JP5601788B2 - Ketreductase mutant - Google Patents

Description

  The present invention relates to a ketoreductase mutant used in a fermentation production method by microorganisms of a daunorubicin derivative produced semisynthetically.

  Anthracycline antibiotics are a group of compounds of aromatic polyketides, including aglycone part composed mainly of 7,8,9,10-tetrahydro-5,12-naphthacenequinone (below) and sugars mainly composed of amino sugars. It is a pigment glycoside (glycoside) composed of

  Anthracycline antibiotics bind to DNA and generate radicals that break the DNA strand or inhibit topoisomerase II. Topoisomerase has DNase and ligase activity and catalyzes the temporary cleavage and recombination of DNA strands, but anthracycline antibiotics inhibit topoisomerase II, thereby impairing DNA replication and exhibiting antitumor activity. To do. Anthracycline antibiotics are positioned as useful anticancer agents because they exhibit antitumor activity against a wide range of cancer cells, although accumulative cardiotoxic effects are observed.

  The main anthracycline anticancer agents currently in use include those derived from fermentation products such as daunorubicin and semi-synthetic products derived from daunorubicin such as doxorubicin and epirubicin.

  Epirubicin is superior to daunorubicin and doxorubicin in terms of antitumor activity and toxicity, but the yield of the chemical synthesis process for reversing the 4-position hydroxyl group of the amino sugar moiety is low, and currently, epirubicin is the starting material for daunorubicin. Although it is produced, there are many problems in terms of manufacturing cost. On the other hand, by introducing a gene encoding ketoreductase (epi-type ketoreductase) whose stereospecificity is different from that of ketoreductase involved in daunorubicin biosynthesis, the biosynthesis pathway of daunorubicin is altered and epithelial A study of direct fermentation production of daunorubicin has been reported (Non-patent Document 1). Since epidaunorubicin has the same conformation of the hydroxyl group of the amino sugar moiety as epirubicin, epidaunorubicin can be a very useful starting material in producing epirubicin. Although it was reported that it was the highest when the epi type ketoreductase gene (avrE) involved in avermectin biosynthesis was introduced, its production amount was only about 54 μg / mL, and it did not reach a practical level. In addition, a patent application has been filed that improves the productivity of epidaunorubicin to 100 μg / mL or more by treating the epidaunorubicin-producing bacterium obtained in this study with a mutagen (Patent Document 1). Details regarding the mutants are not described in the examples. On the other hand, when the present inventors introduced a ketoreductase gene (evaE) involved in epivancosamine biosynthesis, which is an amino sugar contained in chlororemomycin, the production amount of epidaunorubicin is 2.7 compared to the case where the avrE gene is introduced. A patent application was filed as a result of a double improvement (Patent Document 2).

International Publication No. W02006 / 111561 Pamphlet International Publication No. WO2009 / 035107 Pamphlet

“Mature Biotechnology”, (USA), 1998, Vol. 16, pages 69-74, by Madduri, K. et al. Lipman DJ and Pearson WR, Science, (USA), 1985, 227, 1435-1441 Lipman DJ and Pearson WR, "Proceedings of the National Academy of Sciences" of the United States of America), (USA), 1988, Vol. 85, p.2444-2448 By Schmitt-John, T. and Engels, JW, "Applied Microbiology and Biotechnology", (Germany), 1992, Vol. 36, p.493-498 Bibb, M.J. et al., “Molecular Microbiology”, (UK), 1994, Vol. 14, pp.533-545 "Practical Streptomyces Genetics", "The John Innes Foundation", (UK), Norwick, 2000, p.311-338 Bibb, M.J. et al., “Gene” (UK), 1985, 38, p215-226 Komiyama, T. et al., “The Journal of Antibiotics”, (Japan), 1977, Vol. 30, p.619-621

  An object of the present invention is to provide a ketoreductase variant modified to improve the productivity of a daunorubicin derivative with respect to the ketoreductase used in the direct fermentation production method of a daunorubicin derivative such as epidaunorubicin by a microorganism. .

