JP2006191920A - Method for producing steroidal metabolite using genus comamonas with broken gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing 7αβ-methyl-1,5-dioxo-8aα-hexahydroindan-4α-propionic acid or analogues thereof using microorganisms. <P>SOLUTION: The method for producing a compound of formula (1) or analogues thereof comprise of a steroid-assimilating genus Comamonas bacterium in which a metabolism-relating gene of the compound of the formula (1) or analogues thereof is broken is brought into contact with steroidal compounds in a medium, and the formed compound of the formula (1) or analogues thereof is isolated. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to steroid metabolites, specifically optically active 7a-methyl-1,5-dioxohexahydroindane-4-propion, using a gene disrupted strain of a steroid-utilizing Comamonas bacterium. The present invention relates to a method for producing an acid (hereinafter, also simply referred to as “indandione propionic acid”) or an analog thereof.

Indandione propionic acid is useful as a raw material for producing steroids such as pharmaceuticals.
Regarding the production of this compound by microorganisms, JP-A-53-69885 discloses as microorganisms, Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Nocardia. (Nocardia), Protaminobacter, Serratia, and Streptomyces bacterial mutants, especially Mycobacterium fortuitum NRRL B-8129 As, for example, sitosterol, cholesterol, stigmasterol, campesterol, androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, dehydroepiandrosterone and testosterone, Using steroids with or without 17-alkyl side chains with 2 to 10 carbon atoms, Method of making a down Dan dione propionic acid is disclosed. In this method, it takes about 72 hours to 13 days at a temperature of about 30 ° C. for substance conversion by microorganisms.

  Further, JP-A-60-24190 discloses indandione using a steroid similar to the above as a raw material, using a mutant strain Arthrobacter simplex ID-38-6 obtained using a nitrosoguanidine mutation reagent. A method for producing propionic acid is disclosed, and it is reported that the desired compound can be obtained in a yield of 85 to 97% at 28 ° C. for 120 hours.

The present inventors have analyzed the steroid metabolic enzyme genes of the genus Comamonas, particularly the C. testosteroni strain TA441. Steroid metabolism by bacteria was clarified mainly by analysis of Nocardia by AW Coulter and P. Talalay (J. Biol. Chem. (1968), 243: 3238-3247). The present inventors clarified the steroid metabolic pathway by the TA441 strain with reference to the results. In addition, the sequence of the gene cluster containing the steroid metabolism enzyme gene group of TA441 strain was determined, and this sequence was registered as AB076368 in DDBJ / EMBL / GeneBank. Based on this sequence and genome map information, the present inventors further constructed a disruption strain of the above enzyme gene, for example, TesA2 ⁻ , TesA1 ⁻ , TesD ⁻ , TesH ⁻ , ORF17 ⁻ , ORF18 ⁻ strain, etc. After culturing as a substrate, each metabolite was examined, thereby demonstrating that the gene cluster was exclusively involved in steroid metabolism (Japanese Patent Laid-Open No. 2003-61671; M. Horinouchi, T. Hayashi, T. Yamamoto and T Kudo, Appl. Environ. Microbiol. (2003), 69: 4421-4430). In this document, the intermediate intermediate accumulation of testosterone, particularly by the ORF18 ^- strain, is Androst-4-ene-3,17-dione (4-AD), Androst-1,4-diene-3,17 -Dione (ADD) and 17-hydroxy-1,4-androstadien-3-one (17OH-ADD), which are common accumulators accumulated in many other gene-disrupted strains ( M. Horinouchi et al., Supra, FIG. 7). No deposits characteristic of the ORF18 ^- strain were detected.

JP-A-53-69885 Japanese Unexamined Patent Publication No. 60-24190 Japanese Patent Laid-Open No. 2003-61671 A.W. Coulter and P. Talalay, J. Biol. Chem. (1968), 243: 3238-3247 M. Horinouchi, T. Hayashi, T. Yamamoto and T. Kudo, Appl. Environ. Microbiol. (2003), 69: 4421-4430

  The conventional method for producing indandione propionic acid using a mutant bacterial strain requires a long time for substance conversion. Therefore, when a steroid compound that is difficult to dissolve in an aqueous medium is used as a raw material, it becomes an obstacle to industrial production. In addition, it is difficult to obtain information such as which gene is mutated and how many mutations are present in the mutant strain. There are some problems in supplying mutant strains stably and for a long time for substance production.

  Under such circumstances, there has been a demand for a method for producing indandione propionic acid in a relatively short period of time without relying on mutant strains.

  When the present inventors tried to analyze the function of ORF18 and examined conversion using various steroid compounds as substrates, when 4-AD, ADD or the like was used as a substrate instead of testosterone, an ORF18-disrupted strain was obtained. The present inventors have found that these steroid compounds are converted to indandione propionic acid with high yield.

  Accordingly, an object of the present invention is to provide a method for producing indandione propionic acid or an analog thereof using a strain in which the metabolism-related gene of indandione propionic acid or an analog thereof derived from the genus Comamonas is disrupted. .

The features of the present invention are summarized as follows.
(1) The following formula (1)

の化合物又はその類縁体の代謝関連遺伝子が破壊されたステロイド資化性コマモナス属細菌を、培地中でステロイド化合物と接触させ、生成した前記式（１）の化合物又はその類縁体を単離することを含む、式（１）の化合物又はその類縁体を製造する方法。

A steroid-utilizing Comamonas bacterium in which the metabolism-related gene of the compound or its analog has been disrupted is contacted with a steroid compound in a medium, and the produced compound of the formula (1) or an analog thereof is isolated A method for producing a compound of the formula (1) or an analog thereof.

(2) The method according to (1), wherein the genus Comamonas is Comamonas testosteroni.
(3) The method according to (2), wherein the Comamonas testosteroni is Comamonas testosteroni TA441 strain, ATCC11996 strain or a mutant thereof.

