Title of Invention: Carbonyl reductase, nucleic acid encoding the same, and method for producing optically active compounds using these
Technical field
[0001]
The present invention provides a carbonyl reductase that has the activity of reducing a carbonyl group-containing compound to convert it into an optically active compound that is an industrially useful compound as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc., and a nucleic acid that encodes the carbonyl reductase. , a recombinant vector containing the nucleic acid, a transformant containing the recombinant vector, and a method for producing an optically active compound using the carbonyl reductase or the like.
Background technology
[0002]
Statin compounds such as rosuvastatin, pitavastatin, atorvastatin, fluvastatin and pravastatin or salts thereof are HMG-CoA reductase inhibitors and are useful for treating hypercholesterolemia, mixed dyslipidemia, and the like. In recent years, pharmaceuticals using these statin compounds or salts thereof have been marketed as generic pharmaceuticals, and a method for industrially producing them at a lower cost is desired.
[0003]
These statin compounds have the following structures
[0004]
[Chemical 1]

[0005]
An optically active alcohol or a salt thereof having
Structures with consecutive carbonyl groups such as the following
[0006]
[Chemical 2]

[0007]
A method for stereoselectively reducing a carbonyl group-containing compound having is produced by an organic synthetic or biochemical method is known.
[0008]
For example, in Patent Document 1, by reacting a carbonyl group-containing compound with a specific carbonyl reductase (hereinafter sometimes referred to as "OCR1"), an optically active alcohol such as an optically active alcohol with high optical purity and high concentration is obtained. Methods of making compounds are described. Patent Document 2 describes a method for producing rosuvastatin calcium using OCR1, and Patent Document 3 describes a method for producing pitavastatin calcium using OCR1.
[0009]
Here, OCR1 has poor thermostability, and it is necessary to use a large amount of OCR1 because the stability of the enzyme decreases during the reaction (during heating), so mutations aimed at improving the thermostability of OCR1 Studies on the body have been conducted (Non-Patent Document 1).
[0010]
However, in order to industrially produce optically active compounds, especially statin compounds, which are useful as intermediate raw materials for pharmaceuticals, agricultural chemicals, etc. with high purity and high optical purity at low cost, it is desirable to further improve the performance of OCR1. It is rare.
prior art documents
patent literature
[0011]
Patent Document 1: Patent No. 4270918
Patent Document 2: International Publication No. 2015/119261
Patent Document 3: International Publication No. 2017/022846
Non-patent literature
[0012]
Non-Patent Document 1: Journal of Bioscience and Bioengineering, VOL. 123 No. 6, 673-678, 2017
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0013]
The problem to be solved in the present invention is to develop a carbonyl reductase with higher carbonyl reductive activity, thermal stability, stereoselectivity, optical selectivity, etc. than OCR1. Further, another problem to be solved by the present invention is to obtain an optically active compound with high purity and high optical purity at low cost by using the carbonyl reductase.
Means to solve problems
[0014]
In order to solve the above problems, the present inventors have made intensive studies and found that by substituting a specific amino acid in OCR1 with another specific amino acid, carbonyl reduction activity, thermal stability, stereoselectivity, optical selection The inventors have found that a carbonyl reductase with high properties can be obtained, and completed the present invention. Furthermore, the inventors have found that by using the carbonyl reductase, it is possible to industrially obtain an optically active compound with high purity and high optical purity at a low cost, thereby completing the present invention.
[0015]
That is, the gist of the present invention is as follows.
[1] A carbonyl having a polypeptide consisting of an amino acid sequence having at least one mutation selected from the group consisting of the following (a) to (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homologue of the amino acid sequence reductase.
(a) A mutation in which the 54th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is replaced with valine
(b) a mutation in which the 157th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with valine
(c) a mutation in which the 170th alanine in the amino acid sequence of SEQ ID NO: 1 is replaced with serine
(d) a mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine or asparagine
(e) a mutation in which the 214th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine
(f) a mutation in which the 249th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine
[2] The carbonyl reductase of [1], which has at least two mutations selected from the group consisting of (a) to (f).
[3] A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or a homologue of said amino acid sequence, and further comprising (g) a mutation in which valine at position 166 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine. The carbonyl reductase according to [1] or [2].
[4] A carbonyl group-containing compound selected from the group consisting of compounds represented by the following general formulas (I), (II) and (III),
[0016]
[Chemical 3]

[0017]
[Chemical 4]

[0018]
[Chemical 5]

[0019]
(In the above formulas (I), (II) and (III), R represents a hydrogen atom, an alkyl group or an aryl group,
[0020]
[Chemical 6]

[0021]
represents a substituent having an aromatic ring and/or a heterocyclic ring. )
The carbonyl reductase according to any one of [1] to [3], a microorganism or cell capable of producing the enzyme, a processed product of the microorganism or cell, and/or obtained by culturing the microorganism or cell The following general formula (IV), which is characterized by asymmetric reduction of a carbonyl group-containing compound by contact with a culture solution containing the enzyme
[0022]
[Chemical 7]