  As a ketoreductase used in the direct fermentation production method of a daunorubicin derivative by a microorganism, the present inventors have intensively studied the modification of ketoreductase (EvaE) comprising the amino acid sequence described in SEQ ID NO: 1, and the 42nd and 149th positions. Discovered that the productivity of daunorubicin derivatives is improved by using a ketoreductase mutant in which at least one amino acid selected from the 153rd, 270th, and 306th amino acid groups is substituted with another amino acid. The present invention has been completed.

  That is, the present invention provides a ketoreductase mutant that can be used for direct fermentation production of an efficient daunorubicin derivative by a microorganism and a polynucleotide (particularly DNA) encoding the mutant. The present invention also includes a step of providing a transformant with DNA encoding the ketoreductase mutant of the present invention, culturing the transformant, and collecting a daunorubicin derivative from the obtained culture medium. A method for producing a daunorubicin derivative is provided. Furthermore, the present invention provides a daunorubicin derivative produced by the transformant of the present invention.

Accordingly, the present invention provides the following:
(1) A mutant of a ketoreductase enzyme that can be used for the fermentation production of a daunomycin derivative, wherein the mutation is an insertion, substitution, or deletion of one or more amino acids in the amino acid sequence of the parent ketoreductase, or A ketoreductase variant that is an addition to one or both ends and improves the productivity of a daunomycin derivative as compared to the use of the parent ketoreductase.
(2) The ketoreductase variant according to (1), wherein the parent ketoreductase comprises an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1.
(3) One or two or more amino acid residues selected from the group consisting of amino acids at positions corresponding to amino acids 42, 149, 153, 270 and 306 in the amino acid sequence described in SEQ ID NO: 1, The ketoreductase mutant according to (2), which is substituted with an amino acid residue of
(4) The ketoreductase variant according to (3), wherein the amino acid at the position corresponding to the 42nd amino acid in the amino acid sequence described in SEQ ID NO: 1 is substituted with leucine.
(5) The ketoreductase variant according to (3), wherein the amino acid at the position corresponding to the 149th amino acid in the amino acid sequence described in SEQ ID NO: 1 is substituted with serine.
(6) The ketoreductase variant according to (3), wherein the amino acid at the position corresponding to the 153rd amino acid in the amino acid sequence described in SEQ ID NO: 1 is substituted with an amino acid residue other than proline.
(7) The ketoreductase variant according to (3), wherein the amino acid at the position corresponding to the 270th amino acid in the amino acid sequence described in SEQ ID NO: 1 is substituted with arginine.
(8) The ketoreductase variant according to (3), wherein the amino acid at the position corresponding to the 306th amino acid in the amino acid sequence described in SEQ ID NO: 1 is substituted with aspartic acid.
(9) The ketoreductase mutant according to any one of (1) to (8), wherein the daunomycin derivative is epidaunomycin.
(10) A polynucleotide encoding the ketoreductase variant according to any one of (1) to (9).
(11) A transformant in which the DNA according to (10) is introduced into an actinomycete host originally having the ability to produce daunomycin and imparted with the ability to produce a daunomycin derivative.
(12) The transformant according to (11), wherein the actinomycete of the host is Streptomyces coeruleorubidus .
(13) A method for producing a daunomycin derivative, comprising a step of culturing the transformant according to (12) and collecting the daunomycin derivative from the culture solution.
(14) A daunomycin derivative obtained by the production method according to (13).

  According to the ketoreductase mutant of the present invention, the productivity of the daunorubicin derivative can be improved.

  The ketoreductase mutant of the present invention can be obtained as a result of mutating the parent ketoreductase. Further, in the present invention, this mutation is an insertion, substitution, or deletion of one or more amino acids, or addition to one or both ends in the amino acid sequence of ketoreductase, and This means that when the reductase mutant is used in a method for fermentative production of a daunorubicin derivative, the productivity of the daunorubicin derivative is improved as compared with the case where the parent ketoreductase is used.

