Patent Publication Number: US-2022213514-A1

Title: Genetically modified microorganism for producing 3-hydroxyhexanedioic acid, (e)-hex-2-enedioic acid and/or hexanedioic acid, and production method for said chemicals

Description:
TECHNICAL FIELD 
     The present invention relates to a genetically modified microorganism in which a nucleic acid encoding a polypeptide involved in the production of a substance of interest is introduced or the expression of the polypeptide is enhanced, and to a method of producing the substance by using the microorganism. 
     BACKGROUND ART 
     3-Hydroxyadipic acid (IUPAC name: 3-hydroxyhexariediiiic acid), α-hydromuconic acid (IUPAC name: (E)-hex-2-enedioic acid), and adipic acid (IUPAC name: hexanedioic acid) are dicarboxylic acids containing six carbon atoms. These dicarboxylic acids can be polymerized with a polyhydric alcohol or a polyfunctional amine, to be used as raw materials for the production of polyesters or polyamides, respectively. Additionally, these dicarboxylic acids can be used alone after ammonia addition at a terminal position in these chemicals to form lactams as raw materials for the production of polyamides. 
     The following documents related to the production of 3-hydroxyadipic acid or α-hydromuconic acid using a microorganism are known. 
     Patent Document 1 describes a method of producing 1,3-butadiene by using a microorganism in which a relevant metabolic pathway is modified, wherein 3-hydroxyadipic acid (3-hydroxyadipate) is described to be a metabolic intermediate in the metabolic pathway for biosynthesis of 1,3-butadiene from acetyl-CoA and succinyl-CoA. 
     Patent Document 2 describes a method of producing muconic acid by using a microorganism in which a relevant metabolic pathway is modified, wherein α-hydromuconic acid (2,3-dehydroadipate) is described to be a metabolic intermediate in the metabolic pathway for biosynthesis of trans,trans-muconic acid from acetyl-CoA and succinyl-CoA. 
     Patent Documents 3 and 4 describe a method of producing adipic acid and hexamethylene diamine (HMDA) by using a non-natural microorganism, wherein the biosynthetic pathways for these substances are described to share a common reaction to synthesize 3-oxoadipyi-CoA from acetyl-CoA and succinyi-CoA but diverge after the synthesis of 3-oxoadipyl-CoA. Furthermore, Patent Document 3 describes the pyruvate kinase gene as a candidate gene that is additionally deleted from the metabolic pathway to improve the HMDA formation coupled with proliferation for the IIMDA production, but a potential relationship between pyruvate kinase deficiency and increased adipic acid production is not mentioned in this document. 
     Additionally, all the biosynthetic pathways mentioned in Patent Documents 1 to 4 are described to share a common enzymatic reaction that reduces 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. 
     Patent Documents 5 and 6 describe methods of producing 3-hydroxyadipic acid and α-hydromuconic acid by using a microorganism of the genus  Serratia  , respectively. The patent documents disclose that the efficiency of producing 3-hydroxyadipic acid and α-hydromuconic acid can be increased particularly by enhancing the activity of an acyl transferase that catalyzes a reaction to produce 3-oxoadipyl-CoA from acetyl-CoA and succinyl-CoA, but these documents have no description related to pyruvate kinase. 
     Moreover, a method of modifying a microorganism based on an in silky) analysis is disclosed in Patent Document 7, in which the production of succinic acid is increased by deleting genes encoding pyruvate kinase and a phosphotransferase system enzyme in  Escherichia coli  ( E. coli ), pykF, pykA, and ptsG, and culturing the resulting  E. coli  bacteria under anaerobic conditions. 
     PRIOR ART DOCUMENTS 
     Patent Documents 
     
         
         Patent Document 1: JP 2013-535203 A 
         Patent Document 2: US 20110124911 A1 
         Patent Document 3: JP 2015-146810 A 
         Patent Document 4: JP 2011-515111 A 
         Patent Document 5: WO 2017209102 
         Patent Document 6: WO 2017209103 
         Patent Document 7: JP 2008-527991 A 
       
    
     SUMMARY OF THE INVENTION 
     Problems to be Solved by the Invention 
     Patent Documents 1 and 2 describe metabolic pathways by which the microorganisms can produce 3-hydroxyadipic acid and α-hydromuconic acid, but have no description about interruption of the metabolic pathways to allow the microorganisms to secrete 3-hydroxyadipic acid or α-hydromuconic acid into culture medium. Moreover, the prior studies described in Patent Documents 1 to 4 have not examined whether or not 3-hydroxyadipic acid, α-hydromuconic acid, or adipic acid can be actually produced by using a non-natural microorganism in which a nucleic acid encoding an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA has been introduced. Accordingly, it is not known whether the enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, as described in Patent Documents 1 to 4, also exhibits excellent activity in the production of 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid. 
     Accordingly, an object of the present invention is to provide a genetically modified microorganism for producing 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid in high yield and a method of producing a substance by using the modified microorganism, wherein the modified microorganism is based on a genetically modified microorganism in which a nucleic acid encoding an enzyme that exhibits excellent activity in 3-oxoadipyl-CoA reduction reaction is introduced or the expression of the enzyme is enhanced, and wherein the modified microorganism is further modified to have an altered metabolic pathway. 
     Means for Solving the Problem 
     The inventors intensively studied in order to achieve the above-described object and consequently found that 3-hydroxyadipic acid, a-hydromuconic acid, andor adipic acid can be produced in high yield by a genetically modified microorganism in which a nucleic acid encoding an enzyme that exhibits excellent activity in 3-oxoadipyl-CoA reduction reaction is introduced or the expression of the enzyme is enhanced and the function of pyruvate kinase is further impaired, to complete the present invention. 
     That is, the present invention provides the following:
     (1) A genetically modified microorganism in which a nucleic acid encoding any one of the polypeptides described in (a) to (c) below is introduced or the expression of the polypeptide is enhanced and the function of pyruvate kinase is impaired:   

