Abstract:
The application provides a Diketoreductase (DKR) mutant, its nucleotide coding sequence, and an expression cassette, recombinant vector and host cell containing the sequence, as well as a method for application of the mutant to the preparation of 3R,5S-dicarbonyl compound. An ee value of the obtained 3R,5S-dicarbonyl compound is higher than 99%, and a de value is about 90%. The DKR mutant is a key pharmaceutical intermediate, and particularly provides an efficient catalyst for synthesis of a chiral dicarbonyl hexanoic acid chain of a statin drug.

Description:
TECHNICAL FIELD 
       [0001]    The application relates to a Diketoreductase (DKR) mutant and an application thereof, and in particular to a DKR mutant obtained by site-directed saturation mutagenesis and of which properties such as catalytic activity are improved, and an application of the DKR mutant to preparation of a 3R,5S-dihydroxy compound. 
       BACKGROUND 
       [0002]    Carbonyl reductase is a kind of oxidoreductase, and plays an important role in many biotransformation processes of biological organisms. Based on its capability in catalytically generating chiral alcohols with high enantioselectivity, the carbonyl reductase is usually applied as a very important biocatalyst to the synthesis of chiral intermediates in the chemical and pharmaceutical industries. DKR may stereoselectively reduce two carbonyls of a diketo acid ester simultaneously to give the corresponding hydroxyl, and it may be used for synthesizing key drug intermediates, particularly synthesizing a chiral dihydroxy hexanoic acid chain of a statin drug such as worldwide saleable cholesterol lowering drugs atorvastatin and rosuvastatin. 
         [0003]    Currently known DKR can be used as a biocatalyst for reducing a diketone substrate in one step to prepare a chiral intermediate 3R,5S-dihydroxy-6-benzyloxy-tert-butyl hexanoate of a statin lipid-lowering drug with approximately single optical purity, thereby simplifying synthetic steps and reducing production pollution. However, application to industrial production still has some problems to be further solved. For example, low enzyme catalytic activity equates to a large amount of enzyme liquid and increased total volume of a reaction system, which increases production batches and production costs. These problems may be solved by directed evolution to improve catalytic activity of DKR. As a biocatalyst, an enzyme may fully develop its characteristics of high efficiency and high specificity in a biological system. However, there exist the common problems of inadaptability to an industrial production condition, low catalysis capability of an unnatural substrate and the like during industrial applications. Enzyme molecules are required to be modified to meet different application requirements by virtue of protein engineering methods. The protein engineering methods may be summarized into three: rational design, irrational design and semi-rational design. 
         [0004]    Rational design refers to changing individual amino acids in protein molecules by virtue of site-directed mutagenesis or another methods on the basis of knowing the spatial structure of proteins, thereby generating proteins with new characters. This method is theoretically high in pertinence, and is mainly used for modifying catalytic activity of natural apoenzymes, substrate specificity and stability, changing an inhibitor type, coenzyme specificity and the like. 
         [0005]    A site-directed saturation mutagenesis technology is an important technology in protein engineering, belongs to semi-rational design, but combines advantages of rational design and irrational design, overcomes respective shortcomings, and modifies a coding gene of a target protein to acquire a mutant of which an amino acid at a target site is substituted with other 19 amino acids respectively within a short time. This technology not only is a powerful tool for directed modification of proteins, but also is an important means for researching a protein&#39;s structure-function relationship. Researches show that multisite mutagenesis may always obtain an evolution more ideal than that obtained by single-site mutagenesis. Multisite mutagenesis is unlikely to be directly implemented by site-directed mutagenesis. However, site-directed saturation mutagenesis can increase the diversity of mutants and is simple to manipulate. 
         [0006]    Therefore, if a site-directed saturation mutagenesis technology may be utilized to modify DKR to improve its catalytic activity and substrate specificity and/or stability, the problems of large amount of enzyme liquid, high production cost and the like in the prior art may be solved. 
       SUMMARY 
       [0007]    In view of this, the application is intended to modify DKR by virtue of a site-directed saturation mutagenesis technology to improve its catalytic activity and substrate specificity and/or stability. 
         [0008]    The first aspect of the application relates to a DKR mutant, which comprises one of amino acid sequences shown as follows: 
         [0009]    a) SEQ ID NO: 1 to SEQ ID NO:6; 
         [0010]    b) a sequence which has identity of at least 70% with the sequences shown in a) and has improved DKR activity; and 
         [0011]    c) a sequence which is obtained by deleting, adding and/or substituting one or more amino acid residues in the sequences in a) and has improved DKR activity, 
         [0012]    wherein the sequence shown in b) is not a sequence shown as SEQ ID NO: 7. 
         [0013]    In one embodiment, the DKR mutant comprises the amino acid sequence shown as SEQ ID NO: 1, 2, 3, 4, 5 or 6. 
         [0014]    In another embodiment, the DKR mutant comprises the amino acid sequence shown as SEQ ID NO: 1, 2 or 3. 
         [0015]    In another embodiment, the DKR mutant comprises the amino acid sequence shown as SEQ ID NO: 2. 
         [0016]    The second aspect of the application relates to a nucleotide coding sequence of the above mentioned DKR mutant, wherein the nucleotide coding sequence does not contain a sequence shown as SEQ ID NO: 8. 
         [0017]    In an embodiment, the nucleotide coding sequence comprises a sequence shown as follows: 
         [0018]    a) sequences shown as SEQ ID NO: 9-14; 
         [0019]    b) a sequence which has identity of at least 70% with the sequences shown in a) and codes a protein with improved DKR activity; and 
         [0020]    c) a sequence which is hybridized with the sequences shown in a) under a highly stringent condition and codes a protein with improved DKR activity. 
         [0021]    The third aspect of the application relates to an expression cassette comprising the above mentioned nucleotide coding sequence. 
         [0022]    The fourth aspect of the application relates to a recombinant vector effectively linked with the nucleotide coding sequence, preferably, the recombinant vector is a recombinant expression vector. 
         [0023]    The fifth aspect of the application relates to a host cell comprising the above mentioned recombinant vector. 
         [0024]    The sixth aspect of the application relates to a method for producing a 3R,5S-dihydroxy compound, which includes the following steps: causing the above mentioned DKR mutant or a protein coded by the abovementioned nucleotide coding sequence or a protein obtained by virtue of the abovementioned expression cassette or the abovementioned recombinant vector or the abovementioned host cell to contact with a diketone compound under a condition of making DKR to act for a period of time, 
         [0025]    wherein the diketone compound is a commercial raw material on the market or an easy-to-prepare ketone compound with general formula I: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0026]    where R1 is selected from an aromatic group, an alkyl group, a naphthenic base, an alkyl-substituted aromatic base, a halogen-substituted aryl, an aromatic heterocycl, a heterocycloalkyl or an alkyl-substituted heterocycloalkyl; and R2 is selected from an alkyl group, a naphthenic base, a haloalkyl group or a halogen naphthenic base. 
         [0027]    In an embodiment, the diketone compound is selected from 6-benzyloxy-3,5-dioxo-tert-butyl hexanoate, 6-benzyloxy-3,5-dioxo-neopentyl hexanoate, 6-benzyloxy-3,5-dioxo-methyl hexanoate and 6-benzyloxy-3,5-dioxo-ethyl hexanoate. 
         [0028]    In other words, the DKR mutant gene mutation with improved enzyme activity is obtained by a method of performing gene mutation by taking a DKR gene (shown as SEQ ID NO: 7) of a rhodococcuserythropolis SK121 strain as a parent gene and performing directed screening. 
         [0029]    In the application, an amino acid sequence of the DKR mutant derived from the rhodococcuserythropolis SK121 strain includes the following sequences: 
         [0030]    (1) an amino acid sequence shown as SEQ ID NO: 1: a mutation site is F231W: 
         [0000]    
       
