Preparation of (R)-3-Hydroxybutyric Acid or Its Salts by One-Step Fermentation

Abstract

The subject invention relates to a genetically modified microorganism comprising a (R)-3-hydroxybutyric acid pathway and being able of producing (R)-3-hydroxybutyric acid, and a method of preparing (R)-3-hydroxybutyric acid or a salt thereof using the genetically modified microorganism.

What is claimed is: 
     
         1 . A genetically modified microorganism having (R)-3-hydroxybutyrate biosynthesis capability, comprising one or more exogenous nucleic acids encoding at least one (R)-3-hydroxybutyric acid pathway enzyme selected from succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase. 
     
     
         2 . The genetically modified microorganism of  claim 1 , wherein the genetically modified microorganism is able to produce (R)-3-hydroxybutyric acid in a pathway comprising the following steps:
 (i) converting pyruvic acid and coenzyme A to acetyl-CoA,   (ii) converting acetyl-CoA into acetoacetyl-CoA,   (iii) converting acetoacetyl-CoA to acetoacetic acid, and   (iv) converting acetoacetic acid to (R)-3-hydroxybutyric acid.   
     
     
         3 . The genetically modified microorganism of  claim 1 , wherein the exogenous nucleic acid is codon-optimized gene for expression in the genetically modified microorganism. 
     
     
         4 . The genetically modified microorganism of  claim 1 , wherein the pathway enzyme encoded by exogenous nucleic acids is succinyl-CoA transferase, acetoacetyl-CoA synthase, or 3-HB dehydrogenase. 
     
     
         5 . The genetically modified microorganism of  claim 1 , wherein the pathway enzyme encoded by exogenous nucleic acids are succinyl-CoA transferase, acetoacetyl-CoA synthase and 3-HB dehydrogenase. 
     
     
         6 . The genetically modified microorganism of  claim 1 , wherein the exogenous nucleic acid encoding succinyl-CoA transferase is at least 90% identical to the nucleotide sequence of SEQ ID NO:1. 
     
     
         7 . The genetically modified microorganism of  claim 1 , wherein the exogenous nucleic acid encoding acetoacetyl-CoA synthase is at least 90% identical to the nucleotide sequence of SEQ ID NO:2. 
     
     
         8 . The genetically modified microorganism of  claim 1 , wherein the exogenous nucleic acid encoding 3-HB dehydrogenase is at least 90% identical to the nucleotide sequence of SEQ ID NO:3. 
     
     
         9 . The genetically modified microorganism of  claim 1 , wherein the microorganism is nonpathogenic microorganism selected from the group consisting of  Corynebacterium glutamicum, Bacillus subtilis, Brevibacterium lactofermentum, Brevibacterium difficile, Brevibacterium flavum,  and  Brevibacterium breve.    
     
     
         10 . The genetically modified microorganism of  claim 9 , wherein the microorganism is  Corynebacterium glutamicum  or  Bacillus subtilis.    
     
     
         11 . The genetically modified microorganism of  claim 9 , wherein the microorganism is  Corynebacterium glutamicum.    
     
     
         12 . The genetically modified microorganism of  claim 11 , wherein the  Corynebacterium glutamicum  is the the strain deposited at the China General Microbiological Culture Collection Center under the accession number CGMCC No. 13111. 
     
     
         13 . A method for producing (R)-3-hydroxybutyric acid, comprising using the genetically modified microorganism of  claim 1 . 
     
     
         14 . The method of  claim 13 , wherein the method is a one-step fermentation process comprising fermenting the genetically modified microorganism of  claim 1 . 
     
     
         15 . A recombinant nucleic acid segment encoding succinyl-CoA transferase, acetoacetyl-CoA synthase, and 3-HB dehydrogenase.