     (a) a polypeptide composed of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7; 
     (b) a polypeptide composed of the same amino acid sequence as that represented by any one of SEQ ID NOs: 1 to 7, except that one or several amino acids are substituted, deleted, inserted, andor added, and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA; 
     (c) a polypeptide composed of an amino acid sequence with a sequence identity of not less than 70% to the sequence represented by any one of SEQ ID NOs: 1 to 7 and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.
     (2) The genetically modified microorganism according to (1), wherein a polypeptide selected from the above (b) and (c) contains a region composed of an amino acid sequence represented by SEQ ID NO: 173.   (3) The genetically modified microorganism according to (2), wherein the amino acid sequence represented by SEQ ID NO: 173 contains a phenylalanine or leucine residue at the 13th amino acid position from the N terminus, a leucine or glutamine residue at the 15th amino acid position from the N terminus, a lysine or asparagine residue at the 16th amino acid position from the N terminus, a glycine or serine residue at the 17th amino acid position from the N terminus, a proline or arginine residue at the 19th amino acid position from the N terminus, and a leucine, methionine, or valine residue at the 21st amino acid position from the N terminus.   (4) The genetically modified microorganism according to any one of (1) to (3), which is a genetically modified microorganism belonging to a genus selected from the group consisting of Escherichia, Serratia, Hafnia, and Pseudomonas.   (5) The genetically modified microorganism according to any one of (1) to (4), which has an ability to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA and an ability to generate 3-hydroxyadipic acid from 3-hydroxyadipyl-CoA.    (6) The genetically modified microorganism according to any one of (1) to (4), which has an ability to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA, an ability to generate 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA, and an ability to generate α-hydromuconic acid from 2,3-dehydroadipyl-CoA.    (7) The genetically modified microorganism according to any one of (1) to (4), which has an ability to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA, an ability to generate 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA, an ability to generate adipyl-CoA from 2,3-dehydroadipyl-CoA, and an ability to generate adipic acid from adipyl-CoA.   (8) The genetically modified microorganism according to any one of (1) to (7), wherein the function of a phosphotransferase system enzyme is further impaired.   (9) A method of producing 3-hydroxyadipic acid, comprising culturing the genetically modified microorganism according to any one of (1) to (5) and (8) in a culture medium containing a carbon source as a raw material for fermentation.   (10) A method of producing u-hydromuconic acid, comprising culturing the genetically modified microorganism according to any one of (1) to (4), (6) and (8) in a culture medium containing a carbon source as a raw material for fermentation.   (11) A method of producing adipic acid, comprising culturing the genetically modified microorganism according to any one of (1) to (4), (7) and (8) in a culture medium containing a carbon source as a raw material for fermentation.   (12) A method of producing one or more substances selected from the group consisting of 3-hydroxyadipic acid, α-hydromuconic acid, and adipic acid, comprising culturing a genetically modified microorganism in a culture medium containing a carbon source as a raw material for fermentation, wherein a nucleic acid encoding a polypeptide encoded by the 3-hydroxybutyryl-CoA dehydrogenase gene of a microorganism of the genus  Serratia,  which forms a gene cluster with 5-aminolevulinic acid synthase gene in the microorganism, is introduced or the expression of the polypeptide is enhanced and the function of pyruvate kinase is impaired in the genetically modified microorganism.   (13) The method according to (12), wherein the genetically modified microorganism is a microorganism in which the function of a phosphotransferase system enzyme is further impaired.   

