EP1232278A1 - MICROBIAL 9$g(a)-HYDROXYLATION OF STEROIDS - Google Patents
MICROBIAL 9$g(a)-HYDROXYLATION OF STEROIDSInfo
- Publication number
- EP1232278A1 EP1232278A1 EP00977417A EP00977417A EP1232278A1 EP 1232278 A1 EP1232278 A1 EP 1232278A1 EP 00977417 A EP00977417 A EP 00977417A EP 00977417 A EP00977417 A EP 00977417A EP 1232278 A1 EP1232278 A1 EP 1232278A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- gene
- steroid
- dione
- androstene
- kstd2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/06—Hydroxylating
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the invention relates to a method to prepare genetically modified micro-organisms having inhibited capacity for nucleus degradation of steroids, the use of such micr- organism in steroid accumulation as well as such modified micro-organisms.
- the enzyme 3-ketosteroid ⁇ '-dehydrogenase [4-ene-3-oxosteroid:(acceptor)-l- ene-oxidoreductase, EC 1.3.99.4] is involved in cleavage of ring B of the steroid nucleus by introducing a double bond at the C1-C2 position. More particularly, the enzyme is involved in the conversion of 4-androstene-3,17-dione in 1,4-androstadiene- 3,17-dione and of 9 ⁇ -hydroxy-4-androstene-3,17-dione in 9 ⁇ -hydroxy-l,4- androstadiene-3,17-dione (see Figure 1).
- KSTD 3-ketosteroid ⁇ '-dehydrogenase
- the enzyme has been identified in several bacteria: Arthrobacter simplex (Penasse and Peyre, 1968 Rhodococcus. Crit Rev Biotech 14:29-73), Pseudomonas (Levy and Talalay, 1959 J Biol Chem 234:2009- 20013; 1959 J Biol Chem 234:2014-2021 ), Nocardia restrictus (Sih and Bennet, 1962 Biochem Biophys Acta 56:587-592), Nocardia corallina (Itagaki et al., 1990 Biochim Biophys Acta 1038:60-67), Nocardia opaca (Drobnic et al, 1993 Biochim Biophys Res Comm 190:509-515), Mycobacterium fortuitum (Wovcha et al, 1979 Biochim Biophys Acta 574:471-479) and Rhodococcus erythropolis IMET7030 (Kaufinann et al, 1992 J Steroid Biochem Mo lee Biol 43:297-301).
- KSTD of N. opaca has been characterized as a flavoprotein (Lestrovaja et al, 1978 Z Allg Mikrobiol 18:189-196). Only the KSTD encoding genes (kstD: 3-ketosteroid ⁇ 1 Dehydrogenase) of A. simplex, Comamonas testosteroni and Rhodococcus rhodochrous have been fully characterized (Plesiat et al, 1991 J Bacteriol 173:7219-7227; Molnar et al, 1995 Mol Microbiol 15:895-905; Morii et al, 1998 J. Biochem 124:1026-1032).
- Rhodococcus species are able to degrade natural phytosterols, which are inexpensive starting materials for the production of bioactive steroids (Kieslich K., 1986 Drug Res 36: 888-892).
- Rhodococcus and Mycobacterium strains treated with mutagens and/or incubated with enzyme inhibitors convert sterols into 4-androstene-3,17-dione and l,4-androstadiene-3,17-dione (Martin, 1977 Adv Appl Microbiol 22:29-58).
- KSTD activity is essential for steroid nucleus degradation and kstO gene inactivation is needed to accumulate steroid intermediates.
- the nucleotide sequence of the kstO gene of R. erythropolis has been provided.
- KSTD protein is encoded by nucleotides 820-2329 of SEQ ID ⁇ O:l.
- Inactivation of genes is a powerful tool for analysis of gene function and for introduction of metabolic blocks.
- Gene disruption with a non-replicative vector carrying a selective marker is the commonly used method for gene inactivation.
- Construction of strains with desirable properties via metabolic pathway engineering approaches may require the stepwise inactivation or replacement of several genes. This is only possible when a suitable strategy for introduction of unmarked gene deletions or gene replacements, allowing infinite rounds of metabolic engineering without being dependent on multiple markers, is available.
- a stepwise inactivation of genes preferably dehydrogenase genes, involved in steroid degradation.
- the invention applies for an inactivation of genes involved in the accumulation of 9 ⁇ -hydroxy-4-androstene-3,17- dione by growing of micro-organisms on 4-androstene-3,17-dione.