  In the present invention, the parent ketoreductase is not limited as long as it can be used in the fermentation production method of a daunorubicin derivative, but ketoreductase consisting of the amino acid sequence shown in SEQ ID NO: 1 or a homologue thereof is preferable. As used herein, the “homologue thereof” refers to an amino acid sequence represented by SEQ ID NO: 1 in which several amino acid insertions, substitutions or deletions, or additions to one or both ends thereof are made. And having at least 90% identity with the amino acid sequence represented by the formula (1) and retaining its ketoreductase activity. The “identity” mentioned here is a homology search program known to those skilled in the art [FASTA3; http://fasta.ddbj.nig.ac.jp/top-j.html] “Science, 227, 1435. -1441 (1985); Proc.Natl.Acad.Sci.USA, 85,2444-2448 (1988) (Non-Patent Documents 2 and 3) "is a numerical value calculated using default (initial setting) parameters . The presence or absence of ketoreductase activity can be determined by introducing and expressing a gene encoding a homologue in an appropriate host described later and examining the presence or absence of production of a daunorubicin derivative. It is clear that such “homologues” can be selected and produced without difficulty by those skilled in the art with reference to the sequence shown in SEQ ID NO: 1.

  According to a preferred embodiment of the present invention, when the parent ketoreductase consists of the amino acid sequence shown in SEQ ID NO: 1, it is selected from the group consisting of amino acids 42, 149, 153, 270, and 306 in the sequence Specific examples thereof include mutants in which one or two or more amino acid residues are substituted with other amino acid residues.

  According to a preferred embodiment of the present invention, the amino acid at position 42 is leucine, the amino acid at position 149 is serine, the amino acid at position 153 is amino acid other than proline, the amino acid at position 270 is at arginine, and the amino acid at position 306 is asparagine. The thing substituted by the acid is mentioned as a preferable thing. These mutants have an advantageous property that the productivity of the daunorubicin derivative is improved when used in the fermentation production method of the daunorubicin derivative.

  When the parent ketoreductase is a homologue of the amino acid sequence shown in SEQ ID NO: 1, it is selected from the group consisting of amino acids at positions corresponding to the 42nd, 149, 153, 270, and 306th amino acids. Specific examples thereof include those in which one or two or more amino acid residues are substituted with other amino acid residues. Here, the position of the amino acid residue to be substituted in the homologue of the parent ketoreductase consisting of the amino acid sequence shown in SEQ ID NO: 1 can be easily selected by comparing amino acid sequences using a known algorithm. On the other hand, when it is difficult to compare amino acid sequences by a known algorithm, the position of the amino acid residue to be substituted can be specified by comparing the three-dimensional structure of the enzyme.

A transformant producing a daunorubicin derivative can be obtained by introducing a DNA encoding the ketoreductase mutant of the present invention into an appropriate host originally having the ability to produce daunorubicin. A preferred host is actinomycetes, and known actinomycetes that produce daunorubicin include Streptomyces peuceticus and Streptomyces coeruleorubidus , which are ketoreductases of the present invention. It can be used as a host for introducing a DNA encoding a mutant. Baumomycin is a substance in which the amino sugar (L-daunosamine) portion of daunorubicin is modified, and daunorubicin is an intermediate of biosynthesis, so that actinomycetes that produce baumomycin can also be used as hosts. In addition, in the above-mentioned daunorubicin or baumycin-producing bacterium, it is preferable to use a strain that has lost daunorubicin-producing ability due to the lack of the ketoreductase gene involved in the 4-position hydroxyl group biosynthesis of the L-daunosamine part of daunorubicin.