  (4) The method according to (1), wherein the metabolism-related gene is the steroid metabolism enzyme gene ORF18 of the genome of the Comamonas testosteroni TA441 strain, ATCC11996 strain or a mutant strain thereof.

  (5) The method according to (1), wherein the metabolism-related gene is an ORF18 homologue of the genome of the genus Comamonas.

  (6) The method according to any one of (1) to (5), wherein the steroid compound is a testosterone metabolism intermediate compound or a derivative thereof.

  (7) The method according to (6), wherein the testosterone metabolic intermediate compound is androsta-1,4-diene-3,17-dione or androst-4-en-3,17-dione.

  (8) The method according to (6), wherein the steroid compound is cholic acid, chenodeoxycholic acid or deoxycholic acid.

(9) The analog of the compound of the formula (1) is represented by the following formula (11), formula (12) or formula (13):

の化合物である、（１）の方法。

The method of (1), which is a compound of

  (10) The method according to any one of (1) to (9), wherein the genus Comamonas is immobilized on a carrier.

(11) The method according to any one of (1) to (10), wherein the contact between the bacterium and the steroid compound is performed batchwise or continuously.
(12) A compound of the following formula (11) or a diastereomer thereof.

(13) A compound of the following formula (12) or a diastereomer thereof.

(14) A compound of the following formula (13) or a diastereomer thereof.

  Since the method of the present invention uses a gene-disrupted strain of the genus Comamonas, the time required for growth and steroid conversion is remarkably higher than that of the known nitrosoguanidine mutant of the genus Arthrobacter or Mycobacterium. It is shortened, and it is easy to stably maintain a disrupted strain with little or no conservative mutation, and the compound of the above formula (1) or an analog thereof can be obtained in high yield. Since the production of intermediate metabolites derived from steroids other than dionepropionic acid or its analogs is very little or little, it has the advantage that the product can be easily purified.

The present invention will be described more specifically.
As described above, the present invention provides the following formula (1):

の化合物、すなわち光学活性7a-メチル-1,5-ジオキソヘキサヒドロインダン-4−プロピオン酸、又はその類縁体の代謝関連遺伝子が破壊されたステロイド資化性コマモナス属細菌株を、培地中でステロイド化合物と接触させ、生成した式（１）の化合物又はその類縁体を単離することを含む、式（１）の化合物又はその類縁体を製造する方法を提供する。

A steroid-utilizing Comamonas bacterium strain in which the metabolism-related gene of optically active 7a-methyl-1,5-dioxohexahydroindane-4-propionic acid or its analog is disrupted in a medium. There is provided a method for producing a compound of formula (1) or an analog thereof comprising contacting with a steroid compound and isolating the resulting compound of formula (1) or an analog thereof.

In the present invention, any bacterium belonging to the genus Comamonas can be used as long as it is steroid-assimilating and contains a compound of formula (1) or an analog thereof as a metabolic intermediate in the steroid metabolism system. It can be used as a parent strain for the production of the gene disruption strain of the invention. The gene to be disrupted on the parent strain genome is a gene involved in metabolism of the compound of formula (1) or its analog in the steroid metabolism system, that is, a metabolism-related gene. This gene includes at least a structural gene, and can further include its 5 ′ untranslated region, 3 ′ untranslated region and / or transcriptional regulatory region (eg, enhancer or promoter). For the purposes of the present invention, it is preferable to disrupt the structural gene, but even if the untranslated region or the transcriptional regulatory region is disrupted, the transcription is suppressed or prevented and the expression of the metabolism-related gene does not occur. In addition, it is possible to destroy only the region other than the structural gene. A method for producing a gene-disrupted strain will be described later.

  Examples of the genus Comamonas include Comamonas testosteroni (Tamaoka et al., Int. J. Syst. Bacteriol. (1987), 37:58; ATCC 11996, ATCC 1740, DSM 50244, etc.) and related species For example, C. denitrificans (Gumaelius et al., Int. J. Syst. Bacteriol. (2001), 51: 1005; ATCC 700936), C. nitrativorans (Etchebehere et al., Int J. Syst. Bacteriol. (2001), 51: 982; DSM 13191), C. terrigena (DeVos et al., Int. J. Syst. Bacteriol. (1985), 35: 450; ATCC 8461, DSM 7099) and the like, as well as these mutant strains, and steroid-utilizing bacterial species using the compound of the formula (1) or an analog thereof as a metabolic intermediate are used as parent strains for producing gene disruption strains in the present invention. used.

  In the present invention, the preferred genus Comamonas is Comamonas testosteroni. Although not limited to the following strains, for example, TA441 strain (Japanese Patent Laid-Open No. 2003-61671) and related strains thereof, such as ATCC 11996, ATCC 17410, ATCC 15666 strain, ATCC 15667 strain, ATCC 17409 strain, Includes ATCC 17510, ATCC 27911, ATCC 31492, ATCC 33083, etc. Here, ATCC means American Type Culture Collection, which is an American depository, and the number after ATCC is the deposit number of the deposited microorganism. The% identity between TA441 strain and ATCC11996 strain or ATCC 17410 strain is considered to be 99% or more and 80% or more at the genomic DNA level, respectively. Here, the mutant strain refers to a bacterial strain obtained by natural mutation or artificial mutation, and the% identity of the genome sequence including the steroid metabolic enzyme gene group is at least 60% and at least 70% relative to the TA441 strain. Bacterial strains such as at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%.

  Percent identity is expressed as the number of identical overlapping positions / total number of positions × 100, and% identity between two sequences can be determined using known mathematical algorithms. An example of a mathematical algorithm is the BLASTn program (Altschul et al., J. Mol. Biol. (1990), 215: 403-410). For example, a BLAST nucleotide search can be performed using the BLASTn program with a score of 100 and a word length of 12, to obtain a nucleotide sequence that is homologous to the target nucleic acid sequence and to determine% identity. Other examples include methods using Gapped BLAST to obtain gapped alignments (Altschul et al., Nucleic Acids Res. (1997), 25: 3389-3402), for sequence comparisons as described by Pearson and Lipman. FASTA (Proc. Natl. Acad. Sci. (1988), 85: 2444-2448).