         
               
             
           
               
                 MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAKARFDSL 
               
               
                   
               
               
                 AAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAI 
               
               
                   
               
               
                 KRDTYEKLATVAPEHTVFATNSSTLLPSDLKEFTGRPEKFLALHFANHVW 
               
               
                   
               
               
                 VNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLLVP 
               
               
                   
               
               
                 LLNAASDLLIDGIADPDMVDKTWRIGTGAPWGPFQIMDVVGLTTVYNISS 
               
               
                   
               
               
                 QGGEKQREFADYIKKNYIDEGKLGVAVGDGFYNYKG; 
               
             
          
         
       
     
         [0031]    (2) an amino acid sequence shown as SEQ ID NO: 2: mutation sites are I94V+F231W: 
         [0000]    
       
         
               
             
           
               
                 MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAKARFDSL 
               
               
                   
               
               
                 AAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAVPEDIAI 
               
               
                   
               
               
                 KRDTYEKLATVAPEHTVFATNSSTLLPSDLKEFTGRPEKFLALHFANHVW 
               
               
                   
               
               
                 VNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLLVP 
               
               
                   
               
               
                 LLNAASDLLIDGIADPDMVDKTWRIGTGAPWGPFQIMDVVGLTTVYNISS 
               
               
                   
               
               
                 QGGEKQREFADYIKKNYIDEGKLGVAVGDGFYNYKG; 
               
             
          
         
       
     
         [0032]    (3) an amino acid sequence shown as SEQ ID NO: 3: mutation sites are I94V+V151Q+F231W: 
         [0000]    
       
         
               
             
           
               
                 MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAKARFDSL 
               
               
                   
               
               
                 AAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAVPEDIAI 
               
               
                   
               
               
                 KRDTYEKLATVAPEHTVFATNSSTLLPSDLKEFTGRPEKFLALHFANHVW 
               
               
                   
               
               
                 QNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLLVP 
               
               
                   
               
               
                 LLNAASDLLIDGIADPDMVDKTWRIGTGAPWGPFQIMDVVGLTTVYNISS 
               
               
                   
               
               
                 QGGEKQREFADYIKKNYIDEGKLGVAVGDGFYNYKG; 
               
             
          
         
       
     
         [0033]    (4) an amino acid sequence shown as SEQ ID NO:4: mutation sites are V239I+R257K: 
         [0000]    
       
         
               
             
           
               
                 MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAKARFDSL 
               
               
                   
               
               
                 AAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAI 
               
               
                   
               
               
                 KRDTYEKLATVAPEHTVFATNSSTLLPSDLKEFTGRPEKFLALHFANHVW 
               
               
                   
               
               
                 VNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLLVP 
               
               
                   
               
               
                 LLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDIVGLTTVYNISS 
               
               
                   
               
               
                 QGGEKQKEFADYIKKNYIDEGKLGVAVGDGFYNYKG; 
               
             
          
         
       
     
         [0034]    (5) an amino acid sequence shown as SEQ ID NO: 5: mutation sites are V151Q+R257K: 
         [0000]                    MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAKARFDSL               AAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAIPEDIAI               KRDTYEKLATVAPEHTVFATNSSTLLPSDLKEFTGRPEKFLALHFANHVW               QNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLLVP               LLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVVGLTTVYNISS               QGGEKQKEFADYIKKNYIDEGKLGVAVGDGFYNYKG;            
or
 