- at least the gene kstDl is inactivated.
- the second enzyme is a dehydrogenase, more preferably a KSTD isoenzyme.
- a method of site-directed mutagenesis can be used. A method for introduction of unmarked gene deletions is to be preferred for the stepwise inactivation of KSTD genes. The resulting genetically modified strains would be free of heterologous DNA.
- At least the gene _fcstD2 is inactivated. Most preferably, at least both the genes kstDl and _fcytD2 are inactivated.
- Another aspect of the present invention is the nucleotide sequence of the kstD2 gene of R. erythropolis. KSTD2 protein is encoded by nucleotides 1-1678 of SEQ ID NO:5. No methods for introduction of unmarked gene deletions in the genus Rhodococcus have been reported.
- Bacteriol 179:180-186 Corynebacterium (Schafer et al, 1994 Gene 145:69-73) and Mycobacterium (Marklund et al, 1995 J Bacteriol 177:6100-6105; Norman et al, 1995 Mol Microbiol 16:755-760; Sander et al, 1995 Mol Microbiol 16:991-1000; Pelicic et al, 1996 Mol Microbiol 20:919-125; K ipfer et al, 1997 Plasmid 37:129-140).
- Counter-selectable markers may be used to screen for the rare second recombination event resulting in gene deletion or gene replacement.
- both sacB and rpsL proved to be useful reporter genes (Hosted and Baltz, 1997 J Bacteriol 179:180-186; Schafer et al, 1994 J Bacteriol 172:1663-1666; Sander et al, 1995 Mol Microbiol 16:991-1000; Pelicic et al, 1996 Mol Microbiol 20:919-925; Jager et al, 1992 J Bacteriol 174:5462-5465), but other suitable markers can be used as well.
- the B. subtilis levansucrase encoded by the sacB gene, catalyzes hydrolysis of sugars and synthesis of levans (high-molecular weight fructose polymers).
- Expression of sacB in Rhodococcus is lethal in the presence of sucrose.
- the biochemical basis for toxicity of levansucrase action on sucrose is still unknown.
- Conditional lethality (i.e. presence or absence of sucrose) of the sacB gene therefore can be used as a counter-selectable marker. Counter-selection in this context means that expression of the marker is lethal, instead of giving rise to resistance as is the case for selectable markers (e.g. resistance markers).
- Counter-selection is needed to select for those mutants that have undergone a second recombination event, thereby losing the sacB marker and introducing the desired mutatioa
- the advantage of this system is that during selection solely potentially good mutants will survive the selection. Compared to a system in which only one selection marker is used, counter-selection avoids a time consuming screening process for loss of the resistance marker that would be necessary in an one-selection-marker system.
- unmarked mutation allows the repetitive introduction of mutations in the same strain.
- Foreign DNA vector DNA
- Newly introduced vector DNA for the introduction of a second mutation, therefore cannot integrate at the site of the previous mutation (by homologous recombination between vector DNA's). Integration will definitely happen if vector DNA is still present in the chromosome and will give rise to a large number of false-positive integrants.
- the system enables the use of a sole antibiotic gene for the introduction of an infinite number of mutations.
- Unmarked mutation also allows easy use in the industry because of the absence of heterogeneous DNA allowing easy disposal of fermentation broth.
- Gene inactivation by gene deletion enables the construction of stable, non-reverting mutants. Especially small genes ( ⁇ 500 bp) are inactivated more easily by gene deletion compared to gene disruption by a single recombination integration. Gene deletion mutagenesis can also be apphed to inactivate a cluster of several genes from the genome. The gene deletion mutagenesis strategy can be applied also for gene- replacement (e.g. changing wild type into mutant gene).
- Rhodococcus erythropolis The preferred strain for mutagenesis of the catabolic steroid dehydrogenases genes is Rhodococcus erythropolis.
- these species belong to the genus Rhodococcus but also related species such as Nocardia, Mycobacterium and Arthrobacter can be used.
- Rhodococcus Gene inactivation in Rhodococcus is hampered by the occurrence of illegitimate recombination events resulting in random genomic integration of the mutagenic vector (Desomer et al, 1991 Mol Microbiol 5:2115-2124; Barnes et al, 1997 J Bacteriol 179:6145-6153), a phenomenon we encountered when attempting to disrupt the kstDl gene in R. erythropolis SQL Illegitimate recombination is also a well-known phenomenon in some slow-growing species of Mycobacterium (McFadden, 1996 Mol Microbiol 121:205-211).
