experiments, and the error bars indicate ± standard deviations. (c) HPLC comparison of metabolites from the transformation of 2 g liter⁻¹ phytosterols by strains IV and V in the growing cell system at the shake flask level. (d) HPLC comparison of the metabolites from the transformation of 20 g liter⁻¹ phytosterols by strains IV and V in the resting cell system at the shake flask level.

FIG 7

Metabolic pathways of ADD and 9-OHAD synthesis. (a) Metabolic pathway to produce ADD; (b) metabolic pathway to produce 9-OHAD; (c) description of the strains that were established in this study. ADD and 9-OHAD are denoted in red. The key steps catalyzed by Kshs, KstDs, and Hsd4A were labeled by red, purple, and blue arrows, respectively. The symbol X represents the block of the metabolic pathway, and the thick red arrows, purple arrows, and the blue arrow represent the overexpression of Ksh, KstDs, and Hsd4A in the metabolic pathway. The successive thick arrows represent certain unspecified enzymes.