  The gene can be introduced into the host using conventional techniques such as mixing protoplasts with the target DNA, using phage, and using conjugative transfer. Can be implemented. In order to select a strain into which the gene has been introduced, it is desirable to introduce the target epitype ketoreductase gene together with a vector containing a selection marker gene. The selection marker gene is not limited as long as it can select a strain into which an epi-type ketoreductase gene has been introduced, but a kanamycin resistance gene, a streptomycin resistance gene, a hygromycin resistance gene, a biomycin resistance gene, an apramycin resistance gene, or the like is preferable. It is preferable to add a promoter sequence that functions in the host to the epi type ketoreductase gene to be introduced, and an ermE * promoter derived from an erythromycin resistance gene [Schmitt-John, T. and Engels, JW] "Applied Microbiology and Biotechnology", (Germany), 1992, Vol. 36, p.493-498 (Non-Patent Document 4)], [Bibb MJ) et al., “Molecular Microbiology”, (UK), 1994, Vol. 14, pp. 533-545 (Non-patent Document 5)] and the like. The presence state of the epi-type ketoreductase gene in the host may be incorporated into either a plasmid or a chromosome capable of self-replication outside the chromosome. Alternatively, the epi-type ketoreductase gene may be introduced by a method of replacing the host ketoreductase gene involved in the 4-position hydroxyl group biosynthesis of the L-daunosamine part of daunorubicin. Methods commonly used in actinomycetes can be used to replace genes [Practical Streptomyces Genetics], “The John Innes Foundation” ], (UK), Norwick, 2000, p. 311-338 (Non-Patent Document 6)].

  The daunorubicin derivative produced by the transformant of the present invention is a daunorubicin derivative in which the 4-position hydroxyl group of the L-daunosamine part of daunorubicin is inverted. Epidaunorubicin and epirubicin are preferable, and epidaunorubicin is more preferable.

  The transformant of the present invention can be cultured by a conventional method to produce a daunorubicin derivative. As the medium, conventional components such as glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal / vegetable oil, etc. can be used as the carbon source. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, polypeptone, malto extract, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid (dipotassium hydrogen phosphate, etc.), sulfuric acid (magnesium sulfate, etc.) and other inorganic salts that can generate ions are added as necessary. Is also effective. In addition, various vitamins such as thiamine (thiamine hydrochloride, etc.), amino acids such as glutamic acid (sodium glutamate, etc.), asparagine (DL-asparagine, etc.), micronutrients such as nucleotides, antibiotics, etc. are added as necessary. You can also Furthermore, organic substances and inorganic substances that assist the growth of bacteria and promote the production of daunorubicin derivatives can be appropriately added.

  The pH of the medium is, for example, about pH 5.5 to pH 8. As a culture method, a solid culture method under aerobic conditions, a shaking culture method, an aeration and agitation culture method or a deep aerobic culture method can be used, and the deep aerobic culture method is particularly suitable. A suitable temperature for culturing is 15 ° C. to 40 ° C., but in many cases grows at around 25 ° C. to 35 ° C. The production of daunorubicin derivatives varies depending on the medium and culture conditions, or the host used, but in any culture method, the accumulation usually reaches its maximum in 2 to 10 days. When the amount of daunorubicin derivative in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture.

In order to collect the daunorubicin derivative from the culture obtained by culturing the transformant of the present invention, the usual separation means utilizing its properties, for example, solvent extraction method, ion exchange resin method, adsorption or distribution column chromatography Extraction and purification can be performed alone or in appropriate combination with a method, a gel filtration method, a dialysis method, a precipitation method, a crystallization method and the like. In order to further purify the daunorubicin derivative, chromatography using an adsorbent such as silica gel or alumina, Sephadex LH-20 (Pharmacia), Toyopearl HW-40 (Tosoh) or the like may be performed.
Examples of the present invention will be described below, but this is merely an example and is not intended to limit the present invention, and encompasses all of the many variations or modification means not exemplified here.

Introduction of mutations into the ketoreductase gene (evaE) of a chlororemomycin-producing bacterium ( Amycolatopsis orientalis )
Plasmid pIJ4070 [Bibb, MJ et al., “Gene”, (UK), 1985, Vol. 38, p215-226 (Non-patent Document 7)] containing the ermE * promoter was constructed using Eco RI and Bam. After double digestion with HI and fractionation by electrophoresis, an approximately 0.3 kbp Eco RI- Bam HI fragment containing the ermE * promoter was extracted from the gel. This DNA fragment was inserted into the Eco RI and Bam HI sites of plasmid pSET152 to obtain plasmid pSET152-E *.