         [0035]    (6) an amino acid sequence shown as SEQ ID NO: 6: mutation sites are I94V+V151Q: 
         [0000]    
       
         
               
             
           
               
                 MTELKQITVLGTGVLGSQIAYQTACHGFDVVAYDINAEVIEKAKARFDSL 
               
               
                   
               
               
                 AAAYKAENVEGAKEGKADEALQRITYSYDLGEAVAKADLVIEAVPEDIAI 
               
               
                   
               
               
                 KRDTYEKLATVAPEHTVFATNSSTLLPSDLKEFTGRPEKFLALHFANHVW 
               
               
                   
               
               
                 QNNTAEVMGTESTDPAVYREVVEFAKNIGMVPIELKKEKAGYVLNSLLVP 
               
               
                   
               
               
                 LLNAASDLLIDGIADPDMVDKTWRIGTGAPFGPFQIMDVVGLTTVYNISS 
               
               
                   
               
               
                 QGGEKQREFADYIKKNYIDEGKLGVAVGDGFYNYKG; 
               
             
          
         
       
     
         [0036]    a coding DeoxyriboNucleic Acid (DNA) sequence of the DKR mutant includes the following DNA sequences: 
         [0037]    (1) SEQ ID NO: 9, which is obtained by mutating TTC into TGG at the site of 691-693 bp in the DKR gene sequence shown as SEQ ID NO: 8; 
         [0038]    (2) SEQ ID NO: 10, which is obtained by mutating TTC into TGG at the site of 691-693 bp, and mutating ATT into GTT, GTC, GTA or GTG at the site of 280-282 bp in the DKR gene sequence shown as SEQ ID NO: 8; 
         [0039]    (3) SEQ ID NO: 11, which is obtained by mutating TTC into TGG at the site of 691-693 bp, and mutating ATT into GTT, GTC, GTA or GTG at the site of 280-282 bp, and mutating GTC into CΔA or CAG at the site of 451-453 bp in the DKR gene sequence shown as SEQ ID NO: 8; 
         [0040]    (4) SEQ ID NO: 12, which is obtained by mutating GTC into ATT, ATC or ATA at the site of 751-717 bp, and mutating CGC into ΔAA or ΔAG at the site of 769-771 bp in the DKR gene sequence shown as SEQ ID NO: 8; 
         [0041]    (5) SEQ ID NO: 13: which is obtained by mutating GTC into CΔA or CAG at the site of 451-453 bp, and mutating CGC into ΔAA or ΔAG at the site of 769-771 bp in the DKR gene sequence shown as SEQ ID NO: 8; 
         [0042]    (6) SEQ ID NO: 14, which is obtained by mutating GTC into CΔA or CAG at the site of 451-453 bp, and mutating ATT into GTT, GTC, GTA or GTG at the site of 280-282 bp in the DKR gene sequence shown as SEQ ID NO: 8. 
         [0043]    When the mutant of the application is used for preparing the 3R,5S-dihydroxy compound, an ee value of the obtained 3R,5S-dihydroxy compound is higher than 99% and a de value is about 90%. The DKR mutant is a key pharmaceutical intermediate, and particularly provides an efficient catalyst for synthesis of a chiral dihydroxy hexanoate chain of a statin drug, so that the industrial production cost of the 3R,5S-dihydroxy compound is greatly reduced. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0044]      FIG. 1  is a chemical reaction process of synthesis of a 3R,5S-dihydroxy compound according to the application; 
           [0045]      FIG. 2  is a chemical reaction equation of synthesis of 3R,5S-dihydroxy-6-benzyloxy-tert-butyl hexanoate according to the application; 
           [0046]      FIG. 3  is a chemical reaction equation of synthesis of 3R,5S-dihydroxy-6-benzyloxy-neopentyl hexanoate according to the application; 
           [0047]      FIG. 4  is a three-dimensional structure mimic diagram of DKR bound with Nicotinamide Adenine Dinucleotide (NAD); and 
           [0048]      FIG. 5  is a three-dimensional structure mimic diagram of a DKR marking effective mutation sites. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0049]    In the application, a site-directed saturation mutagenesis method is adopted to mutate a parent DKR gene, thereby changing an amino acid sequence of DKR and implement changing of a protein structure and function, then a DKR mutant with greatly improved enzyme activity is obtained by a directed screening method, and the enzyme activity is improved to be more than twice and even triple that of the parent DKR, thereby greatly reducing industrial production cost of a 3R,5S-dihydroxy compound. In some embodiments, an ee value of an obtained product is higher than 99% and a de value is about 90%. 
         [0050]    According to an embodiment of the application, a amount of the DKR mutant of the application used in a process to produce a 3R,5S-dihydroxy compound is only 34% of the amount of DKR coded by a parent gene, and the mutant is suitable for industrial application. 
         [0051]    In an embodiment of the application, the site-directed saturation mutagenesis technology is adopted to perform gene mutation by taking a DKR gene derived from a rhodococcuserythropolis SK121 strain as a starting gene, and then the DKR mutant with improved enzyme activity is obtained by a directed screening method. A mutated amino acid residue of the DKR mutant of the application is positioned at a substrate binding site or an area related to substrate and NAD binding and related to NAD proton transfer. For example, 194 is positioned in a NAD binding area and four amino acids (i.e. V151, F231, V239 and R257) are all positioned in the vicinity of the substrate binding site. Changing these amino acids may improve substrate binding specificity, thereby improving enzyme activity. An experimental result of the application shows that single F231W mutation may remarkably improve the activity of the DKR. Further introducing I94V and/or V151Q mutation on the basis of F231W mutation can further improve the activity of the DKR. Combining R257K mutation and V239I or V151Q mutation can also remarkably improve the activity of the DKR. 
         [0052]    The obtained DKR mutant can be genetically connected to pET-22b(+) and another expression vector by genetic engineering means, and then excessively expressed in  Escherichia coli . A molecular weight of the excessively expressed DKR mutant on SDS-PAGE is about 30KD, and a corresponding di-ketone substrate may be reduced in one step under the conditions of 30° C. and pH6.0 to obtain a 3R,5S-dihydroxy compound with higher optical purity. 
         [0053]    The di-ketone raw material corresponding to the 3R,5S-dihydroxy compound in the application may be a commercial raw material on the market or an easy-to-prepare ketone compound with the general chemical formula 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R1 is selected from an aromatic group, an alkyl group, a naphthenic base, an alkyl-substituted aromatic base, a halogen-substituted aryl, an aromatic heterocycl, a heterocycloalkyl or an alkyl-substituted heterocycloalkyl; and R2 is selected from an alkyl group, a naphthenic base, a haloalkyl group or a halogen naphthenic base. The dihydroxy product is expressed by the following chemical general formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R1 is selected from an aromatic group, an alkyl group, a naphthenic base, an alkyl-substituted aromatic base, a halogen-substituted aryl, an aromatic heterocycl, a heterocycloalkyl or an alkyl-substituted heterocycloalkyl; and R2 is selected from an alkyl group, a naphthenic base, a haloalkyl group or a halogen naphthenic base. 
       DEFINITIONS 