[0023]
(In the formula, R and
[0024]
[Chemical 8]

[0025]
has the same meaning as above. )
A method for producing an optically active compound represented by
[5] The carbonyl group-containing compounds represented by the formulas (II) and (III) are each represented by the following formula (II')
[0026]
[Chemical 9]

[0027]
(In the formula, R and
[0028]
[Chemical 10]

[0029]
has the same meaning as above. )
and the following formula (III')
[0030]
[Chemical 11]

[0031]
(In the formula, R and
[0032]
[Chemical 12]

[0033]
has the same meaning as above. )
The production method according to [4], which is an optically active substance represented by
[6] Substituent
[0034]
[Chemical 13]

[0035]
but,
[0036]
[Chemical 14]

[0037]
(In the formula,
[0038]
[Chemical 15]

[0039]
represents a substituent having an aromatic ring and/or a heterocyclic ring. ),
[0040]
[Chemical 16]

[0041]
,
[0042]
[Chemical 17]

[0043]
or
[0044]
[Chemical 18]

[0045]
The production method according to [4] or [5].
[7] Substituent
[0046]
[Chemical 19]

[0047]
but,
[0048]
[Chemical 20]

[0049]
or
[0050]
[Chemical 21]

[0051]
The manufacturing method according to [6].
[8] The microorganism or cell is a nucleic acid encoding the carbonyl reductase according to any one of [1] to [3], and has a base sequence shown in (p), (q) or (r) below. The production method according to any one of [4] to [7], which is a microorganism or cell transformed with a nucleic acid containing
(p) A base encoding a polypeptide having a base sequence represented by SEQ ID NO: 2 in which 1 to multiple bases are substituted, deleted and/or added and having carbonyl reductase activity arrangement
(q) a nucleotide sequence encoding a polypeptide having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2 and having carbonyl reductase activity
(r) a nucleotide sequence that encodes a polypeptide having a nucleotide sequence that hybridizes under stringent conditions with the complementary strand of the nucleotide sequence represented by SEQ ID NO: 2 and having carbonyl reductase activity
[0052]
In addition, in this specification, the substituent
[0053]
[Chemical 22]

[0054]
is "substituent A", substituent
[0055]
[Chemical 23]

[0056]
may be referred to as "substituent B".
Effect of the invention
[0057]
According to the present invention, a carbonyl reductase having the activity of reducing a carbonyl group-containing compound to convert it into an optically active compound, which is an industrially useful compound as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc., and encoding the carbonyl reductase A nucleic acid, a recombinant vector containing the nucleic acid, and a transformant containing the recombinant vector can be provided. Furthermore, according to the present invention, it is possible to provide a production method for industrially obtaining an industrially useful optically active compound as an intermediate raw material for pharmaceuticals, agrochemicals, etc. with high purity and high optical purity at low cost. .
MODE FOR CARRYING OUT THE INVENTION
[0058]
The present invention will be described in detail below.
[0059]
1. Carbonyl reductase of the present invention
The carbonyl reductase of the present invention comprises an amino acid sequence having at least one mutation selected from the group consisting of the following (a) to (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homologue of the amino acid sequence. A polypeptide having carbonyl reductase activity.
(a) A mutation in which the 54th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is replaced with valine
(b) a mutation in which the 157th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with valine
(c) a mutation in which the 170th alanine in the amino acid sequence of SEQ ID NO: 1 is replaced with serine
(d) a mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine or asparagine
(e) a mutation in which the 214th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine
(f) a mutation in which the 249th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine
[0060]
In the present invention, carbonyl reductase activity means the activity of asymmetrically reducing a carbonyl group in a carbonyl group-containing compound to convert it into an optically active compound. Whether or not a compound has carbonyl reductase activity can be determined by measuring the activity of asymmetrically reducing the carbonyl group in the carbonyl group-containing compound to convert it into an optically active compound by a conventional assay method. For example, a carbonyl group-containing compound represented by general formula (I), (II) or (III) may be combined with a carbonyl reductase to be measured, a microorganism or cell capable of producing the enzyme, or the microorganism or cell. and / or the amount of the compound represented by the general formula (IV) converted from these carbonyl group-containing compounds by acting the culture solution containing the enzyme obtained by culturing the microorganism or cell By directly measuring the carbonyl reductase activity can be confirmed. In the case of a measurement system containing NADPH as a coenzyme, carbonyl reductase activity can be confirmed by measuring the initial rate of NADPH decrease.
[0061]
In the present invention, the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence (OCR1) derived from Ogataea minuta var. be.
[0062]
In the present invention, the homologue of the amino acid sequence represented by SEQ ID NO: 1 is an amino acid sequence in which one to multiple amino acids are deleted, inserted, substituted and/or added in the amino acid sequence represented by SEQ ID NO: 1. have and has carbonyl reductase activity.
[0063]
"1 to several amino acids" means usually 1 to 100, preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably 1 to 5 amino acids.
[0064]
Further, in the present invention, the homologue of the amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence having a sequence identity of 90% or more with the full-length amino acid sequence represented by SEQ ID NO: 1, and carbonyl reductase It is an active polypeptide. Preferably, it has an amino acid sequence having a sequence identity of 95% or more, more preferably 98% or more, and still more preferably 99% or more with the full-length amino acid sequence represented by SEQ ID NO: 1, and has carbonyl reductase activity. It is a polypeptide having
[0065]
Amino acid sequence homology (also referred to as identity or similarity) herein is determined using a homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), for example, under the following conditions (expected value = 10; allow gaps; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993). program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970). [which algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [which is part of the CGC sequence alignment software package. is incorporated in the ALIGN program (version 2.0)], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988). incorporated into the FASTA program], etc., which can also be preferably used.
[0066]
The amino acid sequence represented by SEQ ID NO: 1 and homologues of the amino acid sequence can be obtained by the method described in Japanese Patent No. 4270918.
[0067]
The carbonyl reductase of the present invention is a mutation of (a), (b), (c), (d), (e) or (f) in the amino acid sequence represented by SEQ ID NO: 1 or a homologue of the amino acid sequence. It has excellent properties by having Each mutation will be described below in the order of (d), (c), (f), (a), (b), and (e).
[0068]
The mutation (d) is a mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine or asparagine. This mutation improves stereoselectivity and optical selectivity.
[0069]
Specifically, the performance of selectively reducing the 3-position carbonyl group of the carbonyl group-containing compound represented by the following general formula (I) or (II) is improved. Therefore, the optically active compound represented by general formula (IV) can be obtained in high yield and high chemical purity from the carbonyl group-containing compound represented by general formula (I) or (II). In addition, since the mutation (d) also improves optical selectivity, the carbonyl group-containing compound represented by general formula (I), (II) or (III) can be converted into general formula (IV) with high yield and high optical purity. ) can be obtained.
[0070]
[Chemical 24]