     Effects of the Invention 
     The genetically modified microorganism according to the present invention, which expresses an enzyme that exhibits excellent activity in a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA and, furthermore, has an impaired pyruvate kinase function, can produce 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid in high yield compared to a parental strain of the microorganism in which pyruvate kinase is not impaired. 
     The method of producing a substance according to the present invention uses the genetically modified microorganism which is excellent in the production of 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid via production of 3-hydroxyadipyl-CoA and thus can greatly increase the production of those substances. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         FIG. 1  shows a gene cluster composed of a 3-hydroxybutyryl-CoA dehydrogenase gene and a 5-aminolevulinic acid synthase gene. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The microorganism according to the present invention is a genetically modified microorganism in which a nucleic acid encoding a polypeptide described in (a) to (c) below is introduced or the expression of the polypeptide is enhanced and the function of pyruvate kinase is impaired: 
     (a) a polypeptide composed of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 7; 
     (b) a polypeptide composed of the same amino acid sequence as that represented by any one of SEQ ID NOs: 1 to 7, except that one or several amino acids are substituted, deleted, inserted, andor added, and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA; 
     (c) a polypeptide composed of an amino acid sequence with a sequence identity of not less than 70% to the sequence represented by any one of SEQ ID NOs: 1 to 7 and having activity in reduction of 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. 
     An enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA is hereinafter referred to as “3-oxoadipyl-CoA reductase” in the specification. Additionally, 3-hydroxyadipic acid, α-hydromuconic acid, and adipic acid may be abbreviated as 3IIA, HMA, and ADA, respectively, in this specification. 
     In the present invention, introducing a nucleic acid means introducing a nucleic acid from the outside to the inside of a microorganism to give the microorganism an ability to produce a polypeptide encoded by the nucleic acid. The method of introduction of a nucleic acid is not limited to a particular method, and examples of the method that can be used include a method in which a nucleic acid of interest is integrated into an expression vector capable of autonomous replication in a microorganism and then integrated into a host microorganism, and a method in which a nucleic acid of interest is integrated into the genome of a microorganism. 
     In the present invention, enhancing the expression of a polypeptide means enhancing the expression of a polypeptide which a microorganism originally has. The method of enhancement of expression is not limited to a particular method, and examples of the method include a method in which a nucleic acid encoding a polypeptide of interest is increased in copy number, and a method in which a promoter region or a ribosome-binding sequence upstream of the region coding for a polypeptide of interest is modified. These methods may be carried out individually or in combination. 
     Additionally, one or more of the above nucleic acids may be introduced. Moreover, the introduction of a nucleic acid and the enhancement of polypeptide expression may be combined. 
     For the polypeptide used in the present invention and composed of the same amino acid sequence as that represented by any one of SEQ ID NOs: 1 to 7, except that one or several amino acids are substituted, deleted, inserted, andor added, and having 3-oxoadipyl-CoA reductase activity, the range represented by the phrase “one or several” is preferably 10 or less, more preferably 5 or less, especially preferably 4 or less, and most preferably 1 or 2 or less. In the case of amino acid substitution, the activity of the original polypeptide is more likely to be maintained when an amino acid(s) isare replaced by an amino acid(s) with similar properties (so-called conservative substitution). That is, the physiological properties of the original polypeptide are often maintained when an amino acid(s) isarc replaced by an amino acid(s) with similar properties. Therefore, in the case of substitution, a given amino acid is preferably replaced by another amino acid with similar properties. That is, the 20 amino acids that make up natural proteins can be divided into groups of amino acids with similar properties, such as neutral amino acids with a less polar side chain (Gly, Ile, Val, Leu. Ala, Met, Pro), neutral amino acids with a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), and basic amino acids 
     (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). It is often the case that substitution between amino acids in the same group does not change the properties of the original polypeptide. 
     For the polypeptide used in the present invention and having an amino acid sequence with a sequence identity of not less than 70% to the sequence represented by any one of SEQ ID NOs: 1 to 7 and having 3-oxoadipyl-CoA reductase activity, the sequence identity is preferably not less than 80%, more preferably not less than 85%, further preferably not less than 90%, still further preferably not less than 95%, yet further preferably not less than 97%, and even further preferably not less than 99%. 
     In the present invention, the term “sequence identity” means a ratio (percentage) of the number of identical amino acid or nucleotide residues relative to the total number of amino acid or nucleotide residues over the overlapping portion of an amino acid sequence alignment (including an amino acid corresponding to the translation start site) or a nucleotide sequence alignment (including the start codon), which is obtained by aligning two amino acid or nucleotide sequences with or without introduction of gaps for an optimal match, and is calculated by the following formula (1). In the formula (1), the length of a shorter sequence being compared is not less than 400 amino acids; in cases where the length of the shorter sequence is less than 400 amino acids, the sequence identity is not defined. The sequence identity can be easily determined using BLAST (Basic Local Alignment Search Tool), an algorithm widely used in this field. For example, BLAST is publicly available on a website, such as that of NCB1 (National Center for Biotechnology Information) or KEGG (Kyoto Encyclopedia of Genes and Genomes), on which the sequence identity can be easily determined using default parameters. Additionally, the sequence identity can also be determined using a similar function implemented in a software program such as Genetyx. 
     
       
         
           
             
                 
             
             ⁢ 
             
               ( 
               1 
               ) 
             
           
         
       
       
         
           
             
               Sequence 
               ⁢ 
               
                   
               
               ⁢ 
               identity 
               ⁢ 
               
                   
               
               ⁢ 
               
                 ( 
                 % 
                 ) 
               
             
             = 
             
               the 
               ⁢ 
               
                   
               
               ⁢ 
               number 
               ⁢ 
               
                   
               
               ⁢ 
               of 
               ⁢ 
               
                   
               
               ⁢ 
               
                 matches 
                 ⁢ 
                 
                     
                 
                 ( 
                 
                   without 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   counting 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   the 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   number 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   of 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   gaps 
                 
                 ) 
               
               ⁢ 
               
                 / 
               
               ⁢ 
               the 
               ⁢ 
               
                   
               
               ⁢ 
               length 
               ⁢ 
               
                   
               
               ⁢ 
               of 
               ⁢ 
               
                   
               
               ⁢ 
               a 
               ⁢ 
               
                   
               
               ⁢ 
               shorter 
               ⁢ 
               
                   
               
               ⁢ 
               sequence 
               ⁢ 
               
                   
               
               ⁢ 
               
                 ( 
                 
                   excluding 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   the 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   terminal 
                   ⁢ 
                   
                       
                   
                   ⁢ 
                   gaps 
                 
                 ) 
               
               × 
               100 
             
           
         
       
     
     By using a function of Genetyx (% Identity Matrix) to calculate sequence identities based on the formula (1) among the amino acid sequences represented by SEQ ID NOs: 1 to 7, the lowest sequence identity of 71.51% is found between the sequences represented by SEQ ID NOs: 2 and 4, and the sequence identities among the amino acid sequences represented by SEQ ID NOs: 1 to 7 are found to be at least not less than 70%. The results of calculation of sequence identity using Genetyx are presented in Table 1. In Tables 1 to 5 below, the numbers in the leftmost column represent SEQ ID NOs. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 [GENETYX: identity Matrix] 
               
               
                 *Gaps are NOT taken into account. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 1  Serratia   
                 2  Serratia   
                 3  Serratia   
                 4  Serratia   
                 5  Serratia   
                 6  Serratia   
                 7  Serratia   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 [%] 
                   
                   
                   
                   
                   
                   
                   
               
               
                 1  Serratia marcescens  ATCC13880 
                 * 
               
               
                 2  Serratia nematodiphila  DSM21420 
                 98.23 
                 * 
               
               
                 3  Serratia plymuthica  NBRC102599 
                 72.10 
                 71.51 
                 * 
               
               
                 4  Serratia proteamaculans  568 
                 72.29 
                 71.51 
                 86.24 
                 * 
               
               
                 5  Serratia ureilytica  Lr5/4 
                 90.76 
                 90.76 
                 72.88 
                 73.28 
                 * 
               
               
                 6  Serratia  sp. BW106 
                 72.29 
                 71.90 
                 87.03 
                 92.33 
                 73.67 
                 * 
               