       [0054]    Term “identity” used in the application has a meaning usually known in the field, and those skilled in the art know well rules and standards for measuring identity of different sequences. In the application, sequences limited by different degrees of identity are also required to have improved DKR activity. Those skilled in the art know well a method and means for measure activity of DKR and screen a mutant sequence. Those skilled in the art may easily obtain such a mutant sequence under the guidance of contents of the application. In some embodiments, a sequence of the DKR mutant is an amino acid sequence has identity of at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5% or 99.6% with the sequence shown as SEQ ID NO: 7 or 8 and has or codes a protein with improved DKR activity. For example, one or more amino acid residues in the amino acid sequence may be substituted with conservative amino acids, and for example, the one or more amino acid residues are amino acid residues 1, 2, 3, 4, 5, 6, 8, 9, 10, 15, 20, 30, 40 or 50. Conservative amino acids of amino acids are known in the field. 
         [0055]    Term “improved DKR activity” used in the application refers to that bioactivity of DKR obtained by the site-directed saturation mutagenesis technology is improved compared with that of starting DKR, for example, catalytic activity improvement, substrate spectrum broadening, thermal stability improvement, pH stability improvement or expression amount increase, and for example, is improved by at least 5%, 10%, 20%, 30%, 40%, 50%, 100%, 150%, 200%, 500% or more compared with that of the starting DKR. 
         [0056]    Term “highly stringent condition” used in the application may be defined as follows: (1) weak ionic strength and high temperature are adopted for washing, for example, 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium dodecyl sulfate, 50° C.; (2) a denaturant such as formamide is adopted in a hybridization process, for example, 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer solution with pH6.5, including 750 mM sodium chloride and 75 mM sodium citrate, 42° C.; or (3) 50% formamide, 5×SSC (0.75M NaCl and 0.075M sodium citrate), 50 mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5×Denhardt solution, ultrasonically treated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate are adopted, 42° C., washing is performed in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 42° C., 55° C., and highly stringent washing is performed in 0.1×SSC containing EDTA at 55° C. 
         [0057]    Term “expression cassette” used in the application refers to a linear or ring-shaped nucleic acid molecule, covers DNA and RiboNucleic Acid (RNA) sequences capable of guiding specific nucleotide sequences to be expressed in appropriate host cells, and generally includes a promoter effectively linked with a target polynucleotide, which is randomly effectively linked with a termination signal and/or other regulation elements. The expression cassette may further include sequences required by correct translation of the nucleotide sequences. A coding area usually codes target proteins, and also codes a target functional RNA in a sense or antisense direction, for example, an antisense RNA or a non-translated RNA. An expression cassette including a target polynucleotide sequence may be embedded, referring to that at least one component and at least another component are heterogeneous. An expression cassette may also naturally exist, but is obtained in form of effective recombination for heterogeneous expression. 
         [0058]    Term “effectively linked” used in the application refers to such a link manner that a coding nucleotide is at a proper position of a vector to make the coding nucleotide correctly and smoothly copied, transcribed or expressed. 
         [0059]    Term “vector” used in the application includes any plasmid, cosmid, bacteriophage or  agrobacterium tumefaciens  binary nucleic acid molecule in a double-stranded or single-stranded linear or ring-shaped form, is preferably a recombinant expression vector, may be a prokaryotic expression vector, may also be a eukaryotic expression vector, and is preferably a prokaryotic expression vector. In some embodiments, the recombinant vector is selected from pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31 b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19. In some embodiments, the vector is pET-22b(+). 
         [0060]    Term “host cell” used in the application includes a prokaryotic cell, a yeast or a high eukaryotic cell. A proper prokaryotic cell includes, but not limited to, a  eubacterium , such as gram-negative or gram-positive organism, such as  Escherichia coli  of enterobacteriaceae. Various  Escherichia coli  strains can be obtained publicly. 
         [0061]    Term “condition of making DKR to act” used in the application refers to a condition capable of making the DKR to catalyze its substrate to be transformed into a corresponding product. In some embodiments, the “condition of making the DKR to act” includes the DKR, the substrate of the DKR, a coenzyme and a proper buffer system. 
         [0062]    Term “contact for a period of time” used in the application refers to reacting the DKR mutant with its reaction substrate for enough time under the condition of making the DKR to act to at least partially transform the substrate into a corresponding product. 
         [0063]    Those skilled in the art know that although “include” is adopted for expression when the polynucleotide is defined in the application, it does not mean that other sequences unrelated to the function of the polynucleotide may be freely added to two ends of the polynucleotide. Those skilled in the art know that it is necessary to add proper enzyme digesting sites of a restriction enzyme to the two ends of the polynucleotide or add initiator codons, termination codons and the like. Therefore, defining a bispecific tetravalent antibody by adopting a closed expression may not really cover these conditions. 
         [0064]    Those skilled in the art all know that one or more codons in a nucleotide sequence may be equivalently substituted under the condition of not changing coded amino acids, and for example, the one or more codons are codons, such as 1, 2, 3, 4, 5, 6, 8, 9, 10, 15, 20, 30, 40 or 50 condons. The codon application table is well-known in the field. 
         [0065]    The application will be further described below with nonrestrictive embodiments, those skilled in the art know that many modifications may be made to the application without departing from the spirit of the application, and these modifications also fall within the scope of the application. 
         [0066]    The following experimental methods are all conventional methods if there are no special annotations, and adopted experimental materials may all be easily obtained from commercial companies if there are no special annotations. Various antibodies adopted in the following embodiments of the application are all commercial standard antibodies. 
       EMBODIMENT 
     Embodiment 1: Site-Directed Saturation Mutagenesis of DKR (SEQ ID NO: 7) Derived from a  Rhodococcus erythropolis  SK121 Strain 
       [0067]    An amino acid sequence of the DKR simulates a three-dimensional structure of a protein on website Swiss-model, then binding of a substrate and the protein is simulated by Docking, and Pymol analysis is finally performed to select an amino acid related to substrate and NAD binding and related to NAD proton transfer as a mutated amino acid ( FIG. 4 ). 
         [0068]    A corresponding mutation primer (Table 1) is designed by Primmer5.0 according to the mutated amino acid and its flanking sequences (the mutated amino acid refers to mutation site in Table 1). A complete linear fragment is obtained by whole-plasmid Polymerase Chain Reaction (PCR) by taking a pET22b(+) expression vector (purchased from Novagen and with a product number 69744) containing a DKR gene as a template, and after the parent template is removed by DPn I digestion, the PCR product is transferred into  Escherichia coli  BL21 (DE3) and coated in an LB culture dish containing 50 μg/ml ampicillin for culture overnight at 37° C. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Site-directed saturation mutagenesis primer sequence 
               