  In particular, in a preferred embodiment of the present invention, the Comamonas testosteroni is Comomanas testosteroni TA441 strain, ATCC11996 strain or a mutant thereof.

  As used herein, a “mutant strain” is a gene derived from a natural mutation, or obtained by causing mutation by UV, γ-ray irradiation, mutagen treatment, transposon, etc., and compared to a parent strain. It means a bacterial strain in which the type has changed, and is a bacterial strain that produces the compound of the above formula (1) or an analog thereof as a metabolic intermediate.

Gene-disrupted strain A gene-disrupted strain that can be used in the present invention is an optically active 7a-methyl-1,5-dioxohexahydroindane-4-propionic acid of the formula (1), which is a steroid metabolism intermediate, or an analog thereof. It is characterized by the destruction of metabolism-related genes.

  The metabolic intermediate represented by the formula (1) has two asymmetric carbons at positions 7a and 4 and may be any diastereomer, but preferably 7aβ-methyl-1 , 5-dioxo-8aα-hexahydroindane-4α-propionic acid.

As used herein, an “analog” refers to one or more substituents at any position of the structure of Formula (1), including but not limited to —OH, alkyl, substituted alkyl, cycloalkyl, Derivatives of compounds of formula (1) comprising a substituent selected from phenyl, substituted phenyl, halogen, -OR (R is alkyl or substituted alkyl), -OCOR (R is alkyl or substituted alkyl), and the like. Examples of analogs are those in which the 1-position> C═O group is another optional group such as —O—CO—, —C (OH) (CH ₃ ) —,> C—CH (OH) —CH ₃ , The 1- and 2-position —CO—CH ₂ — is —O—CO— group, the 7-position is any group such as OH, or the propionic acid group is 3-position hydrogen atom is any group such as OH, respectively. A compound having a substituted structure. Examples of the substituent are not limited to the following, but are, for example, halogen (F, Cl, Br, etc.), a lower alkyl group (C ₁ -C ₄ ), halogen, OH or OR (where R is lower alkyl). ) And the like substituted with a lower alkyl group. Examples of analogs are 8aβ-methyl-6-ketoperhydrocoumarin-5α-propionic acid (formula (2)), 1β-oxy-1α, 7aβ-dimethyl-5-ketoperhydroindane represented by the following formula: -4α-propionic acid (formula (3)), 1β- (1-oxyethyl) -7aβ-methyl-5-ketoperhydroindane-4α-propionic acid (formula (4)), etc. .

Further examples of analogs are compounds of formula (11), formula (12) and formula (13) below.

  In the present invention, a bacterial strain in which the metabolism-related gene of the compound of formula (1) or its analog in the steroid-utilizing Comamonas genus genome is disrupted by a DNA recombination technique, particularly a homologous recombination technique, can be used.

  Preferred metabolic-related genes are the steroid metabolism enzyme gene ORF18 of the genome of Comamonas testosteroni TA441, ATCC11996 or their mutants, and the ORF18 homologue of other genomes of the genus Comamonas. The ORF18 homologue means a homologous gene having a function equivalent to that of ORF18 derived from a genus Comamonas other than the Comamonas testosteroni strain TA441, ATCC11996, or mutants thereof.

Below, preparation of the gene disruption strain of the present invention is explained.
In the gene-disrupted strain of the present invention, among the steroid metabolism enzyme gene group of the genus Comonas, more specifically, the testosterone metabolism enzyme gene group, an enzyme involved in metabolism of the compound of the formula (1) or an analog thereof The genomic genes of are destroyed.

  A specific example of a metabolism-related gene to be destroyed is ORF18 of Comamonas testosteroni strain TA441 (M. Horinouchi, T. Hayashi, T. Yamamoto and T. Kudo, Appl. Environ. Microbiol. (2003), 69: 4421). -4430; especially Figure 2). The nucleotide sequence of ORF18 of TA441 strain is shown as SEQ ID NO: 1. The present invention is not limited to this specific example, and the percent identity with the sequence shown in SEQ ID NO: 1 is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least Metabolism-related genes containing sequences that are 97%, at least 98%, at least 99% can also be used for the purposes of the present invention. However, the homologous metabolism-related gene encodes an enzyme involved in the metabolism of indandione propionic acid or its analog of formula (1). All the bacteria of the genus Comamonas having such a homologous gene can be used for the production of a gene-disrupted strain in the present invention. The preferred genus Comamonas is Comamonas testosteroni, such as TA441 strain and related strains thereof, such as ATCC 11996, ATCC 17410, ATCC 15666, ATCC 15667, ATCC 17409, ATCC 17510, ATCC 27911, ATCC 31492. Strains such as ATCC 33083 strains, and gene disruption strains derived from these bacterial strains can be preferably used in the present invention.

In order to investigate that the bacteria of the genus Comamonas have metabolic related genes homologous to SEQ ID NO: 1, radioisotopes such as ³² P and ¹²⁵ I, rhodamine, fluorescamine, their derivatives, and fluorescence such as Cy3 and Cy5 A probe derived from the sequence of SEQ ID NO: 1 labeled with a dye or the like can be prepared, and hybridization with a genomic DNA, mRNA, cDNA or a fragment thereof derived from a genus Comamonas bacterium can be performed.

  The probe is a DNA molecule comprising a sequence complementary to the sequence of SEQ ID NO: 1 or a fragment sequence thereof, and is at least 30 nucleotides (nt), at least 50 nt, at least 70 nt, at least 100 nt, at least 150 nt from the sequence of SEQ ID NO: 1. Is the size of

  Hybridization is not particularly limited, but can be performed by known hybridization techniques such as Southern hybridization, Northern hybridization, in situ hybridization, DNA microarray, etc. (by Hiroki Nakayama et al., Cell Engineering Supplement, Bio-Experimental Illustration) Rated, Basics of Gene Analysis, Shujunsha, 1995; Supervision by Masaaki Matsumura et al., Cell Engineering Separate Volume, DNA Microarray and Latest PCR Method, Shujunsha, 2000). In order to confirm that a highly homologous metabolism-related gene is retained, it is preferable to perform hybridization under stringent conditions.