               
                 7  Sierratia liquefaciens  FK01 
                 72.29 
                 71.70 
                 84.67 
                 86.83 
                 73.47 
                 87.81 
                 * 
               
               
                 [Match Count/Length] 
               
               
                 1  Serratia marcescens  ATCC13880 
                 * 
               
               
                 2  Serratia nematodiphila  DSM21420 
                 500/509 
                 * 
               
               
                 3  Serratia plymuthica  NBRC102599 
                 367/509 
                 364/509 
                 * 
               
               
                 4  Serratia proteamaculans  568 
                 368/509 
                 364/509 
                 439/509 
                 * 
               
               
                 5  Serratia ureilytica  Lr5/4 
                 462/509 
                 462/509 
                 371/509 
                 373/509 
                 * 
               
               
                 6  Serratia  sp. BW106 
                 368/509 
                 366/509 
                 443/509 
                 470/509 
                 375/509 
                 * 
               
               
                 7  Serratia liquefaciens  FK01 
                 368/509 
                 365/509 
                 431/509 
                 442/509 
                 374/509 
                 447/509 
                 * 
               
               
                   
               
            
           
         
       
     
     When each of the amino acid sequences represented by SEQ ID NOs: 1 to 7 as queries was compared using BLASTP to all the amino acid sequences registered in the NCBI amino acid database (non-redundant protein sequences) to determine sequence identities, all sequences with a sequence identity of not less than 70% were found to be from bacteria of the genus Serrano. 
     All the polypeptides represented by SEQ ID NOs: 1 to 7 as described above in (a) contain a common sequence 1 composed of 24 amino acid residues and represented by SEQ ID NO: 173 within a region from the 1 5th to the 38th amino acid residues from the N terminus (hereinafter, an amino acid residue at the n-th position from the N terminus may conveniently be represented by n “a.a.”; for example, the region from the 15th to the 38th amino acid residues from the N tei tinus may be thus simply represented by “15 to 38 a.a.”). In the common sequence 1, Xaa represents an arbitrary amino acid residue, and the 13 a.a. is preferably a phenylalanine or leucine, and the 15 a.a. is preferably a leucine or glutamine, and the 16 a.a. is preferably a lysine or asparagine, and the 17 a.a. is a glycine or serine, more preferably a glycine, and the 19 a.a. is preferably a proline or arginine, and the 21 a.a. is preferably a leucine, methionine, or valine. The common sequence 1 corresponds to the region including the NADtbinding residue and the surrounding amino acid residues. In the NAD ± -binding residues, the 24th amino acid residue in the common sequence 1 is an aspartic acid, as described in Biochimie., 2012 Feb. 94 (2): 471-8., but in the common sequence 1, the residue is an asparagine, which is characteristic. It is thought that the presence of the common sequence 1 causes the polypeptides represented by SEQ ID NOs: 1 to 7 to show excellent enzymatic activity as 3-oxoadipyl-CoA reductases. 
     The polypeptides as described above in (b) and (c) also preferably contain the common sequence 1 composed of 24 amino acid residues and represented by SEQ ID NO: 173 within a region from 1 to 200 a.a. The common sequence is more preferably located within a region from 1 to 150 a.a., and further preferably within a region from 1 to 100 a.a. Specific examples of the polypeptides include those with the amino acid sequences represented by SEQ ID NOs: 8 to 86. The amino acid sequences represented by SEQ ID NOs: 8 to 86 contain the common sequence 1 composed of 24 amino acid residues and represented by SEQ ID NO: 173 within a region from 15 to 38 a.a. The amino acid sequences represented by SEQ ID NOs: 8 to 86 have a sequence identity of not less than 90% to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 7. The results of calculation of sequence identity using Genetyx are presented in Tables 2-1 to 2-3 and Tables 3-1 to 3-3. 
     
       
         
           
               
             
               
                 TABLE 2-1 
               
             
            
               
                   
               
               
                 [GENETYX: 1 Identity Matrix] 
               
               
                 *Gaps are NOT taken into account. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 [%] 
                 1  Serratia   
                 2  Serratia   
                 3  Serratia   
                 4  Serratia   
                 5  Serratia   
                 6  Serratia   
                 7  Serratia   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                   Serratia marcescens  ATCC13880 
                 * 
                   
                   
                   
                   
                   
                   
               
               
                 2 
                   Serratia nematodiphila  DSM21420 
                 98.23 
                 * 
               
               
                 3 
                   Serratia plymuthica  NBRC102599 
                 72.10 
                 71.51 
                 * 
               
               
                 4 
                   Serratia proteamaculans  568 
                 72.29 
                 71.51 
                 86.24 
                 * 
               
               
                 5 
                   Serratia ureilytica  Lr5/4 
                 90.76 
                 90.76 
                 72.88 
                 73.28 
                 * 
               
               
                 6 
                   Serratia  sp. BW106 
                 72.29 
                 71.90 
                 87.03 
                 92.33 
                 73.67 
                 * 
               
               
                 7 
                   Serratia liquefaciens  FK01 
                 72.29 
                 71.70 
                 84.67 
                 86.83 
                 73.47 
                 87.81 
                 * 
               
               
                 8 
                   Serratia  sp. S119 
                 94.89 
                 94.30 
                 72.88 
                 72.49 
                 91.55 
                 73.08 
                 72.88 
               
               
                 9 
                   Serratia  sp. YD25 
                 92.33 
                 92.33 
                 72.49 
                 72.49 
                 93.51 
                 72.69 
                 72.88 
               
               
                 10 
                   Serratia  sp. FS14 
                 98.62 
                 99.60 
                 71.70 
                 71.70 
                 91.15 
                 72.10 
                 72.10 
               
               
                 11 
                   Serratia  sp. HMSC15F11 
                 94.89 
                 94.30 
                 73.28 
                 73.28 
                 91.35 
                 73.47 
                 73.47 
               