Chloro about Remo mycin-producing bacterium ketoreductase gene (Amycolatopsis orientalis) (evaE) is a Bam HI- Xba I fragment comprising the base sequence represented by SEQ ID NO: 2 and total synthesis, plasmid pSET152-E * of Bam HI and The plasmid pEVA-E was obtained by inserting into the Xba I site. Using this plasmid pEVA-E as a template and using GeneMorph II Random Mutagenesis Kit (Stratagene) according to the attached manual, primer pSET153-R (5'-GCGGATAACAATTTCACA-3 ', SEQ ID NO: 3) and pSETermE-R (5'- A random mutation was introduced by a combination of GTGCGGGCCTCTTCGCTATT-3 ′ and SEQ ID NO: 4).

The evaE fragment mutation is double digested with Bam HI and Xba I, and a genomic DNA library cloned into the Bam HI and Xba I sites of pSET152-E *.

Escherichia coli ET12567 / pUZ8002 carrying this genomic DNA library was prepared in 100 mL of LB liquid medium containing chloramphenicol, kanamycin and apramycin at concentrations of 25 μg / mL, 25 μg / mL and 50 μg / mL, respectively ( 1% diffcobact tryptone, 0.5% diffco yeast extract, 0.5% NaCl, 0.1% glucose) and cultured at 37 ° C. overnight to prepare a preculture solution. Inoculate the pre-culture solution in the same liquid medium as the pre-culture at a final concentration of 1%, incubate at 37 ° C for about 4 hours, wash twice with LB liquid medium, and finally 10 mL of LB E. coli solution was suspended in a liquid medium.

A dnmV gene-disrupted strain of Streptomyces coeruleorubidus (Patent Document 2: International Publication No. WO2009 / 035107 pamphlet) of a daunorubicin-producing bacterium, MS agar medium (2% S soy flour, 2% mannitol, 2% agar) ) And cultured at 28 ° C. for 4 days. After culturing, spores were scraped off with 3 mL of 20% glycerin solution to prepare a host spore solution.

The cells were collected by mixing 500 μL of the host spore solution and 500 μL of the E. coli solution prepared as described above, and then applied to an MS agar medium supplemented with MgCl ₂ to a final concentration of 10 mmo1 / L. After culturing at 28 ° C. for 20 hours, 1 mL of sterile water containing 1 mg of apramycin and 1.5 mg of nalidixic acid was overlaid, and further cultured at 28 ° C. for 5 days to obtain an apramycin resistant strain.

In order to confirm the productivity of epidaunorubicin for these strains, liquid production medium prepared in 8-minute test tubes [Komiyama, T. et al., “The Journal of Antibiotics ], (Japan), 1977, Vol. 30, p.619-621 (Non-patent Document 8)] Inoculate 10 mL, incubate at 28 ° C for 2 days, and then prepare 1 mL of the culture solution in a 250 mL Erlenmeyer flask Was inoculated into 20 mL of the same liquid production medium and cultured with shaking at 32 ° C. for 7 days. To extract the bacterial cell product, put 1 mL of culture solution, 1 mL of methanol, 70 μL of 50% H ₂ SO ₄ in a 15 mL centrifuge tube, shake for 1 hour, and cool overnight, 2000 × g The supernatant was centrifuged for 10 minutes and subjected to HPLC analysis.

