Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione
© Yeh et al.; licensee BioMed Central Ltd. 2014
Received: 25 April 2014
Accepted: 23 August 2014
Published: 9 September 2014
The gene encoding the putative reductase component (KshB) of 3-ketosteroid 9α-hydroxylase was cloned from Rhodococcus equi USA-18, a cholesterol oxidase-producing strain formerly named Arthrobacter simplex USA-18, by PCR according to consensus amino acid motifs of several bacterial KshB subunits. Deletion of the gene in R. equi USA-18 by a PCR-targeted gene disruption method resulted in a mutant strain that could accumulate up to 0.58 mg/ml 1,4-androstadiene-3,17-dione (ADD) in the culture medium when 0.2% cholesterol was used as the carbon source, indicating the involvement of the deleted enzyme in 9α-hydroxylation of steroids. In addition, this mutant also accumulated 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ1,4-BNC). Because both ADD and Δ1,4-BNC are important intermediates for the synthesis of steroid drugs, this mutant derived from R. equi USA-18 may deserve further investigation for its application potential.
Industrial processes use mutant microbial strains or add chemicals to inhibit the relevant enzymatic activities to accumulate AD, 9OHAD or ADD in the culture medium . Modifying the sterol degradation pathway by genetic engineering may provide an alternative to allow the creation of potent strains for the production of those catabolic intermediates. Recent completions of the genomic sequences of bacterial strains such as R. erythropolis XP , R. jostii RHA1  and R. equi can provide valuable information when the rationally genetic approach is undertaken. Among the genes related to sterol catabolism, KSTD and KSH represent the most interesting targets for gene disruption because of their critical roles in the ring opening. Two KSTD isozymes were found in R. erythropolis SQ1 ,. Loss of these two KSTD genes disabled R. erythropolis SQ1 from growing on steroid substrates; however, the mutant strain could efficiently convert AD into 9OHAD. Deletion of the KSTD gene in M. neoaurum NwIB-01 resulted in accumulation of AD from soybean phytosterols in the culture medium . KSH is a two-component iron-sulfur-containing monooxygenase, consisting of the terminal oxygenase component (KshA) and the reductase component (KshB) -. Three KshA isozymes (KshA1 ~ KshA3) and one KshB have been found in R. erythropolis SQ1 . The mutant SQ1 strain with either KshA1 or KshB deletion was unable to grow in medium containing AD or ADD as the sole carbon and energy sources. Intriguingly, the mutant strain with null KshB was impaired in sterol degradation, suggesting that KshB of R. erythropolis SQ1 plays a role in not only ring opening but also in side chain degradation .
A. simplex B-7 was isolated as a cholesterol oxidase-producing strain from soil in Taiwan . Cholesterol oxidase productivity of this Arthrobacter strain had been improved via UV-induced mutagenesis by 9 fold  and further increased via protoplast fusion by 20-60% . A cholesterol oxidase gene, with accession number AY963570, was cloned from one of the improved strains, and its nucleotide sequence shows 99.6 and 98.9% identity to that of R. equi 103S and Brevibacterium sterolicum, respectively . In order to learn more about the enzymes critical for cholesterol catabolism in strains derived from A. simplex B-7 and to explore the feasibility of producing AD or ADD by using genetically modified strains, we set out to clone KshA and KshB genes from A. simplex USA-18, a UV-induced mutant derived from A. simplex B-7 , in this study. Two hypothetical KshA and one putative KshB genes were cloned by PCR using degenerate primers, followed by inverse PCR and DNA walking. Subsequently, the effect of the putative KshB deletion on sterol catabolism was investigated. In addition, the 16S ribosomal RNA sequence and the metabolic profile of A. simplex USA-18 suggested that the strain should be renamed as R. equi USA-18.
Materials and methods
Cholesterol was purchased from Tokyo Kasei Kogyo Co., Ltd (Japan). Phytosterols, isolated from soybean, were purchased from a local food ingredient supplier (Ngya, Taiwan). AD and ADD standards were purchased from Sigma-Aldrich (USA).
The gene deletion mutant, R. equi USA-18ΔB8, created in this study was deposited in the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) with stock number BCRC 910611.
