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Previous Article | Next Article 
Applied and Environmental Microbiology, October 2006, p. 6554-6559, Vol. 72, No. 10
0099-2240/06/$08.00+0 doi:10.1128/AEM.00941-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
-Hydroxylase in Mycobacterium smegmatis mc2155
David A. Hopwood,2
Ferenc Jeanplong,1
Éva Ilk
y,1
Attila Kónya,1
István Kurucz,1 and
Gábor Ambrus1
Institute for Drug Research Ltd., 47-49 Berlini St., H-1045 Budapest, Hungary,1 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom2
Received 20 April 2006/ Accepted 22 July 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9-hydroxylase in M. smegmatis mc2155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9 position. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-hydroxy-4-androstene-3,17-dione, and 1,4-androstadiene-3,17-dione, are produced worldwide by selective removal of the sitosterol side chain (14, 15, 26). Economic production of these intermediates is an important goal for pharmaceutical companies. So far, mutation-selection and in vivo genetic recombination have been used for strain improvement (11). Here we describe the use of in vitro DNA recombination for this purpose.
The microbial degradation of sitosterol proceeds on both the skeleton and the side chain (Fig. 1.). First, a 
4-3-keto structure is formed in ring A, and then a double bond is introduced between C-1 and C-2 and ring B is hydroxylated at the 9 position. The resulting structure is unstable, and ring B cleavage produces a 9,10-secophenol derivative (3). Blocking the 1-dehydrogenation or 9-hydroxylation or both reactions leads to industrially valuable intermediates with an intact steroid skeleton.
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van der Geize et al. characterized two components (terminal oxygenase and ferredoxin reductase) of 3-ketosteroid 9-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1 (25). A new bacterial steroid degradation gene cluster in Comamonas testosteroni TA441 was reported by Horinouchi et al. (8), and it contained genes for degradation of seco-steroids, as well as 3-ketosteroid dehydrogenase genes. This cluster also contained a gene probably encoding the ferredoxin reductase component of the 9-hydroxylase. Recently, Brzostek et al. identified a gene encoding the 3-ketosteroid 1-dehydrogenase in Mycobacterium smegmatis (1).
Here we describe a method to generate insertionally blocked mutants with mutations in the sterol degradation pathway in Mycobacterium using pCG79, a temperature-sensitive plasmid carrying Tn611. This transposon is a derivative of Tn610 from Mycobacterium fortuitum, which contains two 880-bp IS6100 insertion sequences flanking an antibiotic resistance gene, sul3, which is replaced by the kanamycin resistance gene of Tn903 in Tn611. IS6100 has been shown to transpose by a replicative mechanism (16). One of the two copies of IS6100 is duplicated during the transposition event, and it generates cointegrates with three copies of IS6100, two in one orientation and one in the other orientation (6). In addition to obtaining mutants producing a useful intermediate for steroid drug synthesis, we used a mutant with blocked 9-hydroxylation of the steroid skeleton to clone and express the terminal oxygenase of 3-ketosteroid 9-hydroxylase in M. smegmatis mc2155.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Isolation of chromosomal DNA from M. smegmatis.
Chromosomal DNA was isolated from M. smegmatis mc2 155 and 10A12 as follows. Bacteria were harvested from 10-ml 24-h cultures by centrifugation and suspended in 550 µl Tris-EDTA buffer containing 10 mg/ml lysozyme. After 1 h of incubation at 37°C, 70 µl of 10% sodium dodecyl sulfate (SDS) and 6 µl of a 10-mg/ml proteinase K solution were added, mixed, and incubated for 10 min at 65°C. Then 5 M NaCl (100 µl) was mixed in, and 80 µl of hexadecyltrimethylammonium bromide (CTAB)-NaCl was added, mixed thoroughly, and incubated at 65°C for 10 min (CTAB-NaCl was prepared as follows: 4.1 g NaCl was dissolved in 80 ml distilled water with stirring, and then 10 g CTAB was added; the solution was heated to 65°C, and the volume was adjusted to 100 ml with distilled water). After chloroform-isoamyl alcohol (24:1) extraction, the DNA was precipitated with 0.6 volume of 2-propanol, collected by centrifugation, washed, and resuspended in 12 µl Tris-EDTA buffer.
Construction and screening of Mycobacterium insertional mutant libraries.