             
          
           
               
                 SEQ ID NO. 
                 Mutation site 
                 Primer name 
                 Primer sequence 
               
               
                   
               
               
                 15 
                 194 
                 194-Forward 
                 GGTCATCGAGGCANNNCCCGAGGACATCG 
               
               
                 16 
                   
                 194-Reverse 
                 CGATGTCCTCGGGNNNTGCCTCGATGACC 
               
               
                   
               
               
                 17 
                 E96 
                 E96-Forward 
                 GTCATCGAGGCAATTCCCNNNGACATCGCCATCAAGCG 
               
               
                 18 
                   
                 E96-Reverse 
                 CGCTTGATGGCGATGTCNNNGGGAATTGCCTCGATGAC 
               
               
                   
               
               
                 19 
                 R102 
                 R102-Forward 
                 GAGGACATCGCCATCAAGNNNGACACCTACGAGAAGCTTG 
               
               
                 20 
                   
                 R102-Reverse 
                 CAAGCTTCTCGTAGGTGTCNNNCTTGATGGCGATGTCCTC 
               
               
                   
               
               
                 21 
                 T124 
                 T124-Forward 
                 CTACCAACTCCTCGNNNCTGCTGCCGAGCG 
               
               
                 22 
                   
                 T124-Reverse 
                 CGCTCGGCAGCAGNNNCGAGGAGTTGGTAG 
               
               
                   
               
               
                 23 
                 S123 
                 S123-Forward 
                 CGCTACCACTCCNNNACGCTGCTGCCGAG 
               
               
                 24 
                   
                 S123-Reverse 
                 CTCGGCAGCAGCGTNNNGGAGTTGGTAGCG 
               
               
                   
               
               
                 25 
                 H148 
                 H148-Forward 
                 CACTTCGCAAATNNNGTGTGGGTCAAC 
               
               
                 26 
                   
                 H148-Reverse 
                 GTTGACCCACACNNNATTTGCGAAGTGG 
               
               
                   
               
               
                 27 
                 V151Q 
                 V151-Forward 
                 CAAATCACGTGTGGNNNAACAACACTGCC 
               
               
                 28 
                   
                 V151-Reverse 
                 GGCAGTGTTGTTNNNCCACACGTGATTTG 
               
               
                   
               
               
                 29 
                 E156 
                 E156-Forward 
                 CAACAACACTGCCNNNGTCATGGGCACCG 
               
               
                 30 
                   
                 E156-Reverse 
                 CGGTGCCCATGACNNNGGCAGTGTTGTTG 
               
               
                   
               
               
                 31 
                 K189 
                 K189-Forward 
                 GAACTCAAGAAGGAGNNNGCGGGCTACGTACTC 
               
               
                 32 
                   
                 K189-Reverse 
                 GAGTACGTAGCCCGCNNNCTCCTTCTTGAGTTC 
               
               
                   
               
               