- the introduction of a second gene inactivation event can be performed using the same methods as is illustrated in the Examples for kstD2.
- the same methods may be used again, or, alternatively, UV irradiation or chemical means such as nitroguanidine or diepoxyethaan may be used. Methods to introduce gene mutations in that way are well known in the art.
- micro-organisms possessing multiple gene inactivation' s can be used to accumulate steroid intermediates.
- the accumulated product is 9 ⁇ -hydroxy-4-androstene-3,17-dione.
- the starting material may depend on the enzyme genes which are inactivated. Suitable starting materials are e.g. phytosterols or 4-androstene-3,17-dione. The preferred starting material is 4- androstene-3 , 17-dione.
- An advantage of the present method is that high conversion yields from the starting steroid into the accumulated product can be obtained.
- the yields may exceed 80%, preferably more than 90% and often reach a value of almost 100%.
- Still another aspect of the invention resides in genetically modified micro-organisms with multiple inactivated genes which are involved in steroid degradation. Especially these genes are dehydrogenases.
- Preferably at least the gene kstDl or kstD2 is inactivated.
- Preferred are micro-organisms belonging to the genus Rhodococcus. Most preferred is the strain Rhodococcus erythropolis RG1-UV29.
- Rhodococcus erythropolis RG1-UV29 and Rhodococcus erythropolis RGl have been deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, Germany under the accession numbers DSM 13157 and DSM 13156, respectively. These deposits have been made under the terms of the Budapest Treaty.
- FIG. 1 Schematic representation of steroid nucleus degradation in R. erythropolis SQL
- the positions of the 3-ketosteroid ⁇ 1 -dehydrogenase (KSTD) isoenzymes are indicated with KSTD 1 and KSTD3
- FIG. 2 Schematic representation of the mutagenic vector pSDH422 with the counter-selectable marker sacB used for construction of Rhodococcus erythropolis strain RGl with an 1062 bp unmarked kstDl gene deletion.
- ORF2 and ORF3 are the flanking genes of kstDl in R. erythropolis SQl.
- Inserted window Southern analysis, using kstDl as a probe, of R. erythropolis chromosomal DNA digested with BamYR of wild type (lane 1), strain SDH422-3 (lane 2), SDH422-4 (lane 3) and two individual kstDl deletion mutants (lanes 4 and 5).
- a degenerated kstD oligonucleotide probe [5' ttcgg(c/g)gg(c/g)ac(c/g)tc(c/g)gc(c/g)tac tc(c/g)gg(c/g)gc(c/g)tc(c/g)atctgg] (SEQ ID NO:2) was developed from an alignment of the N-terminal parts of known KSTD protein sequences of A. simplex, C. testosteroni and N. opaca. Total D ⁇ A of R. erythropolis SQl digested with Bgl ⁇ l was sized by sucrose gradient centrifugation. Southern analysis at 68 °C (stringent washes with
- KstDl deletion strain A mutagenic vector was constructed that contains a R. erythropolis SQl chromosomal DNA fragment with a kstDl deletion. A 1062 bp Bsml fragment of pSDH200, encoding a large internal part of KSTD1, was deleted to construct pSDH200)BsmI. For construction of the mutagenic vector a 2724 bp Smal/EcoRl fragment of pSDH200)BsmI harbouring the remaining 468 bp of kstDl and its flanking regions was cloned into the SmaVEcoBI site of pKl ⁇ mobsacB (pSDH422, see Figure 2).
- the vector pSDH422 encoding kanamycin resistance to select for integration of the mutagenic vector into the chromosome and harbouring the sacB gene of B. subtilis for counter- selection, was introduced into E.coli SI 7-1 and mobilized to R. erythropolis SQl by conjugation as follows. Cells of the R. erythropolis SQl recipient strain were spread on LBP agar supplemented with 30 ⁇ g-ml "1 nalidixic acid and grown for 5 days. The mutagenic vector pSDH422 was first introduced in E.coli SI 7-1 by transformation.
- Transformants (approx. 1000 per plate) grown overnight on selective media (kanamycin 25 ⁇ g ml "1 ) were incubated at room temperature for another 24 h.
- Colonies of both Rhodococcus and E.coli strains were resuspended in a final volume of 1.5 ml of LBP (1% bacto-pepton (Difco), 0.5% yeast extract (BBL) and 1% NaCl). Aliquots of 750 ⁇ l of each strain were mixed and gently pelleted by centrifugation. The pellet was resuspended in 1 ml LBP and cells were spread on non-selective LBP agar in 250 ⁇ l aliquots.