Insertional mutant libraries of sterol-degrading Mycobacterium strains were constructed by introducing pCG79 containing Tn611 into the bacteria by electroporation (10, 22); integration of the plasmid into the chromosome was achieved by raising the temperature from 30°C to 39°C, as previously described (6).
A high-throughput method with microtiter plates was used to screen the libraries by fermentation in the presence of sitosterol to obtain strains with blocked sterol degradation. The fermentation medium was M-ADC-TW broth containing 200 µg/ml ß-sitosterol prepared as follows. Twenty milligrams of ß-sitosterol was dissolved in 1 ml ethanol with boiling, added to 100 ml M-ADC-TW medium heated to 70 to 75°C with vigorous stirring, and then cooled to room temperature. This yielded a very fine-grained suspension of ß-sitosterol that hardly sedimented. Aliquots (200 µl) of this solution were distributed into 96-well microtiter plates (flat-bottom 250-µl wells) with a multichannel pipette. Colonies of individual insertion mutants from the libraries, growing on M-OADC-G agar plates containing 20 µg/ml kanamycin, were transferred into the wells with sterile toothpicks. After the inoculated microtiter plates were covered with their lids, they were wrapped in plastic foil to prevent evaporation. Sitosterol transformation occurred during growth of the mutants in "minifermentors" at 32°C, which was found to be optimal for sitosterol transformation, with shaking at 250 rpm for 5 days. The sitosterol-transforming ability of the strains was determined by thin-layer chromatography (TLC) to reveal intermediate accumulation in the fermentation broth.
TLC analysis.
Fermentation broth (10 µl) from each well of microtiter plates or shaken flask cultures was dropped on TLC sheets (Kieselgel HF254-366; Merck) using an automatic pipette, and the sheets were developed in ethyl acetate-n-hexane (7:3, vol/vol). Detection was performed with UV light and with concentrated H2SO4 treatment to determine the presence of ß-sitosterol (compound I) and the following microbial degradation products of ß-sitosterol: 4-androstene-3,17-dione (compound II), 1,4-androstadiene-3,17-dione (compound III), 9-hydroxy-4-androstene-3,17-dione (compound IV), 17ß-hydroxy-4-androstene-3-one (compound V), 22-hydroxy-23,24-dinor-4-cholen-3-one (compound VI), 3-oxo-23,24-dinor-4,17(20)-choladien-22-oic-acid methyl ester (compound VII), 3-oxo-23,24-dinor-4-cholen-22-oic-acid methyl ester (compound VIII), and 3-hydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (compound IX). The approximate Rf values were as follows: compound I, 0.77; compound II, 0.70; compound III, 0.61; compound IV, 0.41; compound V, 0.56; compound VI, 0.66; compound VII, 0.85; compound VIII, 0.82; and compound IX, 0.78.
HPLC analysis.
Sitosterol transformation products of the insertional mutants and 9-hydroxylation of 4-androstene-3,17-dione (AD) or 1,4-androstadiene-3,17-dione with recombinant strains expressing the terminal oxygenase component of 3-ketosteroid 9-hydroxylase were analyzed by high-performance liquid chromatography (HPLC). An aliquot of a transformation culture was diluted threefold with methanol, homogenized by vortexing, and centrifuged. The supernatant containing the solubilized steroid compounds was injected with an 2695 autosampler (Waters, Milford, MA) onto a reversed-phase column packed with Symmetry RP-18e (3.5 µm; 4.6 by 100 mm; Waters) and onto a Security Guard Cartridge packed with RP-18e (3.5 µm; 2.0 by 4.0 mm; Phenomenex). The column temperature was set at 40°C. Samples were eluted at a rate of 1.2 ml/min for 26 min with 15% tetrahydrofuran and a linear 5 to 50% acetonitrile gradient in water. Compounds II, III, IV, V, VI, VII, and VIII were monitored at 240 nm using a 996 photodiode array detector (Waters). Under these conditions the retention times were as follows: compound II, 8.58 min; compound III, 6.42 min; compound IV, 3.72 min; compound V, 8.85 min; compound VI, 15.18 min; compound VII, 20.05 min; and compound VIII, 20.50 min. Compound IX was monitored at 280 nm using the same detector and eluted at 9.48 min.
Identification of steroid transformation products.