  Stringent conditions can be low, medium or high stringent conditions depending on differences in conditions such as temperature, ionic strength, and washing conditions, but are preferably high stringent conditions. For hybridization conditions and procedures, reagents and the like, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1989; Ausubel et al., Current Protocols In Molecular Biology, John Wiley & Sons, NY, 1998. And can be referred to in the present invention. A commercially available hybridization reagent may be used. As stringent hybridization conditions, for example, hybridization in 2-6 × SSC (NaCl / sodium citrate) at about 45-50 ° C., followed by about 0.1-1 × at about 50-65 ° C. One or more washes in SSC / about 0.1-0.2% SDS.

  If the hybridization is positive, a restriction map of the bacterial genomic DNA fragment is prepared, the desired metabolism-related gene sequence and, if necessary, its peripheral sequence are determined, and if necessary, the sequence of SEQ ID NO: 1 from TA441 strain is determined. Determine% identity to sequence. For sequencing, based on the restriction map, the desired gene is restricted and inserted into a cloning vector such as a plasmid, phage, cosmid, etc., and Escherichia coli, Bacillus bacteria (for example, Bacillus brevis, Bacillus subtilis), etc. The desired gene can be amplified by a method that involves transforming into a host cell. At this time, a desired gene can be efficiently amplified by utilizing the polymerase chain reaction (PCR).

  As a cloning vector, for example, commercially available M13mp (phage), Charomid9 system (cosmid), pMW system, pBluescript system, pBR322, pUC system, pDEL system (hereinafter referred to as a plasmid) and the like can be used. In addition to the desired gene, the vector can appropriately include a promoter, terminator, start codon, stop codon, replication origin, selectable marker, multiple cloning site, and the like.

  The transformation can be performed using a known transformation technique such as a calcium phosphate method, a calcium chloride method, or electroporation.

  Sequencing can be performed using a conventional method such as the Sanger method or the Maxam-Gilbert method. Commercially available kits such as the CUGA® sequencing kit (Nippon Gene) may be used for sequencing.

  PCR consists of denaturation, annealing, and extension reactions of DNA. Denaturation is a stage in which double-stranded DNA is usually treated at 94 ° C for 15 seconds to 1 minute to dissociate into single-stranded DNA, and annealing is performed on template DNA. Primer annealing stage, usually consisting of 55 ° C, 30 seconds to 1 minute treatment conditions. Extension is a heat-resistant property such as Taq DNA polymerase under primer extension conditions usually 72 ° C, 30 seconds to 10 minutes. The step is carried out using a polymerase, and DNA amplification is carried out by repeating this usually as a cycle for 25 to 40 cycles. The primer is preferably 15 to 30 bases in length. The PCR technique is described in, for example, the Protein Nucleic Acid Encyclopedia, PCR Front Line (1996), Vol. 41, No. 5, Kyoritsu Shuppan, and can be referred to in the present invention.

  Techniques such as cloning, sequencing and PCR of the target gene are specifically described in Sambrook et al. (Above) and Ausbel et al. (Above), and these descriptions can be referred to in the present invention.

  In the present invention, gene disruption is disruption of the metabolism-related gene of indandione propionic acid of the formula (1) or its analog in steroid-utilizing comamonas bacteria. These compounds accumulate in the culture medium. In order to disrupt a gene, the present invention can also employ a mutation method using a mutation reagent such as nitrosoguanidine, but preferably a DNA recombination technique, in particular a homologous recombination technique, is used to obtain the desired gene and / or Part or all of the 5 ′ untranslated region, 3 ′ untranslated region or transcriptional regulatory sequence is disrupted to prevent the related metabolic enzyme encoded by the desired gene from being generated or dysfunctional in vivo. The disruption can be carried out in the sequence of the desired gene, for example by deletion of a part of the sequence (for example, deletion, partial degradation), replacement with heterologous DNA, and / or insertion of heterologous DNA. In a specific example of the present invention, the desired gene is destroyed by inserting an antibiotic resistance gene as a heterologous DNA into the desired gene by homologous recombination. Examples of antibiotic resistance genes are kanamycin resistance gene, ampicillin resistance gene, tetracycline resistance gene and the like. In this method, an antibiotic resistance gene sequence is linked to one or more restriction sites within the desired gene, and the desired gene fragment existing on the 5 ′ and 3 ′ sides of the unique restriction site and linked to each end thereof, and Antibiotic resistance gene sequence to the genome using a plasmid for homologous recombination comprising at least about 0.3 kb, preferably at least about 0.6 kb, more preferably at least about 1 kb of each sequence from its adjacent untranslated region Can be inserted. Examples of plasmids include pGT systems, pBluescript KS (+), pUC systems, pCT systems, and the like. Introduction of the plasmid into the microbial cells can be performed by conventional techniques such as calcium phosphate method and electroporation method. An antibiotic-resistant strain is selected by culturing, and a gene-disrupted strain in which the antibiotic-resistant gene is inserted into the desired gene is obtained. Confirmation of the insertion can be performed by Southern hybridization using probes of the antibiotic resistance gene and the desired gene.

  As for gene disruption, the procedure described in JP-A-2003-61671 can be referred to.

Substance production Comamonas gene-disrupted strain obtained by the above-mentioned method is usually pre-cultured in a medium, and a part or all thereof is added to a medium containing a raw material steroid compound and cultured, and the compound of formula (1) Alternatively, the analog is produced and recovered.