[0071]
In particular, if the substituent A is
[0072]
[Chemical 25]

[0073]
or
[0074]
[Chemical 26]

[0075]
, the performance of selectively reducing the carbonyl group at the 3-position is high.
[0076]
Therefore, for example, the optically active compounds shown below can be obtained with high yields, high chemical purities, and high optical purities by the following reactions.
[0077]
[Chemical 27]

[0078]
[Chemical 28]

[0079]
The mutation (c) is a mutation in which the 170th alanine in the amino acid sequence of SEQ ID NO: 1 is replaced with serine. This mutation improves thermostability.
[0080]
In addition, the mutation (f) is a mutation in which methionine at position 249 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine. This mutation improves thermostability.
[0081]
Due to these mutations, the activity of carbonyl reductase does not decrease even in the reduction reaction under heating conditions, and high enzymatic activity is maintained. The optically active compound represented by the general formula (IV) can be obtained from the compound contained in high yield and high chemical purity. Moreover, when both the mutation (c) and the mutation (f) are present, the thermostability is further improved, which is preferable.
[0082]
The mutation (a) is a mutation in which the 54th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is replaced with valine. This mutation improves stereoselectivity.
[0083]
Specifically, since the performance of selectively reducing the carbonyl group at the 3-position of the carbonyl group-containing compound represented by the general formula (I) or (II) is improved, the optically active compounds can be obtained in high yields and high chemical purities.
[0084]
In particular, if the substituent A is
[0085]
[Chemical 29]

[0086]
In that case, the reaction rate and reaction efficiency with respect to the carbonyl group-containing compound are improved.
[0087]
Therefore, for example, the following reactions improve the production rate and production efficiency of the optically active compounds shown below, and the optically active compounds can be obtained with high yields, high chemical purities, and high optical purities.
[0088]
[Chemical 30]

[0089]
The mutation (b) is a mutation in which methionine at position 157 in the amino acid sequence of SEQ ID NO: 1 is replaced with valine. This mutation improves stereoselectivity and optical selectivity.
[0090]
In addition, the mutation (e) is a mutation in which methionine at position 214 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine. This mutation improves stereoselectivity and optical selectivity.
[0091]
Here, the carbonyl reductase having the amino acid sequence represented by SEQ ID NO: 1 is represented by the following general formula (I)
[0092]
[Chemical 31]

[0093]
When acting on a carbonyl group-containing compound represented by, the 3-position carbonyl group is preferentially reduced over the 5-position carbonyl group, so the following general formula (III)
[0094]
[Chemical 32]

[0095]
The compound represented by the following general formula (II)
[0096]
[Chemical 33]

[0097]
produces more than the compound represented by In addition, from the compound represented by the general formula (III)
General formula (IV) below
[0098]
[Chemical 34]