                 33 
                 G191 
                 G191-Forward 
                 GAACTCAAGAAGGAGAAGGCGNNNTACGTACTCAACTCGC 
               
               
                 34 
                   
                 G191-Reverse 
                 GCGAGTTGAGTACGTANNNCGCCTTCTCCTTCTTGAGTTC 
               
               
                   
               
               
                 35 
                 L194 
                 L194-Forward 
                 CGGGCTACGTANNNAACTCGCTCCTGG 
               
               
                 36 
                   
                 L194-Reverse 
                 CCAGGAGCGAGTTNNNTACGTAGCCCG 
               
               
                   
               
               
                 37 
                 W223 
                 W223-Forward 
                 GGTCGACAAGACGNNNCGTATCGGCACCGG 
               
               
                 38 
                   
                 W223-Reverse 
                 CCGGTGCCGATACGNNNCGTCTTGTCGACC 
               
               
                   
               
               
                 39 
                 F231 
                 F231-Forward 
                 TATCGGCACCGGAGCCCCGNNNGGCCCCTTCCAGATCATG 
               
               
                 40 
                   
                 F231-Reverse 
                 CATGATCTGGAAGGGGCCNNNCGGGGCTCCGGTGCCGATA 
               
               
                   
               
               
                 41 
                 M237 
                 M237-Forward 
                 GCCCCTTCCAGATCNNNGACGTCGTCGGGTTG 
               
               
                 42 
                   
                 M237-Reverse 
                 CAACCCGACGACGTCNNNGATCTGGAAGGGGC 
               
               
                   
               
               
                 43 
                 V239 
                 V239-Forward 
                 CTTCCAGATCATGGACNNNGTCGGGTTGACCAC 
               
               
                 44 
                   
                 V239-Reverse 
                 GTGGTCAACCCGACNNNGTCCATGATCTGGAAG 
               
               
                   
               
               
                 45 
                 V240 
                 V240-Forward 
                 GATCATGGACGTCNNNGGGTTGACCAC 
               
               
                 46 
                   
                 V240-Reverse 
                 GTGGTCAACCCNNNGACGTCCATGATC 
               
               
                   
               
               
                 47 
                 N247 
                 N247-Forward 
                 GACCACCGTCTACNNNATCTCCTCCCAGG 
               
               
                 48 
                   
                 N247-Reverse 
                 CCTGGGAGGAGATNNNGTAGACGGTGGTC 
               
               
                   
               
               
                 49 
                 Q251 
                 Q251-Forward 
                 CAACATCTCCTCCNNNGGCGGCGAGAAGC 
               
               
                 50 
                   
                 Q251-Reverse 
                 GCTTCTCGCCGCCNNNGGAGGAGATGTTG 
               
               
                   
               
               
                 Si 
                 R257 
                 R257-Forward 
                 CCCAGGGCGGCGAGAAGCAGNNNGAATTCGCCGACTACATCAAG 
               
               
                 52 
                   
                 R257-Reverse 
                 CTTGATGTAGTCGGCGAATTCNNNCTGCTTCTCGCCGCCCTGGG 
               
               
                   
               
               
                 53 
                 L273 
                 L273-Forward 
                 CATCGACGAGGGCAAGNNNGGCGTTGCTGTC 
               
               
                 54 
                   
                 L273-Reverse 
                 GACAGCAACGCCNNNCTTGCCCTCGTCGATG 
               
               
                   
               
               
                 55 
                 A276 
                 A276-Forward 
                 CGAGGGCAAGCTCGGCGTTNNNGTCGGCGACGGCTTCTAC 
               
               
                 56 
                   
                 A276-Reverse 
                 GTAGAAGCCGTCGCCGACNNNAACGCCGAGCTTGCCCTCG 
               
               
                   
               
             
          
         
       
     
       Embodiment 2: Preliminary Screening of a DKR Mutant 
       [0069]    According to the contents of embodiment 1, a single colony in the culture dish is selected and inoculated to a 96-deep-hole plate, 0.5 millilitre of LB liquid culture medium containing 50 μg/ml ampicillin is pre-added into each hole, shaking culture is performed for 3 h at 37° C., then IPTG is added until a final concentration is 0.2 mM, induced expression is performed for 16 h at 18° C., centrifugation is performed at 6,000 g for 10 min to collect bacteria, cells of the bacteria are disrupted by an ultrasonic disruptor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), centrifugation is performed at 4° C. for 20 min at 10,000 g to obtain supernatant, and activity preliminary screening is performed by virtue of a microplate reader. 30 μl of DMSO is added into the 96-hole plate, 1.5 μl of a main raw material 6-benzyloxy-3,5-dioxo-tert-butyl hexanoate (dissolved in DMSO at 30 mg/ml), 2.5 μl of NADH (20 mg/mL) and 216 μl of a phosphate buffer solution (100 mM, Ph=6.0) are added into a 96-hole plate, background detection is performed at 340 nm, 50 μl of prepared mutant enzyme liquid is added into each hole, and a change of an absorption photometry value at 340 nm is immediately detected at 30° C. 
         [0070]    An enzyme activity calculation formula: enzyme activity (u/mL)=(ΔA×60× V 1 )/(6.22×t×V 2 )
       ΔA: an changed value of absorption photometry in a reaction process;   V 1 : a total volume of a reaction system;   6.22: an extinction coefficient;   t: detection time of ΔA; and   V 1 : the volume of the added enzyme liquid.       
 