Culturing can be performed under known culture conditions for bacteria of the genus Comamonas.
The medium contains a carbon source, a nitrogen source, inorganic salts, and the like, and either a natural medium or a synthetic medium can be used. In general, any solid or liquid medium for bacteria can be used as the medium. Examples of the medium components include peptone, polypeptone, yeast extract, meat extract, and bactotryptone as carbon and nitrogen sources, and NaCl, KH ₂ PO ₄ , K ₂ HPO ₄ , MgSO ₄ , CaCl ₂ and FeSO as inorganic salts. ₄ , MgSO ₄ , MoO ₃ , ZnSO ₄ , CuSO ₄ , MnSO ₄ , CoCl ₂ , H ₃ BO _3, etc. may contain agar if necessary. Examples of the medium are Luria-Bertani (LB) medium, C medium, LB / C mixed medium, and the like. In the present invention, the raw material steroid compound is further contained in the culture medium, but the content of other carbon sources can be reduced and the content of steroid compounds can be increased so that the steroid is efficiently assimilated as a carbon source. The pH of the medium is preferably 7 to 7.3.

  The gene-disrupted strain of the present invention is obtained by culturing and proliferating preserved bacteria in a solid medium containing the above-mentioned medium components, pre-culturing in a liquid medium having the same components, further increasing the number of bacteria, and then the same liquid medium. Used for substance production in. The amount of bacteria is not particularly limited, but is about 1 to about 10% (w / v) per medium.

As a culture method, either batch type or continuous type culture can be used.
In the case of batch culture, a sterilized medium, a cultured gene disrupted strain or its immobilized cells, and a raw steroid compound are placed in a culture tank, and culture methods such as shaking culture, agitation culture, aeration agitation culture, and deep aeration agitation culture The culture can be performed at a culture temperature of about 25 ° C. to about 35 ° C. for about 15 to about 100 hours.

  In the case of continuous culture, various bioreactors such as a membrane type, a fluidized bed type, a packed bed type, and a horizontal type can be used at the same culture temperature. Specifically, for example, a gene-disrupted strain is charged into one compartment of a culture tank partitioned by a semipermeable membrane such as a hollow fiber membrane, and a sterile medium containing a raw material steroid compound is continuously flowed into the other compartment, A method in which a steroid compound is brought into contact with a gene disruption strain through a semipermeable membrane, or an immobilized gene disruption strain in which the gene disruption strain is immobilized on a carrier is prepared, and the immobilized cells are continuously cultured in a column or the like. A method such as a method of filling or flowing in a tank and continuously flowing a sterilized medium containing a raw material steroid compound into the tank to bring the immobilized cells into contact with the steroid compound can be used.

  Carriers for immobilizing gene disruption strains include porous inorganic particles such as pumice, zeolite and silica gel, organic particles such as polymers such as polystyrene, polyvinyl alcohol, polyacrylamide, unsaturated polyester, unsaturated Examples thereof include epoxides, unsaturated acrylic resins, polyesters of polyethylene glycol and (meth) acrylic acid, and hydrophilic photocurable resins such as urethanized adducts of polyethylene glycol and 2-hydroxyethyl methacrylate. Furthermore, an immobilization carrier as described in Japanese Patent Application No. 07-039376, Japanese Patent Application No. 10-296284, and Japanese Patent Application Laid-Open No. 2003-000237 can also be used. As a method for immobilizing the gene disruption strain on these immobilization carriers, general methods such as an adsorption method, a comprehensive method, and an adhesion growth method can be used. The gene-disrupted strain of the present invention is preferably adsorbed or attached to the pore surface of the carrier, or is embedded in the porous carrier and grown on the spot.

  The raw material steroid compound used in the present invention is not limited as long as it is converted into the compound of the formula (1) or its analog by the gene disruption strain of the present invention. Examples of raw steroid compounds are testosterone, sitosterol, campesterol, 1,4-androstadiene-3,17-dione, 4-androstene-3,17-dione, progesterone, bile acids, testosterone metabolizing intermediate compounds, Or a derivative thereof, but is not limited thereto. Here, the derivative may be one or more substituents at any position of the steroid compound, including, but not limited to, —OH, alkyl, substituted alkyl, cycloalkyl, phenyl, substituted phenyl, halogen, —OR ( R represents a derivative of a steroid compound containing a substituent selected from alkyl or substituted alkyl), -OCOR (R is alkyl or substituted alkyl), and the like. Preferably, androsta-1,4-diene-3,17-dione (formula (5)), androst-4-ene-3,17-dione (formula (6)), or a derivative thereof, Examples thereof include, but are not limited to, cholic acid and its derivatives such as chenodeoxycholic acid and deoxycholic acid.

  Examples of other raw material derivatives are testrolactone (formula (7)), 17-methyltestosterone (formula (8)), 3β, 20β-dioxy-5-pregnene (formula (9)) and the like of the following formula .

  The concentration of the raw steroid is about 0.01% to about 2% (w / v), preferably about 0.1% to 1% (w / v) per culture solution, but is not limited to this range.

  When the raw steroid compound is a compound of formula (5) or (6), a compound of formula (1) is generated. A preferred main product is 7aβ-methyl-1,5-dioxo-8aα-hexahydroindane-4α-propionic acid of the following formula (10), although its diastereomers are also included in the product of the present invention. Shall.

  Further, when the starting steroid compound is cholic acid, chenodeoxycholic acid or deoxycholic acid, preferred main products are the compound of formula (11), the compound of formula (12) or the compound of formula (13), respectively. However, the present invention also includes diastereomers of these compounds.

  As used herein, “diastereomers” means all isomers of the stereoisomers except the enantiomers. For example, when two asymmetric carbons exist in one molecule, there are four possible diastereomers: (S, S), (S, R), (R, S) and (R, R).

  Furthermore, when the starting steroid compound is a derivative represented by formula (7), formula (8), or formula (9) as a derivative of the compound of formula (5) or (6), formula (2), formula It is estimated that the compound of (3) or formula (4) is formed.