[0099]
Since the reaction rate to the compound represented by the general formula (II) is slower than the reaction rate to the optically active compound represented by the general formula (IV), the general formula (III) The compound represented by may remain.
[0100]
When a carbonyl reductase having the mutation (b) and the mutation (e) is allowed to act on the carbonyl group-containing compound represented by the general formula (I), the reduction of the carbonyl group at the 3-position is suppressed, and 3 The carbonyl groups at the position and the 5-position are reduced in a well-balanced manner. In addition, since the reaction rate and reaction efficiency for converting the compounds represented by the above general formulas (II) and (III) into the optically active compound represented by the above general formula (IV) are improved, efficient and high-yield It is possible to obtain the optically active compound represented by the general formula (IV) with high yield and high chemical purity.
[0101]
In particular, if the substituent A is
[0102]
[Chemical 35]

[0103]
In that case, the reaction rate and reaction efficiency for converting the carbonyl group-containing compound into the optically active compound are further improved.
[0104]
Therefore, for example, the following reactions improve the production rate and production efficiency of the optically active compounds shown below, and the optically active compounds can be obtained with high yields, high chemical purities, and high optical purities.
[0105]
[Chemical 36]

[0106]
Furthermore, it is more preferable to have both the (b) mutation and the (e) mutation, because the reaction efficiency with respect to the carbonyl group-containing compound is also improved.
[0107]
The carbonyl reductase of the present invention is selected from the group consisting of (a) to (f) above in the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence that is a homologue of the amino acid sequence and has carbonyl reductase activity. A carbonyl reductase having a polypeptide consisting of an amino acid sequence with at least one mutation. Mutations (a), (b), (c), (d), (e), or (f) include one or more mutations depending on the type of the target optically active compound represented by the above formula (IV). They can be selected as appropriate, but preferably have at least two mutations selected from the group consisting of (a) to (f), and more preferably have at least three mutations.
[0108]
In addition, the carbonyl reductase of the present invention may have the mutation (g). The mutation (g) is described in Non-Patent Document 1, and is a mutation in which the 166th valine in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine. This mutation improves the thermostability of carbonyl reductase.
[0109]
The carbonyl reductase of the present invention can be obtained from the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence that is a homologue of the amino acid sequence and has carbonyl reductase activity by a method known to those skilled in the art, such as site-directed mutagenesis. It can be produced by well-known techniques such as method, PCR method, and the like.
[0110]
The carbonyl reductase of the present invention can also be produced by culturing a transformant containing a nucleic acid encoding it, and separating and purifying the carbonyl reductase from the resulting culture. A nucleic acid encoding a carbonyl reductase of the present invention may be DNA, RNA, or a DNA/RNA chimera. DNA is preferred. Also, the nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA or a DNA:RNA hybrid. If single stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).
[0111]
Examples of the DNA encoding the carbonyl reductase of the present invention include synthetic DNA. For example, using as a template total RNA or mRNA fractions prepared from cells or tissues derived from Ogataea minuta var. Using TM-super Express Km (TAKARA BIO INC.), Mutan TM-K (TAKARA BIO INC.), etc., methods known per se such as ODA-LA PCR method, Gapped duplex method, Kunkel method, or methods according to them can be obtained by converting according to Alternatively, from a cDNA library prepared by inserting the above-mentioned total RNA or mRNA fragment into an appropriate vector, colony or plaque hybridization method, PCR method, etc., can also be obtained by transforming the cloned cDNA according to the methods described above. Vectors used for libraries may be bacteriophages, plasmids, cosmids, phagemids, and the like.
[0112]
The nucleic acid encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is not limited as long as it encodes the polypeptide having carbonyl reductase activity of the present invention, particularly the following (p), (q) or Those having the base sequence shown in (r) are included.
(p) a polypeptide having a nucleotide sequence represented by SEQ ID NO: 2 in which one to multiple nucleotides are substituted, deleted and/or added, and having carbonyl reductase activity of the present invention; encoding nucleic acid
(q) a nucleic acid having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2 and encoding a polypeptide having carbonyl reductase activity of the present invention;
(r) a nucleic acid that has a nucleotide sequence that hybridizes under stringent conditions with the complementary strand of the nucleotide sequence represented by SEQ ID NO: 2 and that encodes a polypeptide having carbonyl reductase activity of the present invention;
[0113]
The homologue of the nucleic acid shown in (p) above includes a nucleotide sequence in which 1 to multiple bases are deleted, substituted, inserted and/or added in the nucleotide sequence represented by SEQ ID NO: 2, and the present invention and a nucleic acid encoding a polypeptide having carbonyl reductase activity of In the case of substitution, insertion or addition, one or more bases are preferably substituted, inserted or added. Here, "1 to a plurality of bases" means, for example, 1 to 60, preferably 1 to 30, more preferably 1 to 15, still more preferably 1 to 10, particularly preferably is 1 to 5 bases.
[0114]
The homologue of the nucleic acid shown in (q) above is a polypeptide having a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2 and having carbonyl reductase activity of the present invention. Encoding nucleic acids are included. Preferably, it has a nucleotide sequence having 95% or more, more preferably 98% or more, more preferably 99% or more homology (also referred to as identity) with the nucleotide sequence represented by SEQ ID NO: 2, and the present invention is a nucleic acid encoding a polypeptide having carbonyl reductase activity of