- LBP bacto-pepton
- BBL yeast extract
- kanamycin resistant (kan r ) Rhodococcus transconjugants were sucrose sensitive (suc s ); no growth occurred after replica plating on LBPS (1% bacto-pepton, 0.5% yeast extract, 1% NaCl, 10% sucrose) agar supplemented with 200 ⁇ g-ml "1 kanamycin.
- Late exponential phase R. erythropolis RGl cells (2-10 8 CFUs-ml "1 ) grown in 10 mM glucose mineral medium (K 2 HPO 4 4.65 g-1 "1 , NaH 2 PO 4 H 2 O 1.5 g-1 "1 , NH_tCl 3 g l “1 , MgSO 4 -7H 2 O 1 g-1 "1 , Vishniac trace elements, pH 7.2) were sonicated for a short period of time to obtain single cells. Diluted (10 4 ) samples were spread on glucose mineral agar medium and irradiated for 15-20 sec with an UV lamp (Philips TAW 15W) at a distance of 27 cm, on average resulting in 95% killing of cells.
- Example 4 Microbiological 9 ⁇ -hydroxylation of 4-androstene-3,17dione with UV-mutant Rhodococcus erythropolis UV-29.
- Rhodococcus erythropolis SQl UV-29 is a UV-mutant which is capable of conversion of 4-androstene-3,17-dione (AD) into 9 -hydroxy-4-androstene-3,17-dione (9 ⁇ OH-AD) with concentration of 10 to 20 g/1. This conversion was performed using the following method:
- a 10 liter fermentor with 6 liter in situ sterilized fermentation broth (1.5% yeast extract, 1.5% glucose, 0.01% antifoaming agent polypropylene glycol; pH 7.5) was inoculated with preculture (1%) and incubated at 28°C for 16 hours under sparging with sterile air and the culture was agitated to induce submerged growth.
- a gene library of R. erythropolis RGl was introduced into competent R. erythropolis strain RG1-UV29 by electrotransformation. Colonies obtained were replica plated onto mineral agar medium containing 4-androstene-3,17-dione (0.5 g/1) as sole carbon and energy source. Complementation of the strain RG1-UV29 phenotype was scored after three days of incubation at 30 °C. Colonies growing on 4-androstene-3,17-dione mineral agar medium were cultivated in LBP medium for isolation of plasmid DNA, that was subsequently re- introduced into strain RG1-UV29 to check for genuine complementation.
- Plasmid pKSDIOl isolated from a transformant that showed restored growth on 4-androstene-3,17-dione mineral medium, was introduced into E. coli DH5 ⁇ for further analysis. An insert of approximately 6.5 kb rhodococcal DNA was identified in pKSDIOl and subjected to restriction mapping analysis, subcloning and subsequent complementation experiments. A 3.6 kb EcoRI DNA fragment of pKSDIOl was still able to restore the strain RG1-UV29 phenotype and thus was subcloned in pBluescript(II) KS (pKSD105) for nucleotide sequencing.
- Nucleotide sequence analysis revealed the presence of a large open reading frame (ORF) of 1,698 nt, encoding a putative protein of 565 amino acids with a calculated molecular weight of 60.2 kDa.
- This ORF was designated kstDl (SEQ ID NO :5)( which is identical to the previously described kstDl - see Example 3).
- the deduced amino acid sequence of kstD2 showed high similarity to known 3-ketosteroid ⁇ '-dehydrogenases (KSTD) indicating that kstD2 encodes a second KSTD enzyme in R. erythropolis RGl .
- KSTD 3-ketosteroid ⁇ '-dehydrogenases
- R. erythropolis strain RG7 is a mutant strain, obtained from wild type R. erythropolis strain SQl, containing a single kstD2 gene deletion.
- R. erythropolis strain RG8 is constructed by the successive deletion of two genes encoding 3-ketosteroid ⁇ 1 - dehydrogenase activity, i.e. kstDl and kstD2, from wild type R. erythropolis strain SQl.
- Strain RG8 was obtained by deletion of the kstD2 gene from the genome of the kstDl deletion mutant R. erythropolis strain RGl.
- the method used for kstD2 gene deletion was analogous to the method described for kstDl gene deletion in example 2, except for the fact that a different mutagenic vector was used (pKSD201 versus pSDH422).