       Embodiment 3: Secondary Screening of a DKR Mutant 
       [0076]    A mutant of which enzyme activity is higher than that of the parent strain in embodiment 2 is inoculated to 500 ml of LB liquid culture medium containing 50 μg/ml ampicillin, shaking culture is performed at 37° C. until OD 600 =0.6, IPTG is added until a final concentration is 0.2 mM, and induced expression is performed at 18° C. After induction is performed for 16 h, centrifugation is performed at 6,000 g for 10 min to collect bacteria. Cells of the bacteria are disrupted by an ultrasonic disruptor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), and centrifugation is performed at 4° C. for 20 min at 10,000 g to obtain supernatant for activity detection. 0.05 g of main raw material 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (6-benzyloxy-3,5-dioxo-tert-butyl hexanoate) and 0.5 ml of polyethylene glycol PEG-400 are added into a 10 ml reaction flask, 4.0 ml of phosphate buffer solution (100 mM, pH=6.0) is added after the raw material is dissolved, and the main raw material is uniformly dispersed in the buffer solution; and 1.5 mg of NAD + , 20.6 mg of ammonium formate, 10 mg of coenzyme formate dehydrogenase and 0.5 ml of DKR are added, the pH of the system is 6.0, and the system temperature is preserved at 30+/−3° C. for 16 h, then Thin Layer Chromatography (TLC) tracking is performed, a system with an obvious transformation product point and an unobvious main raw material point is selected for ethyl acetate extraction, standing is performed for liquid separation, and an organic phase is extracted for High Performance Liquid Chromatography (HPLC) analysis. 
         [0077]    A mutant of which catalytic activity is higher than that of the parent strain is selected for sequencing, mutation site analysis and scale-up culture, repeated measurement of catalytic activity to confirm mutants, and the catalytic activity of mutants F231W (SEQ ID NO: 1), I94V+F231W (SEQ ID NO: 2), I94V+V151Q+F231W (SEQ ID NO: 3), V239I+R257K (SEQ ID NO: 4), V151Q+R257K (SEQ ID NO: 5) and I94V+V151Q (SEQ ID NO: 6) is remarkably improved compared with that of the parent strain, and secondary screening results are shown in Table 2. Computer simulation analysis is performed on a three-dimensional structure of the DKR by adopting software, wherein 194 is positioned in a NAD binding area, four amino acids V151, F231, V239 and R257 are all positioned in the vicinity of substrate binding sites, and changing these amino acids may improve substrate binding specificity, thereby improving enzyme activity ( FIG. 5 ). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Activity comparison of 3R,5S-dihydroxy-6-benzyloxy-tert- 
               
               
                 butyl hexanoate prepared by DKR parent strain and mutants 
               
             
          
           
               
                 SEQ ID 
                   
                 Enzyme 
                 Transfor- 
                   
                   
               
               
                 NO 
                 Site 
                 content  a   
                 mation 
                 DE % 
                 EE % 
               
               
                   
               
               
                 1 
                 F231W 
                 3 wt 
                 82.70 
                 87.66 
                 100.00 
               
               
                 2 
                 I94V + F231W 
                 2 wt 
                 72.91 
                 89.89 
                 100.00 
               
               
                 3 
                 I94V + V151Q + 
                 2 wt 
                 78.31 
                 89.44 
                 100.00 
               
               
                   
                 F231W 
               
               
                 4 
                 V239I + R257K 
                 2 wt 
                 68.26 
                 85.24 
                 100.00 
               
               
                 5 
                 V151Q + R257K 
                 2 wt 
                 62.27 
                 88.69 
                 100.00 
               
               
                 6 
                 I94V + V151Q 
                 3 wt 
                 68.46 
                 88.14 
                 100.00 
               
               
                 7 
                 parent strain 
                 6 wt 
                 62.26 
                 87.45 
                 100.00 
               
               
                   
               
               
                   a  refers to a wet weight of recombinant cells of each DKR mutant required by transformation of 1 g substrate; and 1 wt refers to that transformation of 1 g of main raw material requires 1 g of DKR mutant recombinant wet cells. 
               
             
          
         
       