After culturing, the culture solution is adjusted to acidic (for example, pH of about 2) with an acid such as hydrochloric acid, and then an ester such as ethyl acetate, a ketone such as acetone and methyl ethyl ketone, and a halogenated hydrocarbon such as chloroform, dichloromethane, and methylene chloride. The desired product can be purified through extraction with an organic solvent such as ether such as ethyl ether and dioxane, concentration and crystallization. Alternatively, the culture solution may be passed through an ion exchange column (H ⁺ ), and after the product is adsorbed on the resin, the product may be eluted with an acid such as acetic acid or hydrochloric acid. Alternatively, the target product can be purified by appropriately combining chromatographic techniques such as HPLC, silica gel chromatography, and reverse phase chromatography.

  The present invention will be described more specifically in the following examples, but the present invention is not limited to these specific examples.

Construction of ORF18 gene disruption strain derived from Comamonas testosteroni TA441 Gene disruption of ORF18 gene (SEQ ID NO: 1) of Comamonas testosteroni TA441 strain by inserting a kanamycin resistance gene (Km ^r ) into the unique XhoI restriction site of the gene did.

Km ^r plasmid for insertion PORF18-Km ^r was constructed in accordance with the procedure of FIG. Specifically, after inserting the plasmid pUC19 to ORF18 gene carrying carbenicillin resistance gene Cb ^r, the ORF18 gene was digested with XhoI, and blunt, which kanamycin resistance gene Km ^r Sma insert (SEQ ID NO: 2) Inserted. This kanamycin resistance gene is derived from transposon Tn5, and in order to make it easy to make a gene cassette, SmaI sites are linked before and after it, and the restriction enzyme sites of PstI, EcoRI, EcoRV, and HindIII are added at the beginning of the resistance gene. Have. The plasmid was introduced into the TA441 strain by electroporation to perform homologous recombination. Homologously recombined genes ORF18-Km ^r (ORF18 sequences inserting the Km ^r in) is shown in SEQ ID NO: 3.

  The ORF18 disrupted strain was cultured on a separate LB medium solid medium containing kanamycin or carbenicillin, and TA441 mutant strain resistant to kanamycin and sensitive to carbenicillin was selected.

Insertion of the Km ^r gene into the ORF18 gene was confirmed by Southern hybridization using the Km ^r Sma insert (SEQ ID NO: 2) and ORF18 (SEQ ID NO: 1) as probes. The hybridization conditions used are as follows.

(reagent)
10% blocking reagent storage solution Blocking reagent 10 g / buffer 100 ml (autoclaved)
Buffer 1: 0.1M maleic acid
0.15M NaCl, pH 7.5 (adjusted with NaOH) (autoclave sterilization)
High SDS hybridization solution
SDS 35g
100% formamide (deionized) 250ml
30 x SSC 83ml
1M sodium phosphate, pH 7.0 25ml
N-lauroyl sarcosine 0.5g
10% blocking reagent stock solution 100ml / distilled water 500ml
2 x cleaning solution: 2 x SSC, 0.1% SDS
0.1 x cleaning solution: 0.1 x SSC, 0.1% SDS

(operation)
Pre-hybridize in high SDS hybridization solution (20 ml / 100 cm ² membrane) at 42 ° C for at least 1 hour, and then perform hybridization at 42 ° C overnight in high SDS hybridization solution (3.5 ml / 100 cm ² membrane). went. The membrane was washed twice with 2 × SSC and 0.1% SDS at room temperature for 5 minutes, and further washed twice with 0.1 × SSC at 42 ° C. for 15 minutes.

(detection)
Rinse the membrane lightly with buffer 1 (0.1M maleic acid, 0.15M NaCl, pH 7.5), and gently shake with 100ml buffer 2 (10% (w / v) blocking reagent stock solution diluted 10-fold) for 30 minutes at room temperature. In the end, blocking was performed. Subsequently, the mixture was gently shaken with a diluted labeled antibody solution (alkaline phosphatase labeled anti-digoxigenin antibody 150 mU / ml buffer 2) at room temperature for 30 minutes. It was washed twice with Buffer 1 for 15 minutes each and equilibrated with 20 ml of Buffer 3 (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl) for 2 minutes. 10 ml of a coloring reagent (NBT solution 45 μl + X-phosphate solution 35 μl / Buffer 3 10 ml) was added and left in the dark. When a band was detected, the reaction was stopped by washing with 50 ml of TE for 5 minutes. The NBT solution is nitroblue tetrazolium salt 75 mg / ml 70% DMF, and the X-phosphate solution is 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt 50 mg / ml DMF.

  The ORF18 gene-disrupted strain derived from Comamonas testosteroni TA441 was prepared by the above method.

Production of 7aβ-methyl-1,5-dioxo-8aα-hexahydroindane-4α-propionic acid by ORF18 gene-disrupted strain The ORF18 gene-disrupted strain obtained in Example 1 was cultured in an LB test tube medium [polypeptone 10 g; NaCl 5 g; yeast extract 5 g; pH 7.0 (adjusted with NaOH) / 1 L distilled water] 10 ml × 3 pieces were cultured at 30 ° C. overnight. The culture solution was placed in a 500 ml Erlenmeyer flask without baffle with LB medium and C medium [(NH ₄ ) ₂ SO ₄ 5.0 g; K ₂ HPO ₄ 5.87 g; KH ₂ PO ₄ 2.93 g; MgSO ₄ · 7H ₂ O (× 100 10 ml; NaCl 2 g; yeast extract 0.2 g; 10 g / L CaCl ₂ solution 1 ml (final 10 mg / L); 10 g / L FeSO ₄ solution 1 ml (final 10 mg / L); trace element solution (× 100) 20 μl; pH 7.0 (adjusted with NaOH) / 1 L distilled water; MgSO ₄ .7H ₂ O (× 100): MgSO ₄ .7H ₂ O 30 g / L; Trace element solution (× 100): MoO ₃ 0.1 g, ZnSO ₄ · 5H ₂ O 0.7 g, CuSO ₄ · 5H ₂ O 0.05 g, H ₃ BO ₃ 0.1 g, MnSO ₄ · 5H ₂ O0.1 g, CoCl ₂ · 6H ₂ O 0.1 g / 1L distilled water) Inoculate 6 ml of 5 medium each containing 50 ml of 100 ml, add 0.1% (w / v) 1,4-androstadiene-3,17-dione, shake speed 80 rpm, culture temperature 30 Culturing was performed at 4 ° C for 4 days.