- the mutagenic vector pKSD201 was constructed as follows. A 1,093 bp internal DNA fragment of the kstD2 gene was deleted by Mlul digestion and subsequent self-ligation of pKSD105, resulting in construction of pKSD200. A 2.4 kb EcoRI fragment of pKSD200 harboring the mutated kstD2 gene was ligated into EcoRI digested pK18mobsacB, thereby constructing pKSD201. Plasmid pKSD201 was introduced into E. coli SI 7-1 and mobilized by conjugation to R. erythropolis strain SQl (to construct strain RG7), or strain RGl (to construct strain RG8).
- Transconjugants resulting from targeted integration of pKSD201 into the genome appeared after 3 days of growth at 30 °C. Deletion of kstD2 was achieved by growth of one selected transconjugant (suc s kan r ) overnight under non-selective conditions (i.e. LBP medium) and subsequent plating on selective LBPS agar medium.
- Rhodococcus erythropolis RG8 is a kstDl and kstD2 double deletion mutant which is capable of conversion of 4-androstene-3,17-dione (AD) into 9 ⁇ -hydroxy-4-androstene- 3,17-dione (9 ⁇ OH- AD) with a concentration of 10 g/1.
- a 10 liter fermentor with 6 liter in situ sterilized fermentation broth (1.5% yeast extract, 1.5% glucose, 0.01% antifoaming agent polypropylene glycol; pH 7.5) was inoculated with preculture (1%) and incubated at 28°C for 16 hours under sparging with sterile air and the culture was agitated to induce submerged growth.
- the microorganism identified under I. above was accompanied by
- This International Depositary Authority accepts the microorganism identified under 1 above, which was received by it on 1999 - 11 - 25 (Date of the original deposit)'.
- microorganism idenufied under I above was received by this International Depositary Authority on (date of original deposit) and a request to conven the original deposit to a deposit under the Budapest Treatv was received by it on (date of receipt of request for conversion)
- the microorganism identified under I above was accompanied by
- microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion)
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Abstract
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Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00977417A EP1232278A1 (en) | 1999-10-22 | 2000-10-17 | MICROBIAL 9$g(a)-HYDROXYLATION OF STEROIDS |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99203470 | 1999-10-22 | ||
EP99203470 | 1999-10-22 | ||
EP99204449 | 1999-12-22 | ||
EP99204449 | 1999-12-22 | ||
PCT/EP2000/010223 WO2001031050A1 (en) | 1999-10-22 | 2000-10-17 | MICROBIAL 9α-HYDROXYLATION OF STEROIDS |
EP00977417A EP1232278A1 (en) | 1999-10-22 | 2000-10-17 | MICROBIAL 9$g(a)-HYDROXYLATION OF STEROIDS |
Publications (1)
Publication Number | Publication Date |
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EP1232278A1 true EP1232278A1 (en) | 2002-08-21 |
Family
ID=26153382
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EP00977417A Ceased EP1232278A1 (en) | 1999-10-22 | 2000-10-17 | MICROBIAL 9$g(a)-HYDROXYLATION OF STEROIDS |
Country Status (11)
Country | Link |
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EP (1) | EP1232278A1 (en) |
CN (1) | CN1224716C (en) |
AR (1) | AR026186A1 (en) |
AU (1) | AU775476B2 (en) |
CA (1) | CA2396879A1 (en) |
CZ (1) | CZ20021784A3 (en) |
IL (1) | IL149715A0 (en) |
NO (1) | NO20022449L (en) |
NZ (1) | NZ519039A (en) |
RU (1) | RU2268935C2 (en) |
WO (1) | WO2001031050A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015128534A1 (en) | 2014-02-27 | 2015-09-03 | Consejo Superior De Investigaciones Científicas | Selective recombinant mutants of mycobacterium smegmatis mc2 155 and use thereof for producing 1,4-androstadiene-3,17-dione or 4-androstene-3,17-dione from natural sterols |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
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AU2003219134A1 (en) | 2002-02-21 | 2003-09-09 | Akzo Nobel N.V. | Identification of 3-ketosteroid 9-alfa-hydroxylase genes and microorganisms blocked in 3-ketosteroid 9-alfa-hydroxylase activity |