  Genomic DNA was prepared from the resulting highly productive clone using a MagExtractor genomic DNA extraction device (manufactured by Toyobo Co., Ltd.) according to the protocol, and primers pSET153-R (SEQ ID NO: 3) and pSETermE-R (SEQ ID NO: 4) were prepared. ) And PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) under the following cycle conditions (98 ° C., 10 seconds, 60 ° C., 5 seconds, 72 ° C., 1 minute × 25 times). As a result, an amplified DNA fragment of about 1 kbp was obtained. As a result of analyzing the nucleotide sequence of this DNA fragment, clones showing high productivity were identified as Q42L (42th glutamine was replaced with leucine), K153T (153th lysine was replaced with threonine), C270R (270th). It was revealed that there was an amino acid substitution of cysteine replaced with arginine.

Saturation Mutation Introduction into Plasmid pEVA-E Using the plasmid pEVA-E prepared in Example 1 as a template, PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) with the following primer set (98 ° C, PCR reaction was performed by 10 times, 68 ° C., 7 minutes) × 25 cycles. The capital letters in the primer base sequence indicate the introduction site of saturation mutation.
[1] NAC-F
5'-gcggaacagatcctcaagNACgccacggcaaatggccag-3 '(SEQ ID NO: 5)
NAC-R
5'-ctggccatttgccgtggcGTNcttgaggatctgttccgc-3 '(SEQ ID NO: 6)
[2] NCC-F
5'-gcggaacagatcctcaagNCCgccacggcaaatggccag-3 '(SEQ ID NO: 7)
NCC-R
5'-ctggccatttgccgtggcGGNcttgaggatctgttccgc-3 '(SEQ ID NO: 8)
[3] NGC-F
5'-gcggaacagatcctcaagNGCgccacggcaaatggccag-3 '(SEQ ID NO: 9)
NGC-R
5'-ctggccatttgccgtggcGCNcttgaggatctgttccgc-3 '(SEQ ID NO: 10)
[4] NTC-F
5'-gcggaacagatcctcaagNTCgccacggcaaatggccag-3 '(SEQ ID NO: 11)
NTC-R
5'-ctggccatttgccgtggcGANcttgaggatctgttccgc-3 '(SEQ ID NO: 12)
[5] VAG-F
5'-gcggaacagatcctcaagVAGgccacggcaaatggccag-3 '(SEQ ID NO: 13)
VAG-R
5'-ctggccatttgccgtggcCTBcttgaggatctgttccgc-3 '(SEQ ID NO: 14)
[6] TGG-F
5'-gcggaacagatcctcaagTGGgccacggcaaatggccag-3 '(SEQ ID NO: 15)
TGG-R
5'-ctggccatttgccgtggcCCActtgaggatctgttccgc-3 '(SEQ ID NO: 16)
[7] ATG-F
5'-gcggaacagatcctcaagATGgccacggcaaatggccag-3 '(SEQ ID NO: 17)
ATG-R
5'-ctggccatttgccgtggcCATcttgaggatctgttccgc-3 '(SEQ ID NO: 18)

To the obtained PCR product, 1 μL of Dpn I (20 units or less) was added and digested at 37 ° C. for 1 hour. Thereafter, Escherichia coli DH5α (manufactured by Takara Bio Inc.) was transformed with 1 μL of the Dpn I digested product, applied to an apramycin- added LB agar plate, and cultured at 37 ° C. overnight.

  Using LaTaq polymerase (manufactured by Takara Bio Inc.), the colonies that grew grew at 94 ° C for 5 minutes (94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute 30 seconds) x 25 cycles Colony PCR reaction was performed. The PCR reaction product was purified using a high-purity PCR product purification kit (Roche) and the mutation site was confirmed. After confirming that the mutation corresponding to all amino acid substitutions was obtained for the 153rd amino acid, a plasmid containing the mutated gene was prepared using QIAprep Spin Miniprep Kit (manufactured by QIAGEN).

When these plasmids were used to transfer to the dnmV- disrupted strain described in Example 1 by conjugation transfer, and the productivity of epidaunorubicin was confirmed for the resulting strain, productivity was improved by amino acid substitution other than proline compared to the wild strain. It was confirmed that there was an improvement.