[0115]
The homology (also referred to as identity) of the nucleotide sequences herein uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), for example, under the following conditions (expected value = 10; gap filtering = ON; match score = 1; mismatch score = -3). Other algorithms for determining nucleotide sequence homology are similarly preferably exemplified by the above-described amino acid sequence homology calculation algorithms.
[0116]
The homologue of the nucleic acid shown in (r) above is a nucleic acid that hybridizes under stringent conditions with the complementary strand of the base sequence of SEQ ID NO: 2 as long as it encodes the polypeptide having carbonyl reductase activity of the present invention. may Here, the "stringent conditions" can be appropriately set with reference to previously reported conditions (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.16.3.6, 1999). is, for example, normal Southern hybridization washing conditions of 60°C, 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS, more preferably 65°C, 0.1 x SSC, 0.1% SDS and 68° C., 0.1×SSC, 0.1% SDS, etc. (highly stringent conditions) at salt concentration and temperature corresponding to washing once, more preferably 1 to 3 times.
[0117]
A person skilled in the art would be familiar with site-directed mutagenesis (Nucleic Acids Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning) for the nucleic acid represented by SEQ ID NO: 2. , PCR A Practical Approach IRL Press pp.200 (1991)), etc., by appropriately performing substitutions, deletions, insertions and/or additions to introduce desired mutations, thereby creating homologs of the above nucleic acids It is possible to obtain
[0118]
The nucleic acid of the present invention can encode the polypeptide having carbonyl reductase activity of the present invention. When the nucleic acid of the present invention has the nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence highly identical to the nucleotide sequence represented by SEQ ID NO: 2, carbonyl reduction containing the polypeptide encoded by the nucleic acid Although the degree of carbonyl reductase activity of the enzyme may be quantitatively equivalent to that comprising a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or a polypeptide having a homologue of the amino acid sequence, It may differ within an acceptable range (eg, about 0.1 to about 5 times, preferably about 0.3 to about 3 times).
[0119]
Further, based on the amino acid sequence in the amino acid sequence of SEQ ID NO: 1 or a part thereof and the nucleotide sequence represented by SEQ ID NO: 2 or a part thereof, homology data can be obtained in a database such as DNA Databank of Japan (DDBJ). It is also possible to obtain amino acid sequence information of a polypeptide having carbonyl reductase activity or nucleotide sequence information of the DNA encoding it by conducting a search.
[0120]
In the production method of the present invention, which will be described later, the above carbonyl reductase may be used for the direct reaction with the carbonyl group-containing compound that is the substrate, but microorganisms or cells having the ability to produce the enzyme, or the microorganisms or cells and/or a culture medium containing the enzyme obtained by culturing the microorganism or cell.
[0121]
As the microorganism or cell having the ability to produce the carbonyl reductase of the present invention, a microorganism or cell originally having the ability to produce the carbonyl reductase may be used, or a microorganism or cell to which the above production ability has been imparted by breeding. may be As microorganisms or cells, whether they are alive or dead, for example, dormant cells and the like can be preferably used. Types of microorganisms or cells capable of producing the carbonyl reductase of the present invention include those described below as “host microorganisms” or “host cells”.
[0122]
Known methods such as genetic recombination processing (transformation) and mutation processing can be adopted as means for imparting the above-mentioned production capacity through breeding. Transformation methods include a method of introducing a DNA of interest, a method of modifying an expression regulatory sequence such as a promoter on the chromosome to enhance expression of the DNA of interest, and the like.
[0123]
Of these, it is preferable to use microorganisms or cells transformed with the DNA encoding the polypeptide of the present invention.
[0124]
A nucleic acid (DNA) encoding the polypeptide (carbonyl reductase) of the present invention can be obtained by PCR using the chromosomal DNA derived from the Ogataea minuta var. Can be cloned.
[0125]
As described above, the nucleic acid (DNA) encoding the polypeptide (carbonyl reductase) of the present invention is directly amplified by RT-PCR using total RNA or mRNA derived from Ogataea minuta var. nonfermentans NBRC1473 strain as a template. After preparing the full-length carbonyl reductase cDNA, it can be cloned by performing PCR using appropriate primers.
[0126]
For example, the polypeptide gene expression vector of the present invention is provided by inserting the DNA encoding the polypeptide of the present invention obtained as described above into a known expression vector in an expressible arrangement. Then, by transforming a host cell with the expression vector, a transformant into which the DNA encoding the polypeptide of the present invention has been introduced can be obtained. A transformant can also be obtained by integrating the DNA encoding the polypeptide of the present invention into the chromosomal DNA of the host by a technique such as homologous recombination so that the DNA can be expressed.
[0127]
As used herein, an "expression vector" is used to replicate and express a protein having a desired function in an integrating host organism by introducing a polynucleotide encoding a protein having a desired function into the host organism. It is a genetic factor that can be Examples include, but are not limited to, plasmids, viruses, phages, cosmids, and the like. Preferably, the expression vector is a plasmid.
[0128]
As used herein, the term "transformant" refers to a microorganism or organism into which a gene of interest has been introduced using the expression vector or the like, and which has become capable of exhibiting a desired trait associated with a protein having a desired function. means cell.
[0129]