               
                 12 
                   Serratia  sp. JKS000199 
                 90.76 
                 90.76 
                 72.69 
                 73.08 
                 99.41 
                 73.47 
                 73.28 
               
               
                 13 
                   Serratia  sp. TEL 
                 90.56 
                 90.56 
                 72.88 
                 73.28 
                 99.80 
                 73.67 
                 73.47 
               
               
                 14 
                   Serratia  sp. ISTD04 
                 90.56 
                 90.56 
                 72.49 
                 73.08 
                 99.41 
                 73.47 
                 73.28 
               
               
                 15 
                   Serratia  sp. SCB1 
                 90.76 
                 90.76 
                 72.88 
                 73.28 
                 99.60 
                 73.47 
                 73.47 
               
               
                 16 
                   Serratia  sp. S4 
                 72.10 
                 71.31 
                 86.44 
                 98.62 
                 73.08 
                 91.94 
                 86.64 
               
               
                 17 
                   Serratia  sp. C-1 
                 72.49 
                 71.90 
                 98.03 
                 86.05 
                 73.28 
                 86.64 
                 84.08 
               
               
                 18 
                   Serratia marcescens  532 
                 99.80 
                 98.03 
                 72.29 
                 72.10 
                 90.56 
                 72.10 
                 72.10 
               
               
                 19 
                   Serratia marcescens  2880STDY5683033 
                 99.60 
                 97.83 
                 72.10 
                 72.29 
                 90.37 
                 72.10 
                 72.29 
               
               
                 20 
                   Serratia marcescens  WW4 
                 98.42 
                 99.41 
                 71.90 
                 71.90 
                 90.96 
                 72.29 
                 71.90 
               
               
                 21 
                   Serratia marcescens  K27 
                 98.23 
                 99.21 
                 71.31 
                 71.31 
                 90.96 
                 71.70 
                 71.70 
               
               
                 22 
                   Serratia marcescens  280 
                 98.42 
                 99.41 
                 71.70 
                 71.70 
                 90.96 
                 72.10 
                 72.10 
               
               
                 23 
                   Serratia marcescens  19F 
                 98.42 
                 99.41 
                 71.51 
                 71.70 
                 90.96 
                 72.10 
                 72.10 
               
               
                 24 
                   Serratia marcescens  1185 
                 98.23 
                 99.60 
                 71.31 
                 71.31 
                 90.37 
                 71.70 
                 71.51 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 2-2 
               
               
                   
               
             
            
               
                 25 
                   Serratia marcescens  S217 
                 98.23 
                 99.21 
                 71.31 
                 71.51 
                 90.96 
                 71.90 
                 71.90 
               
               
                 26 
                   Serratia marcescens  KHCo-24B 
                 98.03 
                 99.80 
                 71.31 
                 71.31 
                 90.56 
                 71.70 
                 71.90 
               
               
                 27 
                   Serratia marcescens  Z6 
                 98.03 
                 99.01 
                 71.70 
                 71.90 
                 90.56 
                 72.29 
                 71.90 
               
               
                 28 
                   Serratia marcescens  546 
                 97.83 
                 99.21 
                 71.51 
                 71.70 
                 90.37 
                 72.10 
                 71.70 
               
               
                 29 
                   Serratia nematodiphila  HB307 
                 98.03 
                 99.80 
                 71.31 
                 71.51 
                 90.56 
                 71.90 
                 71.70 
               
               
                 30 
                   Serratia marcescens  VGH107 
                 98.03 
                 99.01 
                 71.31 
                 71.51 
                 90.56 
                 71.90 
                 71.90 
               
               
                 31 
                   Serratia marcescens  MCB 
                 95.48 
                 95.28 
                 72.29 
                 72.69 
                 91.15 
                 72.88 
                 72.69 
               
               
                 32 
                   Serratia marcescens  AH0650 
                 95.67 
                 95.48 
                 72.29 
                 72.69 
                 90.76 
                 73.28 
                 72.69 
               
               
                 33 
                   Serratia marcescens  UMH12 
                 95.48 
                 95.28 
                 72.10 
                 72.49 
                 90.56 
                 73.08 
                 72.49 
               
               
                 34 
                   Serratia  sp. OMLW3 
                 95.48 
                 95.28 
                 72.29 
                 72.49 
                 90.76 
                 73.28 
                 72.69 
               
               
                 35 
                   Serratia marcescens  UMH11 
                 95.28 
                 95.08 
                 72.10 
                 72.69 
                 90.56 
                 73.47 
                 72.49 
               
               
                 36 
                   Serratia marcescens  UMH1 
                 95.08 
                 94.89 
                 72.29 
                 72.49 
                 90.17 
                 73.08 
                 72.29 
               
               
                 37 
                   Serratia marcescens  2880STDY5683020 
                 95.48 
                 94.89 
                 73.08 
                 72.69 
                 92.14 
                 73.28 
                 73.08 
               
               
                 38 
                   Serratia marcescens  99 
                 95.48 
                 94.69 
                 73.28 
                 72.88 
                 91.55 
                 73.67 
                 73.28 
               
               
                 39 
                   Serratia marcescens  374 
                 94.89 
                 94.69 
                 72.29 
                 72.29 
                 90.17 
                 73.08 
                 72.29 
               
               
                 40 
                   Serratia marcescens  2880STDY5683036 
                 95.28 
                 94.49 
                 73.08 
                 72.69 
                 91.35 
                 73.47 
                 73.08 
               
               
                 41 
                   Serratia marcescens  2880STDY5683034 
                 95.28 
                 94.69 
                 73.08 
                 72.69 
                 91.94 
                 73.28 
                 73.08 
               
               
                 42 
                   Serratia marcescens  2880STDY5682892 
                 95.28 
                 94.69 
                 73.28 
                 72.88 
                 91.94 
                 73.47 
                 73.28 
               