Preparation and Evaluation of K153T and Q149S Double Mutants Primers pSET153-R (SEQ ID NO: 3) and pSETermE-R (from the genomic DNA of the clone with the most productive K153T amino acid substitution isolated in Example 1) PCR reaction was performed under the following cycle conditions using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) with the combination of SEQ ID NO: 4) (98 ° C · 10 seconds, 60 ° C · 5 seconds, 72 ° C · 1 minute) ) X 25 times. As a result, an amplified fragment of about 1 kbp was obtained. The amplified fragment was double-digested with Bam HI and Xba I, yielding plasmid pEVA-E-1 containing inserted into Bam HI and Xba I sites of plasmid pSET152-E * is amino acid substitution K153T evaE . From this plasmid pEVA-E-1, primers pSET153-R (SEQ ID NO: 3) and Q149S-R (5'-TTCCGCTGTCAGCTTCTG-3 ', SEQ ID NO: 19), Q149S-F (5'-TCGATCCTCAAGACGGCCACGGC-3', SEQ ID NO: 20 ) And pSETermE-R (SEQ ID NO: 4) using PrimeSTAR HS DNA polymerase (manufactured by TAKARA BIO INC.) To carry out the PCR reaction as described above, and this PCR reaction product is subjected to high-purity PCR product purification. It refine | purified using the kit (made by Roche), and obtained two types of DNA solutions. These two DNA solutions were phosphorylated using T4 polynucleotide kinase (Nippon Gene), then inserted into the Bam HI and Xba I sites of plasmid pSET152-E * and Q149S (149th glutamine was converted to serine). And plasmid pEVA-E-2 containing evaE with amino acid substitution of K153T .

Using this plasmid, it was introduced into the dnmV- disrupted strain described in Example 1 by conjugation transfer, and the productivity of epidaunorubicin was confirmed in the same manner as in Example 1 for the obtained strain evaE-2. It was confirmed that this is a strain with improved productivity.

Random mutation introduction into K153T mutant gene Random mutation was introduced using the plasmid pEVA-E-1 described in Example 3 as a template and GeneMorph II Random Mutagenesis Kit (Stratagene) according to the attached manual.

The evaE fragment mutation is double digested with Bam HI and Xba I, and a genomic DNA library cloned into the Bam HI and Xba I sites of pSET152-E *.

These cloned plasmids were introduced into the dnmV- disrupted strain described in Example 1 by conjugation transfer, and the productivity of epidaunorubicin was confirmed in the same manner as in Example 1 for the obtained strain. In addition to K153T, the strain had E306D (306th glutamic acid was replaced by aspartic acid) amino acid substitution, and this clone was designated evaE-3.

Preparation and evaluation of evaE triple mutant (K153T, C270R, E306D) and quadruple mutant (Q42L, K153T, C270R, E306D) Primer pSET153-R (SEQ ID NO :) from the genomic DNA of evaE-3 isolated in Example 4 3) and T808C-R (5'-GTTCCACACGGGTCACCTCG-3 ', SEQ ID NO: 21), T808C-F (5'-CGAGGTGACCCGTGTGGAAC-3', SEQ ID NO: 22) and pSETermE-R (SEQ ID NO: 4) in combination with PrimeSTAR A PCR reaction was performed in the same manner as in Example 3 using HS DNA polymerase (Takara Bio Inc.), and the PCR reaction product was purified using a high-purity PCR product purification kit (Roche). Different types of DNA solutions were obtained. Using a mixture of these two DNA solutions as a template, PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) with a combination of primers pSET153-R (SEQ ID NO: 3) and pSETermE-R (SEQ ID NO: 4) PCR was carried out in the same manner as in example 3, the amplified fragment was double-digested with Bam HI and Xba I, K153T and C270R and E306D amino acids was inserted into Bam HI and Xba I sites of plasmid pSET152-E * Plasmid pEVA-E-4 containing the substituted evaE was obtained.

