Process for producing optically active-4-halo-3-hydroxybutanoate

Abstract

There are provided a polynucleotide sequence coding for an amino acid sequence capable of preferentially producing (S)-4-bromo-3-hydroxybutanoate by asymmetrically reducing 4-bromo-3-oxobutanoate, A DNA construct having a promoter in operative linkage with the polynucleotide sequence, a recombinant vector containing the polynucleotide sequence, a transformant, a recombinant vector and the like.

1 . An isolated polynucleotide sequence having: 
 a) a polynucleotide sequence coding for an amino acid sequence represented by SEQ ID NO: 1;    b) a polynucleotide sequence that hybridizes with a polynucleotide sequence coding for an amino acid sequence represented by SEQ ID NO: 1 to form a hybrid, under stringent conditions, the amino acid sequence being an amino acid sequence of an enzyme capable of preferentially producing (S)-4-bromo-3-hydroxybutanoate by asymmetrically reducing 4-bromo-3-oxobutanotate;    c) a polynucleotide sequence represented by SEQ ID NO: 2,    d) a polynucleotide coding for an amino acid sequence of SEQ ID NO: 1 having additional 6 amino acids of Trp-Ile-Ser-Thr-Lys-Leu (SEQ ID NO:37) at the C-terminal of the amino acid sequence;    e) a polynucleotide sequence having 80% or more sequence identity with the polynucleotide sequence coding for an amino acid sequence of SEQ ID NO: 1, or    f) a polynucleotide sequence that hybridizes with a polynucleotide sequence coding for an amino acid sequence represented by SEQ ID NO: 1 to form a hybrid under high ion concentrations of 450 to 900 mM sodium chloride and 45 to 90 mM sodium citrate at 65° C. and the hybride formed is maintained as a hybrid after being kept at 65° C. for 30 minutes under a low ion concentration of 15 to 300 mM sodium chloride and 1.5 to 30 mM sodium citrate and 0.1 to 1.0 wt % of SDS, the amino acid sequence being an amino acid sequence of an enzyme capable of preferentially producing (S)-4-bromo-3-hydroxybutanoate by asymmetrically reducing 4-bromo-3-oxobutanoate.    
     
     
         2 . canceled  
     
     
         3 . canceled  
     
     
         4 . canceled  
     
     
         5 . canceled  
     
     
         6 . canceled  
     
     
         7 . canceled  
     
     
         8 . canceled  
     
     
         9 . A recombinant vector containing 
 A) a polynucleotide construct as defined in  claim 1 , and    B) a polynucleotide coding for an enzyme capable of converting oxidized β-nicotinamide-adenine dinucleotide phosphate into a reduced form, wherein the enzyme is glucose dehydrogenase.    
     
     
         10 . (canceled).  
     
     
         11 . A transformant having the vector according to  claim 9 .  
     
     
         12 . A transformant according to  claim 11 , wherein the host is a microorganism.  
     
     
         13 . A transformant according to  claim 12 , wherein the microorganism is  E. coli.    
     
     
         14 . canceled  
     
     
         15 . A protein having: 
 i) an amino acid sequence of SEQ ID NO: 1;    ii) an amino acid sequence encoded by a polynucleotide sequence that hybridizes under stringent conditions with a polynucleotide sequence of SEQ ID NO: 2 coding for an amino acid sequence of a protein capable of preferentally producing (S)-4-bromo-3-hydroxybutanoate by asymmetrically reducing 4-bromo-3-oxobutanoate; or    iii) an amino acid sequence of SEQ ID NO: 1, wherein one or more amino acids are deleted, replaced or added, said amino acid sequence being an amino acid sequence of a protein capable of preferentially producing (S)-4-bromo-3-hydroxybutanoate by asymmetrically reducing 4-bromo-3-oxobutanoate.    
     
     
         16 . A process for producing (S)-4-halo-3-hydroxybutanoate, which comprises reacting 4-halo-3-oxobutanoic acid ester with the protein as defined in  claim 15 , a transformant, which produces said protein or a treated product thereof.  
     
     
         17 . A process according to  claim 16 , which comprises allowing the coexistence of an enzyme capable of converting the oxdized β-nicotinamide-adenine dinucleotide phosphate into a reduced form.  
     
     
         18 . A process according to  claim 17 , wherein the enzyme capable of converting an oxidized β-nicotinamide-adenine dinucleotide phosphate into a reduced form is a glucose dehydrogenase.  
     
     
         19 . A process according to  claim 17 , wherein the 4-halo-3-oxobutanoic acid ester is contacted with the transformant as defined in any one of  claims 11  to  14  or a treated product thereof.  
     
     
         20 . A process according to  claim 16 ,  17 ,  18  or  19 , wherein the 4-halo-3-oxobutanoic acid ester is represented by a formula (1):  
       
         
           
           
               
               
           
         
       
       wherein R 1  represents an alkyl group, and R 2  represents a methyl group which is substituted with a halogen atom, which process comprises reacting 4-halo-3-oxobutanoic acid ester of formula (2):  
       
         
           
           
               
               
           
         
         wherein R 1  and R 2  represent the same as defined above.  
       