               
                 43 
                   Serratia marcescens  SM39 
                 95.08 
                 94.49 
                 73.28 
                 72.69 
                 92.14 
                 73.28 
                 73.28 
               
               
                 44 
                   Serratia marcescens  189 
                 95.08 
                 94.49 
                 73.28 
                 72.88 
                 92.14 
                 73.47 
                 73.28 
               
               
                 45 
                   Serratia marcescens  SMB2099 
                 95.08 
                 94.49 
                 73.47 
                 72.69 
                 91.74 
                 73.67 
                 73.47 
               
               
                 46 
                   Serratia marcescens  2880STDY5682862 
                 94.89 
                 94.30 
                 73.47 
                 72.88 
                 91.55 
                 73.47 
                 73.47 
               
               
                 47 
                   Serratia marcescens  SE4145 
                 94.89 
                 94.30 
                 73.08 
                 72.49 
                 91.94 
                 73.08 
                 73.08 
               
               
                 48 
                   Serratia marcescens  2880STDY5682876 
                 95.08 
                 94.49 
                 73.28 
                 72.88 
                 91.74 
                 73.47 
                 73.28 
               
               
                 49 
                   Serratia marcescens  709 
                 95.08 
                 94.49 
                 73.08 
                 72.69 
                 91.74 
                 73.28 
                 73.08 
               
               
                 50 
                   Serratia marcescens  MGH136 
                 94.89 
                 94.30 
                 72.88 
                 72.49 
                 91.94 
                 73.08 
                 72.88 
               
               
                 51 
                   Serratia marcescens  2880STDY5682884 
                 94.69 
                 94.10 
                 72.88 
                 72.49 
                 91.74 
                 73.08 
                 73.08 
               
               
                 52 
                   Serratia marcescens  D-3 
                 95.08 
                 94.49 
                 73.08 
                 72.69 
                 91.74 
                 73.28 
                 73.08 
               
               
                 53 
                   Serratia marcescens  2880STDY5682957 
                 94.89 
                 94.30 
                 72.88 
                 72.69 
                 91.55 
                 73.28 
                 72.88 
               
               
                 54 
                   Serratia marcescens  YDC563 
                 94.69 
                 94.10 
                 72.88 
                 72.69 
                 91.35 
                 73.28 
                 72.88 
               
               
                 55 
                   Serratia marcescens  2880STDY5683035 
                 94.80 
                 94.30 
                 73.08 
                 72.69 
                 91.55 
                 73.28 
                 73.08 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 2-3 
               
               
                   
               
             
            
               
                 56 
                   Serratia marcescens  2880STDY5682930 
                 94.69 
                 94.10 
                 72.88 
                 72.49 
                 91.35 
                 73.08 
                 72.88 
               
               
                 57 
                   Serratia marcescens  790 
                 94.49 
                 94.30 
                 73.28 
                 72.88 
                 91.35 
                 73.47 
                 73.28 
               
               
                 58 
                   Serratia marcescens  UMH5 
                 93.51 
                 92.92 
                 72.69 
                 72.88 
                 90.37 
                 72.69 
                 72.49 
               
               
                 59 
                   Serratia marcescens  288OSTDY5682988 
                 93.32 
                 92.73 
                 72.69 
                 72.88 
                 90.17 
                 72.69 
                 72.49 
               
               
                 60 
                   Serratia marcescens  945154301 
                 94.89 
                 94.30 
                 73.28 
                 73.28 
                 91.35 
                 73.67 
                 73.47 
               
               
                 61 
                   Serratia marcescens  at10508 
                 94.69 
                 94.10 
                 73.47 
                 73.47 
                 91.15 
                 73.67 
                 73.67 
               
               
                 62 
                   Serratia marcescens  ML2637 
                 94.49 
                 93.90 
                 73.28 
                 73.47 
                 90.96 
                 73.67 
                 73.67 
               
               
                 63 
                   Serratia marcescens  SM1978 
                 94.30 
                 93.71 
                 73.28 
                 73.28 
                 90.76 
                 73.67 
                 73.67 
               
               
                 64 
                   Serratia marcescens  PWN146 
                 94.10 
                 93.51 
                 72.88 
                 72.88 
                 90.96 
                 72.88 
                 73.28 
               
               
                 65 
                   Serratia marcescens  H1q 
                 92.53 
                 92.53 
                 72.49 
                 72.49 
                 93.51 
                 72.69 
                 73.08 
               
               
                 66 
                   Serratia marcescens  UMH6 
                 91.15 
                 91.15 
                 72.69 
                 73.08 
                 99.60 
                 73.47 
                 73.28 
               
               
                 67 
                   Serratia nematodiphila  WCU338 
                 91.15 
                 91.15 
                 72.69 
                 73.08 
                 99.41 
                 73.47 
                 73.28 
               
               
                 68 
                   Serratia  sp. OLEL1 
                 90.96 
                 90.96 
                 72.88 
                 73.28 
                 99.80 
                 73.67 
                 73.47 
               
               
                 69 
                   Serratia marcescens  7209 
                 90.96 
                 90.96 
                 72.49 
                 72.88 
                 99.41 
                 73.28 
                 73.08 
               
               
                 70 
                   Serratia marcescens  sicaria (Ss1) 
                 90.96 
                 90.96 
                 72.69 
                 73.08 
                 99.41 
                 73.28 
                 73.28 
               
               
                 71 
                   Serratia  sp. OLFL2 
                 90.76 
                 90.76 
                 72.69 
                 73.08 
                 99.60 
                 73.47 
                 73.28 
               
               
                 72 
                   Serratia marcescens  BIDMC 81 
                 90.76 
                 90.76 
                 72.88 
                 73.28 
                 99.60 
                 73.67 
                 73.47 
               
               
                 73 
                   Serratia marcescens  BIDMC 50 
                 90.76 
                 90.76 
                 72.69 
                 73.08 
                 99.21 
                 73.47 
                 73.28 
               