  The culture solution (100 ml × 5) was centrifuged, the supernatant was adjusted to pH 2 using 6N HCl solution, and extracted twice with 600 ml of ethyl acetate. The ethyl acetate fractions were combined, dehydrated and concentrated to give 560 mg of a pale blue oily residue.

The HPLC analysis results of the culture supernatant are shown in Fig. 2-1, and the HPLC analysis results of the ethyl acetate fraction are shown in Fig. 2-2. The HPLC analysis conditions are as follows.
Instrument: Waters 2690 HPLC
Column: Inertsil ODS-3 (2.1 x 150 mm)
Developing solvent: Solution A (CH ₃ CN: CH ₃ OH: TFA = 95: 5: 0.05), Solution B (H ₂ O: CH ₃ OH: TFA = 95: 5: 0.05)
Gradient: 20% solution A + 80% solution B to 65% solution A + 35% solution B gradient 10 minutes; hold for 3 minutes; change to 20% solution A in 5 minutes Temperature: 40 ° C
Flow rate: 0.21ml / min

560 mg of a pale blue oily residue was dissolved in a small amount of methanol, and a peak of 322 mg of the desired product was fractionated using HPLC. The HPLC conditions are as follows.
HPLC (preparative conditions):
HPLC: Waters 600 HPLC
Column: Inertsil ODS-3 column (20 x 250 mm)
Flow rate: 8 ml / min
Temperature: 40 ° C
Developing solvent 20% solution A + 80% solution B to 65% solution A + 35% solution B changed in 25 minutes, held for 10 minutes, then changed to 20% solution A in 5 minutes.
Solution A (CH ₃ CN: CH ₃ OH: TFA = 95: 5: 0.05)
Solution B (H ₂ O: CH ₃ OH: TFA = 95: 5: 0.05)
Detection wavelength: 210nm

The results of HPLC analysis of the target product purified by HPLC are shown in Fig. 2-3.
As a result of FAB-MS (apparatus: JEOL JMS-SX 102), NMR (apparatus: JNM-ECP500, JNM-ECA600 (manufactured by JEOL)) and UV analysis, the product obtained was 7aβ- It was methyl-1,5-dioxo-8aα-hexahydroindane-4α-propionic acid (yield 76.8%).

(result of analysis)
NMR analysis (CDCl ₃ or CD ₃ OD):

FAB-MS analysis: Experimental value: m / z 239.1297 [M + H] ⁺ ; Calculated value: m / z 239.1283 (C ₁₃ H ₁₈ O ₄ )
UV analysis: 290 nm (e: 58) (MeOH)

Production of compound of formula (11) from cholic acid by ORF18 gene disruption strain
The ORF18 gene-disrupted strain prepared in Example 1 using 0.1% (w / v) concentration of cholic acid as a substrate was cultured in medium LB + C 100 ml × 5, shaking number 80 rpm, culture temperature 30 ° C. for 3 days. did.

The culture solution (500 ml) was centrifuged (6500 rpm), and the supernatant was extracted once with 550 ml of ethyl acetate, then the aqueous layer was adjusted to pH 2 with 6N HCl solution, and extracted again three times with 550 ml of ethyl acetate. The acetic acid ethyl ester fraction was dehydrated and concentrated to obtain 460 mg of residue. This was fractionated and purified using HPLC.
HPLC (preparative conditions):
HPLC: Waters 600 HPLC
Column: Inertsil ODS-3 column (20 x 250 mm)
Flow rate: 8 ml / min
Temperature: 40 ° C
Developing solvent 20% solution A + 80% solution B to 65% solution A + 35% solution B changed in 25 minutes, held for 10 minutes, then changed to 20% solution A in 5 minutes.
Solution A (CH ₃ CN: CH ₃ OH: TFA = 95: 5: 0.05)
Solution B (H ₂ O: CH ₃ OH: TFA = 95: 5: 0.05)
Detection wavelength: 210nm
The accumulated product was contained in the fractions (Fr) 3 and Fr4 collected, and the weight of the concentrated and dried Fr3 (oily substance) was 68 mg.

  The UV spectrum of Fr3 showed a maximum absorption at 260 nm. As a result of NMR measurement in deuterated methanol, it was confirmed to be an analog having one OH group at the 7-position of the compound of the formula (1) and the 3-position of its propionic acid group. That is, an analog (formula (11)) in which the OH groups at positions 7 and 12 which were substituents of cholic acid used as a substrate remained as it was was obtained.

The analysis results are as follows.
HPLC (analysis conditions, 3D):
HPLC: Waters 2690 HPLC
Column: Inertsil ODS-3 column (4.6 x 250 mm)
Flow rate: 1 ml / min
Temperature: 40 ° C
Developing solvent: 20% solution A + 80% solution B to 65% solution A + 35% solution B changed in 10 minutes, held for 3 minutes, then changed to 20% solution A in 2 minutes. Return to the initial state 20% solution A + 80% solution B from 15 minutes.

The metabolite showed a characteristic peak at RT 3.9 min.
NMR analysis (CDCl ₃ or CD ₃ OD):

HR-FAB (negative) measurement result:
Experimental value: m / z 269.1032 [MH] ^- ; Calculated value: m / z 269.1025 (C ₁₃ H ₁₇ O ₆ )

Production of compound of formula (12) from chenodeoxycholic acid by ORF18 gene disruption strain
Using the chenodeoxycholic acid at a concentration of 0.1% (w / v) as a substrate, the ORF18 gene-disrupted strain prepared in Example 1 was cultured in medium LB + C 100 ml × 10, shaking number 80 rpm, culture temperature 30 ° C. for 4 days. did.