The degradation products of mutant and recombinant Mycobacterium strains were isolated from 5 liters of culture broth (M-ADC-TW medium containing 200 µg/ml ß-sitosterol) prepared in 500-ml shake flasks. An ethyl acetate extract of the fermentation broth was evaporated to dryness, and the degradation products were separated by column chromatography (adsorbent, silicic acid; eluent, ethyl acetate-n-hexane with a gradually increasing content of ethyl acetate) and preparative TLC (adsorbent, Kieselgel HF254-366; developing solvent described above; compounds eluted from the adsorbent with methanol).
The steroid drug synthesis key intermediates compounds II, III, and IV and the testosterone hormone compound V were identified chromatographically and by spectroscopic comparison with standard data. The structures of compounds VI, VII, VIII, and IX were equated to those of previously described compounds (3, 13, 21) by spectroscopic analyses. The characteristic nuclear magnetic resonance (NMR) and mass spectral (MS) data are as follows.
(i) NMR spectroscopy.
The 1H and 13C NMR spectra were recorded at 300 K using a Bruker Avence 500 spectrometer in CDCl3 with tetramethylsilane and CDCl3 (77.2 ppm) as the internal reference standards at frequencies of 500.1 and 125.8 MHz, respectively. For structure elucidation, one-dimensional 1H, 13C, and DEPT and two-dimensional 1H,1H-COSY, 1H,13C-HSQC, and 1H,13C-HMBC methods were used.
(ii) Mass spectroscopy.
Electron ionization (EI) measurements were obtained as follows: Finningan Polaris Q mass spectrometer; inlet system, direct; ionization mode, EI(+); source temperature, 250°C; and electron energy, 70 eV. Atmospheric pressure chemical ionization (APCI) measurements were obtained as follows: Finningan LCQ mass spectrometer; mode of application, direct inlet; ionization mode, APCI(+); and source temperature, 25°C.
(iii) 22-Hydroxy-23,24-dinor-4-cholen-3-one (compound VI).
1H NMR: 
= 5.73 s, br (H-4); = 3.64 dd, J1 = 10.5 Hz, J2 = 3.3 Hz; and = 3.37 dd, J1 = 10.5 Hz, J2 = 6.9 Hz (CH2OH); = 1.6-1.5 m (H-20); = 1.19 s (CH3-19); = 1.05 d, J = 6.9 Hz (CH3-21); = 0.74 s (CH3-18).
13C NMR: = 199.8 (C-3); 171.7 (C-5); 124.0 (C-4); 68.1 (CH2OH); 38.8 (C-20); 17.6 (CH3-19); 16.9 (CH3-21); 12.2 (CH3-18).
MS: EI: 330 [M]+·,124 [ring A + C6 + 2H]+·; APCI: 331 [M + H]+.
(iv) 3-Oxo-23,24-dinor-4,17(20)-choladien-22-oic acid methyl ester (compound VII).
1H NMR: = 5.75 s, br (H-4); = 3.71 s (OCH3); = 2.83 m and 2.65 m (CH2-16); = 1.96 dd, J1 = J2 = 2.0 Hz (CH3-21); = 1.20 s (CH3-19); = 0.98 s (CH3-18).
13C NMR: = 199.6 (C-3); 171.1 (C-5); 170.2 (COOCH3); 163.7 (C-17); 124.1 (C-4); 118.7 (C-20); 51.3 (OCH3); 33.0 (C-16); 17.5 (CH3-19); 15.5 (CH3-18); 14.9 (CH3-21).
MS: EI: 356 [M]+·, 341 [M-·CH3]+, 324 [M-CH3OH]+·, 309 [324-·CH3]+.
(v) 3-Oxo-23,24-dinor-4-cholen-22-oic acid methyl ester (compound VIII).
1H NMR: = 5.73 s, br (H-4); = 3.66 s (OCH3); = 2.4 m (H-20); = 1.20 d, J = 7.0 Hz (CH3-21); = 1.19 s (CH3-19); = 0.74 s (CH3-18).
13C NMR: = 199.7 (C-3); 177.3 (COOCH3); 171.4 (C-5); 124.2 (C-4); 51.5 (OCH3); 42.6 (C-20); 17.6 (CH3-21); 17.3 (CH3-19); 12.3 (CH3-18).
MS: EI: 358 [M]+·, 343 [M-·CH3]+, 316 [M-42]+·, 229 [M-·(C15-C17+H)]+, 124 [ring A + C6 + 2H]+·.
(vi) 3-Hydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (compound IX).