CA2506217A1 (en) * | 2002-12-03 | 2004-07-01 | Robert Van Der Geize | New expression system from rhodococcus |
AT503486B1 (en) * | 2006-04-11 | 2008-05-15 | Iep Gmbh | METHOD FOR THE ENANTIOSELECTIVE REDUCTION OF STEROIDS |
WO2009024572A1 (en) * | 2007-08-21 | 2009-02-26 | N.V. Organon | Method for the production of modified steroid degrading microorganisms and their use |
CN102413876A (en) | 2009-02-23 | 2012-04-11 | 格罗宁根大学 | Pharmaceutical compositions and methods for treating tuberculosis |
CN103361394B (en) * | 2013-08-07 | 2016-08-17 | 中国科学院上海高等研究院 | Utilize the method that microorganism converts preparation 9 Alpha-hydroxies-androstenedione |
CN103805577A (en) * | 2013-08-14 | 2014-05-21 | 济南环亿生物科技有限公司 | Method for efficiently producing hydroxysteroid dehydrogenase with testosterone comamonas |
CN107586762A (en) * | 2017-09-18 | 2018-01-16 | 天津科技大学 | A kind of dehydrogenase mutant of 3 sterone Δ 1 and its application |
US11001871B2 (en) | 2017-12-15 | 2021-05-11 | Jiangnan University | Method for producing 9alpha-hydroxy androstane-4-alkene-3,17-diketone by enzymatic conversion |
CN107955827B (en) * | 2017-12-15 | 2019-07-02 | 江南大学 | A kind of 9 Alpha-hydroxy androstane-4-alkene-3s of enzymatic conversion method production, the method for 17- diketone |
GB2577037A (en) * | 2018-08-09 | 2020-03-18 | Cambrex Karlskoga Ab | Genetically-modified bacteria and uses thereof |
CN110229838B (en) * | 2019-05-28 | 2021-01-08 | 浙江理工大学 | Method for obtaining hydroxylated compound by biotransformation of steroid compound |
CN114621965B (en) * | 2022-02-15 | 2023-10-03 | 复旦大学 | 3-sterone-delta 1 Dehydrogenase mutants and uses thereof |
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HU196627B (en) * | 1986-11-18 | 1988-12-28 | Gyogyszerkutato Intezet | Microbiological process for producing y alpha-hydroxy-4-androstene-3,17-dione |
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2000
- 2000-10-17 IL IL14971500A patent/IL149715A0/en unknown
- 2000-10-17 AU AU15145/01A patent/AU775476B2/en not_active Ceased
- 2000-10-17 NZ NZ519039A patent/NZ519039A/en unknown
- 2000-10-17 RU RU2002113373/13A patent/RU2268935C2/en not_active IP Right Cessation
- 2000-10-17 CN CNB008175675A patent/CN1224716C/en not_active Expired - Fee Related
- 2000-10-17 WO PCT/EP2000/010223 patent/WO2001031050A1/en active IP Right Grant
- 2000-10-17 EP EP00977417A patent/EP1232278A1/en not_active Ceased
- 2000-10-17 CZ CZ20021784A patent/CZ20021784A3/en unknown
- 2000-10-17 CA CA002396879A patent/CA2396879A1/en not_active Abandoned
- 2000-10-20 AR ARP000105523A patent/AR026186A1/en not_active Application Discontinuation
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2002
- 2002-05-23 NO NO20022449A patent/NO20022449L/en not_active Application Discontinuation
Non-Patent Citations (3)
Title |
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KIESLICH (1985) J. Basic Microbiol. 25, 461-474. * |
See also references of WO0131050A1 * |
WOVCHA ET AL. (1979) Biochim. Biophys. Acta 574, 471-479. * |
Cited By (1)
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WO2015128534A1 (en) | 2014-02-27 | 2015-09-03 | Consejo Superior De Investigaciones Científicas | Selective recombinant mutants of mycobacterium smegmatis mc2 155 and use thereof for producing 1,4-androstadiene-3,17-dione or 4-androstene-3,17-dione from natural sterols |
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RU2002113373A (en) | 2004-02-27 |
RU2268935C2 (en) | 2006-01-27 |
NZ519039A (en) | 2004-09-24 |
NO20022449L (en) | 2002-06-19 |
AU775476B2 (en) | 2004-08-05 |
AU1514501A (en) | 2001-05-08 |
CN1224716C (en) | 2005-10-26 |
AR026186A1 (en) | 2003-01-29 |
WO2001031050A1 (en) | 2001-05-03 |
IL149715A0 (en) | 2002-11-10 |
CA2396879A1 (en) | 2001-05-03 |
CN1413260A (en) | 2003-04-23 |
NO20022449D0 (en) | 2002-05-23 |
CZ20021784A3 (en) | 2002-08-14 |
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