     
     
         21 . A process for producing an optically active 3-hydroxybutanoic acid ester of formula (1a):  
       
         
           
           
               
               
           
         
       
       wherein R 1  represents an alkyl group, and R 20  represents a methyl group which may be substituted with a halogen atom, which process comprises reacting 3-oxobutanoic acid ester of formula (2a):  
       
         
           
           
               
               
           
         
       
       wherein R 1  and R 20  represent the same as defined above, with whole cells of a microorganism or a treated product thereof, which microorganism belongs to  Penicillium citrinum, Cryptcoccus humicolus,  or  Bacillus alvei  and is capable of asymmetrically reducing the oxo group at 3-position of the compound of formula (2a) to corresponding 3-hydroxy group.  
     
     
         22 . A process according to  claim 21 , wherein R 2  represents a halomethyl group.  
     
     
         23 . A process according to  claim 21  or  22 , wherein the microorganism is a strain selected from the group of  Penicillium citrinum  (IFO4631),  Cryptcoccus humicolus  (IFO1527), and  Bacillus alvei  (IFO3343t).  
     
     
         24 . A process for producing an optically active 4-bromo-3-hydroxybutanoate of formula (1b):  
       
         
           
           
               
               
           
         
       
       wherein R 1  represents a (C2-C8) alkyl group, which process comprises reacting 4-bromo-3-oxobutanoate of formula (2b):  
       
         
           
           
               
               
           
         
       
       wherein R 1  represents the same as defined above, with an enzyme having: 
 iv) an amino acid sequence of SEQ ID NO: 34;  
 v) an amino acid sequence encoded by a polynucleotide sequence that hybridizes, under stringent conditions, with a polynucleotide sequence of SEQ ID NO: 34, wherein said amino acid sequence is an amino acid sequence of a protein capable of preferentially producing optically active 4-bromo-3-hydroxybutanoate by asymmetrically reducing 4-bromo-3-oxobutanoate; and  
 vi) an amino acid sequence of SEQ ID NO: 3, wherein one or more amino acids are deleted, replaced or added, said amino acid sequence being an amino acid sequence of a protein capable of preferentially producing optically active 4-bromo-3-hydroxybutanoate by asymmetrically reducing 4-bromo-3-oxbutanoate.  
 
     
     
         25 . A process for producing 4-cyano-3-hydroxybutanoic acid, which comprises reacting 4-bromo-3-hydroxybtanoic acid ester with a metal cyanide in the presence of an alkaline earth metal hydroxide and an alkaline earth metal halogenide.  
     
     
         26 . A process according to  claim 25 , which further comprises the step of reacting the 4-cyano-3-hydroxybutanoic acid with dialkyl sulfate to produce 4-cyano-3-hydroxybutanoic acid alkyl ester.  
     
     
         27 . A process according to  claim 25  or  26 , wherein the alkaline earth metal hydroxide is calcium hydroxide, and the alkaline earth metal halogenide is calcium chloride.  
     
     
         28 . A process according to  claim 25 , wherein the 4-bromo-3-hydroxybutanoic acid ester is (C1-C8) alkyl 4-bromo-3-hydroxybutanoate, and the dialkyl sulfate is dimethyl or diethyl sulfate.  
     
     
         29 . A process according to  claim 25  or  26 , wherein the 4-bromo-3-hydroxybutanoic acid and hydroxybutanoic acid are optically active compounds.  
     
     
         30 . A process according to  claim 25  or  26 , wherein 4-bromo-3-hydroxybutanoic acid is (S)-4-bromo-3-hydroxybutanoic acid ester and 4-cyano-3-hydroxybutanoic acid is (R)-4-cyano-3-hydroxybutanoic acid.  
     
     
         31 . A process for producing (R)-4-cyano-3-hydroxybutanoic acid, which comrpises 
 producing (S)-4-bromo-3-hydroxybutanoic acid ester by asymmetrically reducing the 4-bromo-3-oxobutanoic acid ester, and    reacting (S)-4-bromo-3-hydroxybutanoic acid ester with a metal cyanide in the presence of an alkaline earth metal hydroxide and an alkaline earth metal halogenide.    
     
     
         32 . A process according to  claim 31 , wherein the asymmetrical reduction is conducted by a microorganism or treated product thereof capable of asymmetrically reducing the 4-bromo-3-oxobutanoic acid ester to (S)-4-bromo-3-hydroxybutanoic acid ester.  
     
     
         33 . A process according to  claim 32 , wherein the microorganism is a microorganism belong to  Penicillium citrinum.    
     
     
         34 . A process according to  claim 31 ,  32  or  33 , wherein (S)-4-bromo-3-hydroxybutanoic acid ester and 4-bromo-3-oxobutanoic acid ester are (C1-C8) alkyl ester.  
     
     
         35 . A process according to  claim 33 , wherein the microorganism is a strain  Penicillium citrinum  (IFO 4631).  
     
     
         36 . A process according to any one of  claims 31  to  35 , wherein the alkaline earth metal hydroxide is calcium hydroxide and the alkaline earth halogenide is calcium chloride.  
     
     
         37 . A process according to  claim 31 , which further comprises the step of reacting (R)-4-cyano-3-hydroxybutanoic acid with dialkyl sulfate to produce (R)-4-cyano-3-hydroxybutanoic acid alkyl ester.  
     
     
         38 . A process according to  claim 32 , wherein the alkyl group of the dialkyl sulfate is a methyl or ethyl group.