1H NMR: = 7.00 d, J = 8.1 Hz (H-1); = 6.68 d, J = 2.7 Hz (H-4); = 6.60 dd, J1 = 8.1 Hz, J2 = 2.7 Hz (H-2); = 4.72 s, br (OH); = 2.73 m and 2.55-2.45 m (CH2-6); 2.60 m and 2.27-2.21 m (CH2-16); = 2.27 s (CH3-19); = 1.18 s (CH3-18).
13C NMR: = 218.0 (C-17); 210.6 (C-9); 153.7 (C-3); 141.9 (C-5); 131.2 (C-1); 128.0 (C-10); 115.7 (C-4); 112.7 (C-2); 47.5 (C-13); 36.1 (C-16); 31.0 (C-6); 18.4 (C-19); 13.5 (C-18).
MS: EI: 300 [M]+·,134 [ring A + C6-C7-H]+·, 121 [ring A + C6]+· (9).
Preparation of a DNA library from M. smegmatis mc2155.
Genomic DNA from M. smegmatis mc2155 was partially digested with Sau3AI, and 35- to 40-kb fragments were isolated by sucrose gradient centrifugation (7) and then ligated with a 50-fold excess of BamHI-linearized and calf intestinal phosphatase-treated cosmid pYUB18. The ligation mixture was packaged into bacteriophage 
heads using Gigapack packaging extract (Stratagene La Jolla, CA), and the resulting packaged cosmid molecules were transduced into E. coli LE 392 and plated on LB agar containing 25 µg/ml chloramphenicol. Each resulting chloramphenicol-resistant clone contained a large segment of chromosomal DNA with pYUB18 inserted into it.
DNA sequencing.
A genomic DNA fragment of M. smegmatis involved in steroid 9-hydroxylation was subcloned in pBluescript II SK(+) (purchased from Stratagene, La Jolla, CA), and the chromosomal insert was amplified by PCR. After purification of the PCR products from an agarose gel, the purified DNA was used as a template and sequenced by the fluorescence DyeDeoxy method (ABI PRISM dye terminator cycle sequencing core kit; Applied Biosystems, United States).
Other methods.
The methods used for DNA manipulation were based on the methods of either Hopwood et al. (7) or Sambrook et al. (20) unless indicated otherwise. Electroporation of M. smegmatis and M. phlei was performed as described by Jacobs et al. (10). Blast and ORF Finder of the National Center for Biotechnology Information, Bethesda, MD, were used to compare nucleotide or protein sequences to sequences in databases and to analyze possible open reading frames in the DNA region isolated, respectively.
Nucleotide sequence accession number.
The sequence of ORF-1 has been deposited in the GenBank database under accession no. DQ357196.
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Individual mutants were screened by fermentation in the presence of ß-sitosterol to obtain strains with a block in the sitosterol degradation pathway. Selection was based on analysis of intermediates that accumulated in microtiter fermentation broth media. Of approximately 10,000 insertional mutants in each library, 4 had altered sterol-degrading ability compared to the parental strain (Table 1.). The fermentation broth media of these mutants were analyzed by TLC and, for selected strains, by HPLC, and the structures of the isolated degradation products were identified.
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-hydroxylase, so the steroid skeleton remained intact. This strain formed 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione from sitosterol.
One of the mutants derived from M. phlei M51-Ept, 3B7, degraded the sitosterol side chain only partially and accumulated 23,24-dinorcholane and 17(20)-dehydro-23,24-dinorcholane derivatives because of a lack of the late enzymatic steps. These types of sterol degradation products are starting materials for synthesis of compounds having 16-dehydro-20-oxo-pregnane (23) and 17-hydroxy-20-oxo-pregnane (18, 24) structures, which are key intermediates in the synthesis of corticosteroids. We assumed that M. phlei 3B7 is suitable for isolation of the gene for enoyl-CoA hydratase involved in the fourth cycle of ß-oxidation of the sitosterol side chain, which catalyzes addition of water to the 17(20) double bond of 3-oxo-23,24-dinor-4,17(20)-choladien-22-oyl-CoA. This intermediate side chain could not be degraded further, because the gene for enoyl-CoA hydratase was inactivated by the insertion. Instead, it was metabolized into 3-oxo-23,24-dinor-4,17(20)-choladien-22-oic acid methyl ester by hydrolysis and esterification. Interruption of side chain degradation also resulted in the accumulation of 3-oxo-23,24-dinor-4-cholen-22-oyl-CoA, which is the preceding intermediate of the degradation. The latter intermediate was metabolized into 3-oxo-23,24-dinor-4-cholen-22-oic acid methyl ester and 22-hydroxy-23,24-dinor-4-cholen-3-one. Mutants 5G4 and 10G9 formed mainly testosterone, together with a reduced amount of 4-androstene-3,17-dione compared to the amount in the parental strain.