The culture solution (1000 ml) was centrifuged (6500 rpm), the supernatant was adjusted to pH 2 with 6N HCl solution, and extracted twice with 1100 ml of ethyl acetate (EA). The EA fraction was dehydrated and concentrated to obtain 1100 mg of residue. This was separated and purified using HPLC (FIGS. 3-1 and 3-2).
HPLC (preparative conditions):
The same conditions and apparatus as in Example 3 were used except that the conditions of the developing solvent were partially different.
Developing solvent 10% solution A + 90% solution B to 65% solution A + 35% solution B for 25 minutes; hold for 10 minutes; change to 20% solution A in 5 minutes.
Solution A (CH ₃ CN: CH ₃ OH: TFA = 95: 5: 0.05)
Solution B (H ₂ O: CH ₃ OH: TFA = 95: 5: 0.05)

  As a result of concentrating each fraction collected by HPLC and measuring the weight, the largest accumulation was 138 mg of the fraction (oil) of retention time (RT) 5.2 min, followed by 40 mg of RT7.0, and others This fraction was around 10 mg, and the peak RT5.2 having the largest weight was considered to be an accumulation of this strain.

The UV spectrum of RT5.2 in HPLC solvent showed a maximum absorption at 286.6 nm.
Further, the RT5.2 fraction was concentrated, dried, and subjected to NMR measurement in CDCl _3. As a result, it was found that it was the compound (12) (molecular formula C ₁₃ H ₁₈ O ₅ and molecular weight 254) (Table 3). .

The analysis results are as follows.
HPLC (analysis conditions, 3D):
HPLC: Waters 2690 HPLC
Column: Inertsil ODS-3 column (4.6 x 250 mm)
Flow rate: 1ml / min
Temperature: 40 ° C
Developing solvent: Change from 20% solution A + 80% solution B to 65% solution A + 35% solution B in 10 minutes, hold for 3 minutes, then change to 20% solution A in 2 minutes. Return to the initial state 20% solution A + 80% solution B from 15 minutes.

The metabolite showed a characteristic peak at RT 5.2 min.
NMR analysis (CDCl ₃ ):

  As a result of measuring and analyzing the NMR spectrum in the same manner, the RT7.0 accumulation showing the second weight was identified as the same compound of the formula (10) as obtained in Example 2.

  In the above examples, the HPLC charts shown in FIGS. 2-1, 2-2, 2-3, 3-1, and 3-2 are all analytical results obtained by analytical HPLC (three-dimensional Waters 2690). RT in the text indicates the retention time in 3D HPLC.

  The method of the present invention is advantageously used for industrial production of a compound of the above formula (1) or an analog thereof as a steroid synthesis intermediate for pharmaceuticals, health foods, foods, cosmetics, hair restorers, tonics, etc. can do.

Shows construction diagram of plasmid for disruption pORF18-Km ^r. The HPLC analysis chromatogram of the centrifugation supernatant of the culture solution containing the conversion product of 1,4-androstadiene-3,17-dione by the ORF18 gene disruption strain is shown. The arrow indicates the compound of formula (10). The HPLC analysis chromatogram of the fraction obtained by further extracting the centrifugal supernatant with ethyl acetate is shown. The arrow indicates the compound of formula (10). 1 shows an HPLC analysis chromatogram of a purified compound of formula (10) (7aβ-methyl-1,5-dioxo-8aα-hexahydroindane-4α-propionic acid) (arrow). The HPLC analysis chromatogram of the centrifugation supernatant of the culture solution containing the conversion product of chenodeoxycholic acid by the ORF18 gene disruption strain is shown. The RT5.2 arrow indicates the compound of formula (12), and the RT7.0 arrow indicates the compound of formula (10). The HPLC analysis chromatogram of the fraction obtained by further extracting the centrifugal supernatant with ethyl acetate is shown. The RT5.2 arrow indicates the compound of formula (12), and the RT7.0 arrow indicates the compound of formula (10).

SEQ ID NO: 2: Km ^r Sma insert SEQ ID NO: 3: ORF18-Km ^r

Claims (14)

The following formula (1)

A steroid-utilizing Comamonas genus bacterium in which the metabolism-related gene of the compound or its analog is disrupted is contacted with a steroid compound in a culture medium, and the compound of the above formula (1) or its analog is simply produced. A method for producing a compound of the formula (1) or an analogue thereof, comprising releasing.   The method according to claim 1, wherein the genus Comamonas is Comamonas testosteroni.   The method according to claim 2, wherein the Comamonas testosteroni is Comamonas testosteroni TA441 strain, ATCC11996 strain or a mutant thereof.   The method according to claim 1, wherein the metabolism-related gene is the steroid metabolism enzyme gene ORF18 of the Comamonas testosteroni TA441 strain, ATCC11996 strain or a mutant strain thereof.   The method according to claim 1, wherein the metabolic-related gene is an ORF18 homologue of the genome of the genus Comamonas.   The method according to claim 1, wherein the steroid compound is a testosterone metabolic intermediate compound or a derivative thereof.   7. The method of claim 6, wherein the testosterone metabolizing intermediate compound is androsta-1,4-diene-3,17-dione or androst-4-ene-3,17-dione.   The method according to claim 6, wherein the steroid compound is cholic acid, chenodeoxycholic acid or deoxycholic acid. The analog of the compound of the formula (1) is the following formula (11), formula (12) or formula (13)

The method according to claim 1, wherein   The method according to any one of claims 1 to 9, wherein the genus Comamonas is immobilized on a carrier.   The method according to any one of claims 1 to 10, wherein the contact between the bacterium and the steroid compound is carried out batchwise or continuously. A compound of the following formula (11) or a diastereomer thereof.

A compound of the following formula (12) or a diastereomer thereof.

A compound of the following formula (13) or a diastereomer thereof.

Images