From plasmid pEVA-E-4, primers pSET153-R (SEQ ID NO: 3) and A125T-R (5'-ACGGTCGTCTCGGCTAGGCCGGGCG-3 ', SEQ ID NO: 23), A125T-F (5'-GCCCGCGCCCGGCCTAGCCGAGACG-3', SEQ ID NO: 24) And pSETermE-R (SEQ ID NO: 4) in each case using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) and carrying out a PCR reaction in the same manner as in Example 3. Two types of DNA solutions were obtained by purification using an application kit (Roche). Using a mixture of these two DNA solutions as a template, PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) with a combination of primers pSET153-R (SEQ ID NO: 3) and pSETermE-R (SEQ ID NO: 4) PCR was carried out in the same manner as in example 3, the amplified fragment was double-digested with Bam HI and Xba I, and inserted into the Bam HI and Xba I sites of plasmid pSET152-E * Q42L and K153T and C270R and E306D Plasmid pEVA-E-5 containing evaE with the amino acid substitution was obtained.

When this plasmid pEVA-E-5 was introduced into the dnmV- disrupted strain described in Example 1 by conjugation transfer and the resulting strain evaE-5 was confirmed for epidaunorubicin productivity in the same manner as in Example 1, It was confirmed that the strain had improved productivity compared to the strain before the mutation introduction.

以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。

As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

  The ketoreductase mutant of the present invention can be used for the production of daunorubicin derivatives.

The description of “Artificial Sequence” is described in the numerical heading <223> of the sequence listing.
The base sequences represented by the sequences of SEQ ID NOs: 2 to 24 are synthetic DNA. The symbols “n” in SEQ ID NOs: 5 to 12 each represent an arbitrary base.

Claims (11)

Daunorubicin (RTM daunomycin) a variant of the ketoreductase enzymes that can be used for fermentation production of derivatives, in the amino acid sequence of the parent ketoreductase of SEQ ID NO: 1, 4 2,149,153,270 and 306 One or more amino acid residues selected from the group consisting of amino acids at positions corresponding to the th amino acid are substituted with other amino acid residues;
The substitution of the amino acid at the position corresponding to the 42nd amino acid in the amino acid sequence represented by SEQ ID NO: 1 is a substitution with leucine,
Substitution of the amino acid at the position corresponding to the 149th amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substitution with serine;
The substitution of the amino acid at the position corresponding to the 153rd amino acid in the amino acid sequence represented by SEQ ID NO: 1 is a substitution to an amino acid residue other than proline,
Substitution of the amino acid at the position corresponding to the 270th amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substitution with arginine;
Substitution of the amino acid at the position corresponding to the 306th amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substitution with aspartic acid,
A ketoreductase variant in which the productivity of a daunorubicin derivative is improved as compared to when the parent ketoreductase is used.   The ketoreductase variant according to claim 1, wherein the amino acid at the position corresponding to the 42nd amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substituted with leucine.   The ketoreductase variant according to claim 1, wherein the amino acid at the position corresponding to the 149th amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substituted with serine.   The ketoreductase variant according to claim 1, wherein the amino acid at the position corresponding to the 153rd amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid residue other than proline.   The ketoreductase variant according to claim 1, wherein the amino acid at the position corresponding to the 270th amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substituted with arginine.   The ketoreductase variant according to claim 1, wherein the amino acid at the position corresponding to the 306th amino acid in the amino acid sequence represented by SEQ ID NO: 1 is substituted with aspartic acid. The ketoreductase variant according to any one of claims 1 to 6, wherein the daunorubicin derivative is epidaunorubicin (registered trademark: epidaunomycin ) .   A polynucleotide encoding the ketoreductase variant according to any one of claims 1 to 7. A transformant in which the polynucleotide according to claim 8 is introduced into an actinomycete host originally capable of producing daunorubicin, and is given a daunomycin derivative-producing ability. The transformant according to claim 9, wherein the host actinomycete is Streptomyces coeruleorubidus .   A method for producing a daunorubicin derivative comprising culturing the transformant according to claim 10 and collecting a daunorubicin derivative from the culture solution.