The stability of the four insertional mutants was investigated by comparing the numbers of CFU in the presence and in the absence of 20 µg/ml kanamycin on M-OADC-G agar after cultivation of the mutants in antibiotic-free liquid medium until stationary phase. The reversion frequency was found to be at least as low as that determined previously (2 x 10–6) by Guilhot et al. for auxotrophic insertional mutants of M. smegmatis mc2155 (6).
Cloning of DNA encoding the putative terminal oxygenase of 3-ketosteroid 9-hydroxylase in M. smegmatis.
In order to clone the putative 9-hydroxylase gene, chromosomal DNA was isolated from the M. smegmatis 10A12 mutant and digested with NotI to generate large fragments containing the integrated plasmid pCG79 (NotI does not cleave pCG79). The digested DNA was purified by sucrose gradient centrifugation to separate 30- to 40-kb fragments. Southern blot analysis using pCG79 DNA as the probe revealed that the DNA fraction containing the integrated pCG79 vector was the largest fraction. Fractions from the sucrose gradient with the greatest radioactivity were collected, and the linear molecules were circularized with T4 ligase and used to transform E. coli XL1-Blue, with selection on 20 µg/ml streptomycin in LB agar. Plasmid DNAs from eight streptomycin-resistant transformants were analyzed by agarose gel electrophoresis after digestion with PstI. The PstI digestion patterns of all the recombinant plasmid DNAs were similar, and the DNA fragments typical of pCG79 could also be detected. A representative recombinant plasmid was designated pFJ92 (Fig. 2.). Its size, estimated from the digestion fragments, was approximately 26.8 kb.
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-hydroxylase gene, pFJ92 was used to identify the wild-type gene, which was used to complement the mutation in M. smegmatis 10A12. A genomic library was constructed from the parental strain M. smegmatis mc2155 using the cosmid vector pYUB18. Independent E. coli clones bearing mycobacterial chromosomal fragments were screened by colony hybridization using two radiolabeled internal fragments of the Mycobacterium chromosomal region of pFJ92, PstI-PstI and EcoRI-EcoRI fragments, one located near each end of the fragment (Fig. 2.), as probes. Of 960 colonies, 2 gave positive hybridization signals with both probes so could be expected to contain the chromosomal DNA surrounding the integration point of the transposon and therefore the putative 9-hydroxylase gene in the native form. Restriction analysis of the two selected recombinants with NotI or EcoRV resulted in the same fragments, so either cosmid was used in further experiments. The 8.9-kb NotI fragment predicted from the restriction map of pFJ92 was purified from an agarose gel after electrophoretic separation. The NotI chromosomal fragment of M. smegmatis mc2155 isolated in this way was inserted into the NotI site of the polylinker region of pOLYG carrying the hygromycin resistance gene (19). The resulting recombinant plasmid, pAA23, and its orientation isomer, pAA23*, were introduced into the 10A12 insertional mutant, whereupon the metabolic block in 9-hydroxylation was complemented.
In a further experiment, pAA23 was introduced into M. phlei M51-Ept, which lacks 9-hydroxylase activity, with selection for hygromycin-resistant transformants. These transformants were found to convert 4-androstene-3,17-dione into 9-hydroxy-4-androstene-3,17-dione. When 1,4-androstadiene-3,17-dione was added as the substrate, 3-hydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (3) was formed because of spontaneous rearrangement of the unstable compound 9-hydroxy-1,4-androstadiene-3,17-dione. These observations demonstrated the ability of the transformants to introduce a hydroxyl group at the 9 position of the steroid skeleton.
The exact location of the putative 9-hydroxylase gene was determined by deletion mutagenesis, followed by complementation experiments and sequencing. Restriction sites mapped on the NotI fragment were used in making deletion derivatives of pAA23. The size of the NotI chromosomal insert was gradually decreased until the deletions abolished 9-hydroxylase activity, localizing the gene to the EcoRI-EcoRV internal segment (Fig. 2). DNA sequencing of this fragment revealed five open reading frames. Further deletions and sequencing identified ORF-1 as the open reading frame encoding the 9-hydroxylase enzyme. The integration site was located at the 5' end of ORF-1 between the BamHI and SacI restriction sites 62 bp downstream of the translation start site of ORF-1.