     
       Embodiment 4: Cloning and Expression of a DKR Mutant 
       [0078]    In order to facilitate expression and identification of a DKR mutant, compatible restriction enzyme digesting sites are designed at 5′ and 3′ ends of its gene. Enzyme digesting is performed respectively on a target gene and pET-22b(+) (other expression plasmids expressing protein in  Escherichia coli  can also be used) at the same time by adopting Nde I and Xho I, connection reaction is performed on larger fragments of the enzyme-digested target gene and plasmid by virtue of T4 DNA ligase, a connection product is transformed into a competent cell of an  Escherichia coli  DH5α strain, and an LB culture flat plate containing 50 μg/ml ampicillin is coated with the transformed competent cell for culture overnight at 37° C. 
         [0079]    A single colony grown on the culture dish is selected and inoculated to an LB liquid culture medium containing 50 mg/ml ampicillin, shaking culture is performed overnight at 37° C., bacteria are collected for plasmid extraction, PCR identification and dual-enzyme digestion identification, then a correctly cloned vector is named as pET22b(+)-R-M and transformed into  Escherichia coli  BL21 (DE3), and the LB culture flat plate containing the 50 mg/ml ampicillin is coated with the transformed  Escherichia coli  BL21 (DE3) for culture overnight at 37° C. A single colony grown on the culture flat plate is selected and inoculated to 5 ml of LB liquid culture medium containing 50 mg/ml ampicillin for identification with colony PCR, and subsequent induced expression is performed on  Escherichia coli  containing a correct expression vector. The bacteria liquid is transferred to 500 ml of LB liquid culture medium containing 50 mg/ml ampicillin, shaking culture is performed at 37° C. until OD 600 =0.5˜0.6, IPTG is added until the final concentration is 0.2˜1.0 mM respectively, induced expression is performed for 10 to 16 h at 18˜25° C., the bacteria solution is extracted, centrifugation is performed at 6,000 g for 10 min to collect bacteria, and the bacteria are frozen and preserved for later use at −20° C. Cells of the bacteria are disrupted by an ultrasonic disruptor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd.), centrifugation is performed for 20 min at 10,000 g and 4° C. to obtain supernatant and precipitates, and SDS-PAGE detection is performed on the supernant by virtue of a vertical electrophoresis device. A molecular weight of the expressed DKR mutant on SDS-PAGE is about 30KD. 
       Embodiment 5: Application of DKR Mutant I94V+F231W to the Preparation of a 3R,5S-Dihydroxy Compound 
       [0080]    A diketone compound consistent with general formula I 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (general formula 1) is selected as an initial raw material, where R 1  is selected from an aromatic group, an alkyl group, a naphthenic base, an alkyl-substituted aromatic base, a halogen-substituted aryl, an aromatic heterocycl, a heterocycloalkyl or an alkyl-substituted heterocycloalkyl; and R 2  is selected from an alkyl group, a naphthenic base, a haloalkyl group or a halogen naphthenic base. The dihydroxy product is expressed by the following chemical general formula II: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R 1  is selected from an aromatic group, an alkyl group, a naphthenic base, an alkyl-substituted aromatic base, a halogen-substituted aryl, an aromatic heterocycl, a heterocycloalkyl or an alkyl-substituted heterocycloalkyl; and R 2  is selected from an alkyl group, a naphthenic base, a haloalkyl group or a halogen naphthenic base. 
         [0081]    (1) application of the DKR mutant I94V+F231W to the preparation of 3R,5S-dihydroxy-6-benzyloxy-tert-butyl hexanoate 
         [0082]    5 g of main raw material 6-benzyloxy-3,5-dioxo-tert-butyl hexanoate: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and 20 ml of polyethylene glycol PEG-400 are added into a 250 ml reaction flask, 160 ml of phosphate buffer solution (100 mM, pH=6.0) is added after the raw materials are dissolved, and the main raw material is uniformly dispersed in the buffer solution; 0.15 g of NAD + , 20.6 g of ammonium formate, 0.25 g of coenzyme formate dehydrogenase and crude enzyme liquid of 2 wt DKR mutant I94V+F231W are added, the pH of the system is 6.0, and temperature is preserved for 17 h at 30+/−3° C.; and reaction is terminated by 200 ml of ethyl acetate, filtration is performed by 125 g of diatomaceous earth, extraction is performed twice with 200 ml of ethyl acetate, standing is performed for liquid separation, and an organic phase is dried, filtered and concentrated to obtain a crude product, wherein a proportion of 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (6-benzyloxy-3,5-dioxo-tert-butyl hexanoate) in the system of the product 3R,5S-dihydroxy-6-benzyloxy-tert-butyl hexanoate is 86˜91%, the yield is 80˜86%, the ee value is higher than 99.5% and the de value is 88˜95%. 
         [0083]    NMR data of the obtained product is as follows: 400 Hz, CDCl 3 : δ 7.29 (m, 5H), 4.54 (s, 2H), 4.22 (m, 1H), 4.07 (m, 1H), 3.45˜3.40 (m, 4H), 2.41 (d, 2H), 1.65 (t, 2H) and 1.43 (S, 9H). 
         [0084]    Catalytic activity over the main raw material 6-benzyloxy-3,5-dioxo-tert-butyl hexanoate and reaction methods of the other five DKR mutants are similar, so as not to be repeatedly described here. 
         [0085]    (2) application of the DKR mutant I94V+F231W to preparation of 3R,5S-dihydroxy-6-benzyloxy-neopentyl hexanoate 
         [0086]    5 g of main raw material 6-benzyloxy-3,5-dioxo-neopentyl hexanoate: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and 10 ml of polyethylene glycol PEG-400 are added into a 500 ml reaction flask, 160 ml of phosphate buffer solution (100 mM, pH=6.0) is added after the raw materials are dissolved, and the main raw material is uniformly dispersed in the buffer solution; 0.15 g of NAD + , 20.6 g of ammonium formate, 0.25 g of coenzyme formate dehydrogenase and crude enzyme liquid of 9 wt DKR mutant I94V+F231W are added, the pH of the system is 6.0, and temperature is preserved at 30+/−3° C. for 17 h; and reaction is terminated by 200 ml of ethyl acetate, filtration is performed by 125 g of diatomaceous earth, extraction is performed twice with 200 ml of ethyl acetate, standing is performed for liquid separation, and an organic phase is dried, filtered and concentrated to obtain a crude product, wherein a proportion of 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (3R,5S-dihydroxy-6-benzyloxy-neopentyl hexanoate) in the system of the product 3R,5S-dihydroxy-6-benzyloxy-neopentyl hexanoate is 80˜90%, the yield is 75˜85%, the ee value is higher than 99.3% and the de value is 90˜96%. 
         [0087]    NMR data of the obtained product is as follows: 400 Hz, CDCl 3 : 7.26˜7.35 ppm (m, 5H), 4.56 ppm (s, 2H), 4.24 ppm (m, 1H), 4.08 ppm (m, 1H), 3.79 ppm (s, 1H), 3.45 ppm (d, 2H), 3.30 ppm (d, 1H), 2.44 ppm (d, 2H), 1.79 ppm (q, 2H), 1.60˜1.65 ppm (dd, 2H), 1.43 ppm (s, 6H) and 0.88 ppm (t, 3H). 
         [0088]    Catalytic activity over the main raw material 6-benzyloxy-3,5-dioxo-neopentyl hexanoate and reaction methods of the other five DKR mutants are similar, so as not to be repeatedly described here.