               
                 74 
                   Serratia marcescens  UMH7 
                 90.56 
                 90.56 
                 72.88 
                 73.28 
                 99.80 
                 73.67 
                 73.47 
               
               
                 75 
                   Serratia marcescens  RSC-14 
                 90.56 
                 90.56 
                 72.88 
                 73.47 
                 99.21 
                 73.87 
                 73.67 
               
               
                 76 
                   Serratia marcescens  SIMO3 
                 92.33 
                 92.33 
                 72.29 
                 72.29 
                 93.51 
                 72.49 
                 72.88 
               
               
                 77 
                   Serratia marcescens  90-166 
                 90.17 
                 89.78 
                 72.49 
                 73.47 
                 96.66 
                 73.67 
                 73.08 
               
               
                 78 
                   Serratia marcescens  UMH2 
                 90.76 
                 90.76 
                 72.88 
                 73.28 
                 99.21 
                 73.67 
                 73.47 
               
               
                 79 
                   Serratia plymuthica  A30 
                 72.49 
                 71.90 
                 96.66 
                 85.06 
                 73.47 
                 86.05 
                 83.69 
               
               
                 80 
                   Serratia plymuthica  tumart 205 
                 72.69 
                 72.10 
                 98.03 
                 86.24 
                 73.47 
                 86.64 
                 84.28 
               
               
                 81 
                   Serratia plymuthica  A30 
                 72.29 
                 71.70 
                 98.82 
                 85.65 
                 72.88 
                 86.44 
                 84.08 
               
               
                 82 
                   Serratia plymuthica  4Rx13 
                 72.29 
                 71.70 
                 97.83 
                 85.85 
                 73.08 
                 86.44 
                 84.28 
               
               
                 83 
                   Serratia plymuthica  V4 
                 72.29 
                 71.70 
                 98.42 
                 85.85 
                 71.08 
                 86.44 
                 84.28 
               
               
                 84 
                   Serratia plymuthica  3Rp8 
                 72.29 
                 71.70 
                 98.62 
                 86.05 
                 73.08 
                 86.64 
                 84.08 
               
               
                 85 
                   Serratia proteamaculans  MFPA44A14 
                 72.29 
                 71.90 
                 87.03 
                 92.53 
                 73.28 
                 98.82 
                 87.22 
               
               
                 86 
                   Serratia plymuthica  A153 
                 72.10 
                 71.51 
                 99.21 
                 86.05 
                 72.88 
                 86.64 
                 84.47 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 3-1 
               
               
                   
               
               
                 [Match Count/Length] 
                 1  Serratia   
                 2  Serratia   
                 3  Serratia   
                 4  Serratia   
                 5  Serratia   
                 6  Serratia   
                 7  Serratia   
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                   Serratia marcescens  ATCC13880 
                 * 
                   
                   
                   
                   
                   
                   
               
               
                 2 
                   Serratia nematodiphila  DSM21420 
                 500/509 
                 * 
               
               
                 3 
                   Serratia plymuthica  NBRC102599 
                 367/509 
                 364/509 
                 * 
               
               
                 4 
                   Serratia proteamaculans  568 
                 368/509 
                 364/509 
                 439/509 
                 * 
               
               
                 5 
                   Serratia ureilytica  Lr5/4 
                 482/509 
                 462/509 
                 371/500 
                 373/509 
                 * 
               
               
                 6 
                   Serratia  sp. BW106 
                 368/509 
                 366/509 
                 443/509 
                 470/509 
                 375/509 
                 * 
               
               
                 7 
                   Serratia liquefaciens  FK01 
                 368/509 
                 365/509 
                 431/509 
                 442/509 
                 374/509 
                 447/509 
                 * 
               
               
                 8 
                   Serratia  sp. S119 
                 483/509 
                 480/509 
                 371/509 
                 369/509 
                 466/509 
                 372/509 
                 371/509 
               
               
                 9 
                   Serratia  sp. YD25 
                 470/509 
                 470/509 
                 369/509 
                 369/509 
                 476/509 
                 370/509 
                 371/509 
               
               
                 10 
                   Serratia  sp. FS14 
                 502/509 
                 507/509 
                 365/509 
                 365/509 
                 464/509 
                 367/509 
                 367/509 
               
               
                 11 
                   Serratia  sp. HMSC15F11 
                 483/509 
                 480/509 
                 373/509 
                 373/509 
                 465/509 
                 374/509 
                 374/509 
               
               
                 12 
                   Serratia  sp. JKS000199 
                 402/509 
                 462/509 
                 370/509 
                 372/509 
                 506/509 
                 374/509 
                 373/509 
               
               
                 13 
                   Serratia  sp. TEL 
                 461/509 
                 461/509 
                 371/509 
                 373/509 
                 508/508 
                 375/509 
                 374/509 
               
               
                 14 
                   Serratia  sp. ISTD04 
                 461/509 
                 461/509 
                 369/509 
                 372/509 
                 506/509 
                 374/509 
                 373/509 
               
               
                 15 
                   Serratia  sp. SCBI 
                 462/509 
                 462/509 
                 371/509 
                 373/509 
                 507/509 
                 374/509 
                 374/509 
               
               
                 16 
                   Serratia  sp. S4 
                 367/509 
                 363/509 
                 440/509 
                 502/509 
                 372/509 
                 468/509 
                 441/509 
               
               
                 17 
                   Serratia  sp. C-1 
                 369/509 
                 366/509 
                 499/509 
                 438/509 
                 373/509 
                 441/509 
                 428/509 
               
               
                 18 
                   Serratia marcescens  532 
                 508/509 
                 499/509 
                 368/509 
                 367/509 
                 461/509 
                 367/509 
                 367/509 
               
               
                 19 
                   Serratia marcescens  2880STDY5683033 
                 507/509 
                 498/509 
                 367/509 
                 368/509 
                 460/509 
                 367/509 
                 368/509 
               