The bacterial strains used in this study were regularly grown in Luria-Bertani (LB) medium at 37°C. For the production of steroid metabolites, the cells were grown in glycerol minimal medium (1% (v/v) glycerol, 40 mg/L thiamine HCl, 4.65 g/L K2HPO4, 1.5 g/L NaH2PO4 · H2O, 3 g/L NH4Cl, 1 g/L MgSO4 · 7H2O, and 0.1% (v/v) trace element solution) supplemented with 2 g/L sterols (cholesterol or phytosterols) and 0.5% (v/v) detergent (Tween 20 or 80). Trace element solution contained 10 g ethylenediaminetetraacetic acid, 4.4 g ZnSO4 · 7H2O, 1.47 g CaCl2 · 2H2O, 1 g MnCl2 · 7H2O, 1 g FeSO4 · 7H2O, 0.22 g (NH4)6Mo7O24 · 4H2O, 0.315 g CuSO4 · 5H2O and 0.32 g CoCl2 · 6H2O in final 1 L water. To measure the cell density in the sterol-containing broth, an aliquot of the broth was added with glycerol to a final 50% concentration (v/v), thoroughly mixed, and subjected to 15000 rpm centrifugation. After removing the residual sterol that floated on the top, the cell pellet was suspended in a same volume of medium and the resulting optical density was measured by spectroscopy.
The nomenclature of the working strain was confirmed by the Biolog identification system (Biolog, USA). Briefly, the bacterial colonies obtained after 36 h at 37°C on LB agar were transferred into the IF-A solution according to the manufacturer's procedure. A 100-μl aliquot of the bacterial suspension was used to inoculate each of the culture wells of GENIII MicroPlats. The metabolic profile after incubation at 30°C for 5 to 27 h was read by using Biolog's Microbial Identification Systems software (OmniLog® Data Collection).
Primer pairs used in PCR in this study
Nucleotide sequence (5′→3′)
Product size (bp)
An approximately 5.2-kb DNA fragment, encompassing the putative KshB in the middle, was obtained from chromosome of A. simplex USA-18 by PCR using primer 6 and 7 (Table 1), with HindIII and EcoRI sites engineered at the 5′ ends, respectively. The amplified fragment was inserted into HindIII-EcoRI-opened pUC18. The resulting plasmid, pUC-KshB5.2, was linearized by inverse PCR using primer 8 and 9 (Table 1), by which the open reading frame of the putative KshB was deleted. The kanamycin resistant gene plus its promoter (Kan), was amplified by PCR from pK18mobsacB (ATCC® 87097™) using primer 10 and 11 (Table 1). These two fragments were joined to form plasmid pUC-ΔΒ-Kan after both of them had been treated with KpnI and XbaI. Aliquots (200 μL) of A. simplex USA-18 competent cells, suspended in 10% ice-cold glycerol, were mixed with 2 μg NdeI-treated pUC-ΔΒ-Kan in 2 mm gapped cuvettes. Electroporation was performed using the Gene Pulser apparatus (Bio-Rad, USA) under the condition of 12.5 kV/cm, 1000 Ω and 25 μF. After electroporation, the cells were incubated in 1 mL LB medium for 2 h at 37°C, 200 rpm, and subsequently plated on agar plates containing kanamycin (300 μg/mL). Colonies, appeared on the selection plates after about a week, were checked for homologous recombination by PCR using a sense primer matching Kan (primer 10) or KshB (primer 13) and an antisense primer (primer 12) recognizing a sequence downstream of the 5.2-kb KshB-containing fragment.
Biotransformation product analysis
Culture broth was extracted with ethyl acetate at a ratio of 5:2 (v/v). Steroid metabolites in the extraction were regularly analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). TLC was performed using Kieselgel gel 60 F254 plates (Merck, Germany), developed in petroleum ether/ethyl acetate (6:4, v/v) solvent system, and visualized by spraying with 10% sulfuric acid and heating in a hot air oven at 120°C for 10 min. For HPLC, the metabolites were separated by Luna C18 column (250 x 4.6 mm, Phenomenex, CA) using 80% acetonitrile and 20% water as mobile phase at a flow rate of 1.0 mL/min, and detected at OD 254 nm. ADD concentration in the sample was calculated according to the standard curve of the known concentrations of ADD versus the respective peak areas in HPLC profile. Molecular weights of the metabolites and their fragmentation pattern were analyzed by ultra performance liquid chromatography-tandem mass spectrometry (UltiMate 3000 UHPLC, Thermo Scientific, USA) using ionization energy of 70 eV.