Specifically, the method for producing a transformant includes an expression vector constructed by introducing a DNA encoding the polypeptide of the present invention into a plasmid vector, phage vector, or virus vector that stably exists in a host cell. into the host cell, and a method of directly introducing the DNA into the host genome and transcribing/translating its genetic information. In this case, it is preferable to link an appropriate promoter to the 5'-side upstream of the DNA in the host, and more preferably link a terminator to the 3'-side downstream. Such promoters and terminators are not particularly limited as long as they are promoters and terminators known to function in cells used as hosts. The vectors, promoters and terminators detailed can be used.
[0130]
The host microorganism to be transformed to express the carbonyl reductase of the present invention is particularly limited as long as the host itself does not adversely affect the substrate carbonyl group-containing compound or the target optically active compound. For example, the following microorganisms can be mentioned.
[0131]
Escherichia genus, Bacillus genus, Pseudomonas genus, Serratia genus, Brevibacterium genus, Corynebacterium genus, Streptococcus genus, Lactobacillus ) Bacteria with established host-vector systems belonging to the genus.
[0132]
Actinomycetes with established host-vector systems belonging to the genus Rhodococcus, Streptomyces, etc.
[0133]
Saccharomyces genus, Kluyveromyces genus, Schizosaccharomyces genus, Zygosaccharomyces genus, Yarrowia genus, Trichosporon genus, Rhodosporidium genus, Yeast with established host-vector systems belonging to the genus Hansenula, Pichia, Candida, and the like.
[0134]
Genus Neurospora, Aspergillus, Cephalospori Fungi with established host-vector systems belonging to the genus Cephalosporium, Trichoderma, and the like.
[0135]
Procedures for producing transformants, construction of recombinant vectors suitable for the host, and methods for culturing the host can be performed according to techniques commonly used in the fields of molecular biology, bioengineering, and genetic engineering ( For example, the method described in Green et al., Molecular Cloning: A Laboratory Manual (4 thed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012)).
[0136]
Specific examples of preferred host microorganisms, preferred transformation methods, vectors, promoters, terminators, etc. for each microorganism are given below, but the present invention is not limited to these examples.
[0137]
In the genus Escherichia, especially in Escherichia coli, examples of plasmid vectors include pBR, pUC-based plasmids, lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp). fusion), λ phage PL, PR and the like. Examples of the terminator include trpA-derived, phage-derived, and rrnB ribosomal RNA-derived terminators.
[0138]
In the genus Bacillus, vectors include pUB110-based plasmids, pC194-based plasmids, etc., and can also be integrated into the chromosome. Promoters and terminators of enzyme genes such as alkaline protease, neutral protease and α-amylase can be used as promoters and terminators.
[0139]
In the genus Pseudomonas, vectors include common host-vector systems established in Pseudomonas putida, Pseudomonas cepacia, etc., plasmids involved in the decomposition of toluene compounds, and TOL plasmids. A wide-host-range vector based on it (including genes necessary for autonomous replication derived from RSF1010, etc.) pKT240 (Gene, 26, 273-82 (1983)) and the like can be mentioned.
[0140]
In the genus Brevibacterium, particularly Brevibacterium lactofermentum, vectors include plasmid vectors such as pAJ43 (Gene 39, 281 (1985)). As promoters and terminators, various promoters and terminators used in E. coli can be used.
[0141]
In the genus Corynebacterium, particularly Corynebacterium glutamicum, plasmids such as pCS11 (Japanese Unexamined Patent Publication No. 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984)) are used as vectors. vector.
[0142]
In the genus Saccharomyces, particularly Saccharomyces cerevisiae, vectors include YRp, YEp, YCp, and YIp plasmids. In addition, promoters and terminators of various enzyme genes such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, β-galactosidase, phosphoglycerate kinase and enolase can be used.
[0143]
In the genus Schizosaccharomyces, examples of vectors include plasmid vectors derived from Schizosaccharomyces pombe described in Mol. Cell. Biol. 6, 80 (1986). In particular, pAUR224 is commercially available from Takara Bio Inc. and can be easily used.
[0144]
In the Aspergillus genus, Aspergillus niger, Aspergillus oryzae, etc. are the most well-studied fungi, and plasmids and chromosomal integration are available. Promoters derived from ectoprotease and amylase are available (Trendsin Biotechnology 7, 283-287 (1989)).
[0145]
In addition to the above, host-vector systems have been established for various microorganisms, and these can be used as appropriate.
[0146]
In addition to microorganisms, various host/vector systems have been established in plants and animals, particularly in animals such as insects (e.g., silkworms) (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes, etc. and systems using cell-free protein synthesis systems such as E. coli cell-free extracts and wheat germ have been established and can be suitably used.
[0147]
The treated microorganisms or cells having the ability to produce carbonyl reductase of the present invention include, for example, those treated with organic solvents such as acetone, dimethylsulfoxide (DMSO), toluene, or surfactants, Cell preparations such as freeze-dried ones, physically or enzymatically disrupted ones, microorganisms or enzymatic fractions in cells taken out as crude or purified products, further, polyacrylamide gels, carrageenan Examples thereof include those immobilized on carriers represented by gels and the like.
[0148]
Examples of the culture medium containing the enzyme obtained by culturing the microorganism or cell having the ability to produce the carbonyl reductase of the present invention include, for example, a suspension of the cell and a liquid medium, and a secretory expression type of the cell. In the case of cells, the supernatant obtained by removing the cells by centrifugation or the like and a concentrate thereof can be used.
[0149]
2. Compositions of the invention
The composition (enzyme agent) of the present invention is obtained by culturing the carbonyl reductase of the present invention, a microorganism or cell capable of producing the enzyme, a processed product of the microorganism or cell, and/or the microorganism or cell. including a culture medium containing the enzyme obtained by general formula (I), (II) or (III)
[0150]
[Chemical 37]