The sequence of ORF-1, encoding a 383-amino-acid protein, has been deposited in the GenBank database. Homology analysis of the deduced amino acid sequence showed that there was complete identity to MSMEG5884 (The Institute for Genome Research database), which is annotated as an iron-sulfur cluster-binding protein in the M. smegmatis genome that is 82% identical to the protein encoded by Rv3526 of Mycobacterium tuberculosis H37Rv (2) and 61% identical to the terminal oxygenase of the known two-component 3-ketosteroid 9-hydroxylase of R. erythropolis SQ1 (25). All three proteins contain the Rieske [2Fe-2S] domain and a nonheme Fe(II) domain, whose presence is characteristic of class IA monooxygenases (Fig. 3) (12, 17, 25). This supports the hypothesis that ORF-1 encodes the terminal oxygenase component of the 3-ketosteroid 9-hydroxylase that is identical to MSMEG5884 in M. smegmatis mc2155.
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-hydroxylase in E. coli.-hydroxylase was amplified by PCR from pAA23. Primers were designed to allow cloning of ORF-1 into the NcoI and HindIII sites of pET-28a(+). The forward and reverse primers were 5'-CCATGGCTACCGAGACTGTCG-3' and 5'-AAGCTTCAGCTCGACTGCTCC-3', respectively. The DNA fragment was PCR amplified, cloned into the pBluescript SK II(+) vector, and checked by sequencing. A recombinant plasmid carrying the correct insert was digested with NcoI and HindIII and ligated into the appropriate sites of pET-28a(+). The ligated DNA was transformed into E. coli BL21-DE3. One clone was selected and named pOX17.
E. coli BL21/pOX17 was cultured in LB medium containing 25 µg/ml kanamycin. When the optical density at 600 nm reached 0.5 to 1.0, 100 µg/ml AD as the substrate and 0.4 mM isopropyl-ß-D-thiogalactoside (IPTG) for induction of the lac promoter were added. After 24 h of propagation, 9-hydroxylation of AD could be detected in the fermentation broth by chromatography. Protein expression was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Cells from an aliquot of the culture were sonicated, and the inclusion bodies were separated by centrifugation. The supernatant contained the soluble forms of the proteins. SDS-PAGE analysis of protein extracts from the control and IPTG-induced, 9-hydroxylating E. coli BL21/pOX17 cultures revealed a large quantity of the recombinant terminal oxygenase. The recombinant protein appeared mostly as inclusion bodies; a minority was in the soluble form (Fig. 4.). Since E. coli BL21/pOX17 could carry out steroid transformation in vivo despite the lack of the cognate reductase component of the hydroxylase enzyme, it must have relied on one of the reductases of E. coli.
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-hydroxylase in the active form provided further evidence for the correct DNA sequence.
Industrial perspectives.
Insertional mutagenesis with pCG79 carrying Tn611 combined with high-throughput screening for the bioconversion products of sitosterol that accumulate in miniaturized cultures of the mutants is an efficient method to detect genes involved in sitosterol degradation and to determine their exact positions in the genomes of fast-growing mycobacteria. The transposon-disrupted genes can be isolated easily and used for DNA manipulation. For example, expression of the rate-limiting enzymes of sitosterol side chain cleavage can be enhanced in order to improve the side chain degradation power of Mycobacterium strains used industrially. We expect these enzymes to be important tools for strain improvement in these commercially important microorganisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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liwi
ski, A. Rumijowska-Galevicz, M. Korycka-Macha
a, and J. Dziadek. 2005. Identification and targeted disruption of the gene encoding the main 3-ketosteroid dehydrogenase in Mycobacterium smegmatis. Microbiology 151:2393-2402.-hydroxy steroids and related compounds. Biomed. Mass Spectrom. 4:376-380.[CrossRef][Medline]y, and G. Ambrus. 1989. Genetic recombination by spheroplast fusion of sterol-transforming Mycobacterium strains. J. Gen. Microbiol. 135:1727-1733.-Hydroxy-pregna-1,4-dien-3,20-dion. East German patent 142,054.-hydroxy-20-oxo-pregnanes from 17(20)-dehydro-23,24-dinorcholan-22-oic acids. Tetrahedron Lett. 33:5265-5266.[CrossRef]-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1. Mol. Microbiol. 45:1007-1018.[CrossRef][Medline]
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Deceased.