Macrophage infection assay
The THP-1 human monocyte cell line was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 0.45% glucose, 0.15% sodium pyruvate, 4 mm L-glutamine, and 1% PSA (penicillin-streptomycin-amphotericin B). One ml of 5 × 105 cells/ml THP-1 cells was seeded onto each well of 6-well plates and the cells were treated with 300 nM phorbol 12-myristate 13-acetate for about 24 h to induce their differentiation to macrophages. The differentiated cells were washed with phosphate buffered saline (PBS) twice and re-suspended in antibiotic free RPMI 1640 supplemented with 2% FBS. The bacterial culture (108 CFU/ml) was added into each of the wells at a multiplicity of infection of 10. After 1 h incubation, macrophages were washed twice with PBS to remove extracellular bacteria. The infected macrophages were then cultivated in RPMI 1640 supplemented with 10% FBS and 150 μg/ml ampicillin. At the indicated time points, the cells were washed with PBS twice and lysed by treating with 0.1% Tween 20 in PBS. The number of live bacteria released from lysed macrophages was determined by plate counting.
Isolation of putative KshA and KshB genes from A. simplex USA-18
To clone KshA genes from A. simplex USA-18, the amino acid sequences of several bacterial Rieske [2Fe-2S] terminal oxygenases, including those isolated from R. jostii RHA1 (YP_704482), M. smegmatis (YP_890151), Burkholderia cenocepacia J2315 (YP_002234232), Ralstonia eutropha JMP134 (YP_295786) and Comamonas testosteroni KF-1 (WP_003057373), were aligned. Consensus motifs such as (R/T)(Y/F)(A/P)RGW and CP(F/Y)H(G/D)W were chosen for the design of sense degenerate primers (primer 1 and 3, respectively, Table 1), while the (N/I)(C/M)H(Y/V/T)P(I/V) motif was used for the design of an antisense degenerate primer (primer 2, Table 1). PCR using primer 1 and 2 gave rise to a 728-bp DNA (S1A2), while primer 3 and 2 produced a 532-bp DNA (S3A2). These two DNA fragments share 72.3% identity within the overlapped region, suggested that A. simplex USA-18 contains at least two potential KshA genes. The upstream and downstream regions of S1A2 were obtained by inverse PCR and DNA walking, and thus an S1A2-containing open reading frame (ORF) of 1155 nucleotides (accession number KJ598876) was identified. Searching databases using BLASTn algorithms revealed that the ORF exhibits 99.7% identity to a putative iron-sulfur binding oxidoreductase gene (REQ_45190) of R. equi 103S . Similarly, the nucleotide sequences flanking S3A2 were determined and an ORF of 1161 nucleotides (accession number KJ598877) was identified. It shares 99.7% identity with another iron-sulfur binding oxidoreductase gene (REQ_06790) of R. equi 103S . To clone KshB gene, the amino acid sequences of the reductase subunit of 3-ketosteroid 9α-hydroxylases from M. smegmatis (WP_003894254), Pseudovibrio sp. (WP_008550016), R. erythropolis (AAL96830), and R. jostii RHA1 (YP_705768) were aligned. Accordingly, the conserved GSGITP and PYSC(R/Q/K)(E/S)G motifs were chosen to design the sense and antisense degenerate primers, respectively (primer 4 and 5, Table 1). PCR using this pair of primer generated a 530-bp fragment (S4A5). A putative ORF of 1185 nucleotides (accession number KJ598878) was subsequently identified after the flanking regions of S4A5 were obtained by DNA walking. The 1185-bp ORF was found to have an identical nucleotide sequence to REQ_36320 of R. equi 103S that presumably encoding the reductase component of 3-ketosteroid 9α-hydroxylase .
Reclassification of A. simplex USA-18 as R. equi USA-18
The great resemblance of the genes cloned in this study and the cholesterol oxidase gene cloned previously  to those of R. equi 103S raised a suspicion of whether the taxonomic classification of A. simplex USA-18 had been properly determined. The gene encoding for 16S ribosomal RNA was amplified from A. simplex USA-18 by PCR using the universal primer 8 F and U1492R . Blastn showed that the gene, with the accession number KJ598875, is highly similar, with identities over 99%, to the 16S ribosomal RNA genes isolated from a variety of R. equi strains. However, the identities between the gene and those from Arthrobacter strains are about 91-92%, suggesting that A. simplex USA-18 is phylogenetically closer to R. equi than to A. simplex.
To confirm the 16S ribosomal RNA sequence-based suggestion, the metabolic profile of A. simplex USA-18 was checked using the Biolog Identification System, in which the ability of the bacterium to metabolize 71 carbon sources and sensitivity to 23 chemicals were analyzed. The profile identifies the test strain USA18 as Rhodococcus equi, with similarity index between 0.774 and 0.789. Accordingly, A. simplex USA-18 was renamed R. equi USA-18 hereafter.