[0151]
[Chemical 38]

[0152]
[Chemical 39]

[0153]
With a carbonyl group-containing compound represented by the substrate, the general formula (IV)
[0154]
[Chemical 40]

[0155]
It catalyzes the reaction that produces the optically active compound represented by . By using the composition of the present invention as a catalyst, the optically active compound, which is industrially useful as an intermediate raw material for pharmaceuticals, agricultural chemicals, etc., can be industrially produced with high purity and high optical purity at low cost. It is useful because it can
[0156]
The composition of the present invention may contain excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, etc., in addition to active ingredients (enzymes, etc.). Lactose, sorbitol, D-mannitol, sucrose and the like can be used as excipients. Phosphate, citrate, acetate and the like can be used as buffers. Propylene glycol, ascorbic acid and the like can be used as stabilizers. Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. As antiseptics, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used.
[0157]
3. Method for producing an optically active compound of the present invention
According to the present invention, the carbonyl reductase of the present invention is
A carbonyl group-containing compound selected from the group consisting of compounds represented by the following general formulas (I), (II) and (III)
[0158]
[Chemical 41]

[0159]
[Chemical 42]

[0160]
[Chemical 43]

[0161]
There is provided a method for producing an optically active compound represented by the following general formula (IV), which comprises producing an optically active compound represented by the following general formula (IV) by reacting
[0162]
[Chemical 44]

[0163]
In addition, the carbonyl group-containing compounds represented by the formulas (II) and (III) are represented by the following formulas (II') and (III'), respectively.
[0164]
[Chemical 45]

[0165]
[Chemical 46]

[0166]
It is preferable that it is an optically active substance represented by.
[0167]
In this specification, R represents a hydrogen atom, an alkyl group or an aryl group. The alkyl group is preferably a linear or branched alkyl group having 1 to 8 carbon atoms, more preferably a linear alkyl group having 1 to 4 carbon atoms, and particularly preferably an ethyl group or an n-propyl group. .
[0168]
Also, the substituent A represents a substituent having an aromatic ring and/or a heterocyclic ring. Specifically, the substituent A is preferably a substituent having an aromatic ring having a fluorine atom as a substituent, a substituent having a heterocyclic ring having a nitrogen atom, and/or a substituent having a naphthalene ring. Above all,
[0169]
[Chemical 47]

[0170]
,
[0171]
[Chemical 48]

[0172]
,
[0173]
[Chemical 49]

[0174]
or
[0175]
[Chemical 50]

[0176]
is preferred, especially
[0177]
[Chemical 51]

[0178]
is preferred.
Here, the substituent B represents a substituent having an aromatic ring and/or a heterocyclic ring. Specifically, as the substituent B, a substituent having a heterocyclic ring having a nitrogen atom is preferable, and in particular,
[0179]
[Chemical 52]

[0180]
or
[0181]
[Chemical 53]

[0182]
is preferred.
[0183]
In the present invention, the substituent A is
[0184]
[Chemical 54]

or
[0185]
[Chemical 55]