Deletion of the REQ_36320 ortholog in R. equi USA-18
Sterol catabolism in REQ_36320 knockout mutant
Time course of ADD production
Restricted growth of R. equi USA-18 in macrophages
This study cloned the REQ_36320 ortholog from R. equi USA-18 and then deleted it to test whether its gene product participates in the 3-ketosteroid 9α-hydroxylase activity. An unmarked gene deletion approach using pK18mobsacB was taken on the first attempt. Unfortunately, sacB-carrying R. equi USA-18 was insensitive to sucrose up to 20% (w/v) (data not shown). The invalidity of using sacB as a counter-selection marker in R. equi was actually mentioned in literature . A PCR-targeted gene replacement strategy, as described in materials and methods, was then launched to replace the REQ_36320 ortholog with Kan. Dozens of colonies that were resistant to kanamycin were obtained after delivering 2 μg DNA construct into 1 × 109 competent cells by electroporation. Of them, two colonies had the REQ_36320 ortholog replaced correctly by Kan. This method thus proved to be an alternative for gene deletion in a R. equi type of strain.
R. equi USA-18ΔB8, the deletion strain that lacks the REQ_36320 ortholog, survived on the medium that contained sterols as the sole carbon source and accumulated Δ1,4-BNC and ADD in the broth. However, this strain was unable to grow when ADD was supplied as the sole carbon source (data not shown); suggesting that the strain was devoid of 3-ketosteroid 9α-hydroxylase activity. It is logical to assume that the REQ_36320 ortholog encodes the reductase component of 9α-hydroxylase based on the above observations and the similarity of its deduced amino acid sequence to the characterized reductase components isolated from R. erythropolis (78.9%)  and R. rhodochrous (78.3%) .
The reason why Δ1,4-BNC accumulated in the broth remains unclear. A mutant strain of R. rhodochrous DSM 43269, devoid of 3-ketosteroid 9α-hydroxylase activity due to inactivation of all the five KshA gene homologs, transforms cholesterol into ADD and Δ1,4-BNC in molar ratios of 3 and 73%, respectively . Accordingly, accumulation of Δ1,4-BNC seems to be a common phenomenon in Rhodococcus species when their 3-ketosteroid 9α-hydroxylase activity is inactivated. This phenomenon implies that lack of the hydroxylase activity adversely affects the enzymatic steps leading Δ1,4-BNC to ADD. Since ADD could descend from either Δ1,4-BNC after side chain oxidation or AD after dehydrogenation at carbon 1and 2 (Figure 1), we propose that ADD in R. equi USA-18ΔB8 preferentially imposes a feedback inhibition on the enzymes involved in the side chain oxidation of Δ1,4-BNC, but not Δ4-BNC. After all, ADD and Δ1,4-BNC share a common polycyclic ring structure and this similarity may contribute to this presumed preference. Under this inhibition condition, the part of sterol that had been catabolized to Δ1,4-BNC would mostly stop at here, while the rest would be catabolized to ADD via the route involving Δ4-BNC and AD intermediates.
There are five putative KSTD isozymes in R. equi 103S according to Blastp analysis. It is logical to assume that each KSTD has its preferable 3-ketosteroids for dehydrogenation. In other words, transformation of Δ4-BNC, AD, and 9OHAD to Δ1,4-BNC, ADD, and 9OHADD (Figure 1), respectively, may be catalyzed by specific KSTD isozymes. It will be interesting to find out the number of KSTD isozymes in R.equi USA-18 and elucidate their specific roles in the sterol catabolic pathway.
R. equi USA-18ΔB8 was capable of converting sterol to ADD and Δ1,4-BNC. The molar yield of ADD was about 40% when 0.2% cholesterol was included in the culture medium in an uncontrolled small-scale cultivation system. Fermentation technology shall be employed to further evaluate the potential of the strain in the transformation of sterols to ADD or/and Δ1,4-BNC.
CHY selected the KshB-knockout strains and performed taxonomic determination. CHY and YSK analyzed the steroid metabolites accumulated in the culture medium. CHY and CMC cloned KshA and KshB genes from R. equi USA-18. WHL contributed to literature search and discussion and provided the parental strain. MLS participated in the macrophage infection assay. MMH coordinated the works and prepared the manuscript. All authors read and approved the final manuscript.
We are grateful to Professor Sheng-Yang Wang (Department of Forest, National Chung Hsing University) for technical assistance on UHPLC-MS/MS assay and to Dr. Feng-Chia Hsieh (Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture, ROC) for the assistance on the Biolog identification assay.
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