[0186]
is particularly preferred.
[0187]
When the carbonyl reductase of the present invention is brought into contact with the carbonyl group-containing compound represented by the general formula (I), (II) or (III), the purified or partially purified carbonyl reductase of the present invention, the present Microorganisms or cells having the ability to produce the carbonyl reductase of the invention (e.g., transformants having DNA encoding the polypeptide of the invention), processed products of the microorganisms or cells, and/or the microorganisms or cells By contacting the culture solution containing the enzyme obtained by culturing with the carbonyl group-containing compound represented by the general formula (I), (II) or (III), the enzyme represented by the general formula (IV) is obtained. optically active compounds can be produced.
[0188]
The carbonyl reductase of the present invention may be used for a direct reaction, but may be obtained by culturing a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and/or the microorganism or cell. It is preferable to use a culture medium containing the enzyme obtained from the enzyme, and among these, it is preferable to use a transformant having a DNA encoding the polypeptide of the present invention.
[0189]
The amount of the microorganisms or cells added to the reaction solution, the treated products of the microorganisms or cells, and/or the culture solution containing the enzyme obtained by culturing the microorganisms or cells depends on the carbonyl group-containing compound that serves as the substrate. can be selected as appropriate. For example, when adding microorganisms or cells, the concentration of the microorganisms or cells in the reaction solution is usually about 0.1 w/v% to 50 w/v%, preferably 1 w/v% to 20 w/v in wet cell weight. When using a treated product or a culture medium, the specific activity of the enzyme is determined, and the amount added is such that the above cell concentration is reached when added. Here, w/v % represents weight/volume %.

The scope of the claims
[Claim 1]
A carbonyl reductase having a polypeptide consisting of an amino acid sequence having at least one mutation selected from the group consisting of (a) to (f) below in the amino acid sequence represented by SEQ ID NO: 1 or a homologue of said amino acid sequence.
(a) A mutation in which the 54th aspartic acid in the amino acid sequence of SEQ ID NO: 1 is replaced with valine
(b) a mutation in which the 157th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with valine
(c) a mutation in which the 170th alanine in the amino acid sequence of SEQ ID NO: 1 is replaced with serine
(d) a mutation in which the 211th isoleucine in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine or asparagine
(e) a mutation in which the 214th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine
(f) a mutation in which the 249th methionine in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine
[Claim 2]
The carbonyl reductase according to claim 1, which has at least two mutations selected from the group consisting of (a) to (f).
[Claim 3]
1. A polypeptide comprising an amino acid sequence shown in SEQ ID NO: 1 or a homologue of said amino acid sequence, and further comprising (g) an amino acid sequence having a mutation in which valine at position 166 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine. Or the carbonyl reductase according to 2.
[Claim 4]
A carbonyl group-containing compound selected from the group consisting of compounds represented by the following general formulas (I), (II) and (III),
[Chemical 1]

[Chemical 2]

[Chemical 3]

(In the above formulas (I), (II) and (III), R represents a hydrogen atom, an alkyl group or an aryl group,
[Chemical 4]

represents a substituent having an aromatic ring and/or a heterocyclic ring. )
The carbonyl reductase according to any one of claims 1 to 3, a microorganism or cell having the ability to produce the enzyme, a processed product of the microorganism or cell, and / or obtained by culturing the microorganism or cell The following general formula (IV), which is characterized by asymmetric reduction of a carbonyl group-containing compound by contact with a culture solution containing the enzyme
[Chemical 5]

(In the formula, R and
[Chemical 6]

has the same meaning as above. )
A method for producing an optically active compound represented by
[Claim 5]
The carbonyl group-containing compounds represented by the above formulas (II) and (III) are each represented by the following formula (II')
[Chemical 7]

(In the formula, R and
[Chemical 8]

has the same meaning as above. )
and the following formula (III')
[Chemical 9]

(In the formula, R and
[Chemical 10]

has the same meaning as above. )
5. The production method according to claim 4, wherein the optically active substance is represented by:
[Claim 6]
Substituent
[Chemical 11]

but,
[Chemical 12]

(In the formula,
[Chemical 13]

represents a substituent having an aromatic ring and/or a heterocyclic ring. ),
[Chemical 14]

[Chemical 15]

or
[Chemical 16]

The manufacturing method according to claim 4 or 5.
[Claim 7]
Substituent
[Chemical 17]

but,
[Chemical 18]

or
[Chemical 19]

The manufacturing method according to claim 6.
[Claim 8]
The microorganism or cell is a nucleic acid encoding the carbonyl reductase according to any one of claims 1 to 3, the nucleic acid comprising a base sequence shown in (p), (q) or (r) below. The production method according to any one of claims 4 to 7, wherein the microorganism or cell is transformed with.
(p) A base encoding a polypeptide having a base sequence represented by SEQ ID NO: 2 in which 1 to multiple bases are substituted, deleted and/or added and having carbonyl reductase activity arrangement
(q) a nucleotide sequence encoding a polypeptide having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2 and having carbonyl reductase activity
(r) a nucleotide sequence that encodes a polypeptide having a nucleotide sequence that hybridizes under stringent conditions with the complementary strand of the nucleotide sequence represented by SEQ ID NO: 2 and having carbonyl reductase activity