               
                 20 
                   Serratia marcescens  WW4 
                 501/509 
                 506/509 
                 366/509 
                 366/509 
                 463/509 
                 368/509 
                 366/509 
               
               
                 21 
                   Serratia marcescens  K27 
                 500/509 
                 505/509 
                 363/509 
                 363/509 
                 463/509 
                 365/509 
                 365/509 
               
               
                 22 
                   Serratia marcescens  280 
                 501/509 
                 506/509 
                 365/509 
                 365/509 
                 463/509 
                 367/509 
                 367/509 
               
               
                 23 
                   Serratia marcescens  19F 
                 501/509 
                 506/509 
                 364/509 
                 365/509 
                 463/509 
                 367/509 
                 367/509 
               
               
                 24 
                   Serratia marcescens  1185 
                 500/509 
                 507/509 
                 363/509 
                 363/509 
                 460/509 
                 365/509 
                 364/509 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 3-2 
               
               
                   
               
             
            
               
                 25 
                   Serratia marcescens  S217 
                 500/509 
                 505/509 
                 363/509 
                 364/509 
                 463/509 
                 366/509 
                 366/509 
               
               
                 26 
                   Serratia marcescens  KHCo-24B 
                 499/509 
                 508/509 
                 363/509 
                 363/509 
                 461/509 
                 365/509 
                 366/509 
               
               
                 27 
                   Serratia marcescens  Z6 
                 499/509 
                 504/509 
                 365/509 
                 366/509 
                 461/509 
                 368/509 
                 366/509 
               
               
                 28 
                   Serratia marcescens  546 
                 498/509 
                 505/509 
                 364/509 
                 365/509 
                 460/509 
                 367/509 
                 365/509 
               
               
                 29 
                   Serratia nematodiphila  MB307 
                 499/509 
                 508/509 
                 363/509 
                 364/509 
                 461/509 
                 366/509 
                 365/509 
               
               
                 30 
                   Serratia marcescens  VGH107 
                 499/509 
                 504/509 
                 363/509 
                 364/509 
                 461/509 
                 366/509 
                 366/509 
               
               
                 31 
                   Serratia marcescens  MCB 
                 486/509 
                 485/509 
                 368/509 
                 370/509 
                 464/509 
                 371/509 
                 370/509 
               
               
                 32 
                   Serratia marcescens  AH0650 
                 487/509 
                 486/509 
                 368/509 
                 370/509 
                 462/509 
                 373/509 
                 370/509 
               
               
                 33 
                   Serratia marcescens  UMH12 
                 486/509 
                 485/509 
                 367/509 
                 369/509 
                 461/509 
                 372/509 
                 369/509 
               
               
                 34 
                   Serratia  sp. OMLW3 
                 486/509 
                 485/509 
                 368/509 
                 369/509 
                 462/509 
                 373/509 
                 370/509 
               
               
                 35 
                   Serratia marcescens  UMH11 
                 485/509 
                 484/509 
                 367/509 
                 370/509 
                 461/509 
                 374/509 
                 369/509 
               
               
                 36 
                   Serratia marcescens  UMH1 
                 484/509 
                 483/509 
                 368/509 
                 369/509 
                 459/509 
                 372/509 
                 368/509 
               
               
                 37 
                   Serratia marcescens  2880STDY568320 
                 486/509 
                 483/509 
                 372/509 
                 370/509 
                 469/509 
                 373/509 
                 372/509 
               
               
                 38 
                   Serratia marcescens  99 
                 486/509 
                 482/509 
                 373/509 
                 371/509 
                 466/509 
                 375/509 
                 373/509 
               
               
                 39 
                   Serratia marcescens  374 
                 483/509 
                 482/509 
                 368/509 
                 368/509 
                 459/509 
                 372/509 
                 368/509 
               
               
                 40 
                   Serratia marcescens  2880STDY5683036 
                 485/509 
                 481/509 
                 372/509 
                 370/509 
                 465/509 
                 374/509 
                 372/509 
               
               
                 41 
                   Serratia marcescens  2880STDY5683034 
                 485/509 
                 482/509 
                 372/509 
                 370/509 
                 468/509 
                 373/509 
                 372/509 
               
               
                 42 
                   Serratia marcescens  2880STDY5682892 
                 485/509 
                 482/509 
                 373/509 
                 371/509 
                 468/509 
                 374/509 
                 373/509 
               
               
                 43 
                   Serratia marcescens  SM39 
                 484/509 
                 481/509 
                 373/509 
                 370/509 
                 469/509 
                 373/509 
                 373/509 
               
               
                 44 
                   Serratia marcescens  189 
                 484/509 
                 481/509 
                 373/509 
                 371/509 
                 469/509 
                 374/509 
                 373/509 
               
               
                 45 
                   Serratia marcescens  SMB2099 
                 484/509 
                 481/509 
                 374/509 
                 370/509 
                 467/509 
                 375/509 
                 374/509 
               
               
                 46 
                   Serratia marcescens  2880STDY5682862 
                 483/509 
                 480/509 
                 374/509 
                 371/509 
                 466/509 
                 374/509 
                 374/509 
               
               
                 47 
                   Serratia marcescens  SE4145 
                 483/509 
                 480/509 
                 372/509 
                 369/509 
                 468/509 
                 372/509 
                 372/509 
               
               
                 48 
                   Serratia marcescens  2880STDY5682876 
                 484/509 
                 481/509 
                 373/509 
                 371/509 
                 467/509 
                 374/509 
                 373/500 
               
               
                 49 
                   Serratia marcescens  709 
                 484/509 
                 481/509 
                 372/509 
                 370/509 
                 467/509 
                 373/509 
                 372/509 
               
               
                 50 
                   Serratia marcescens  MGH136 
                 483/509 
                 480/509 
                 371/509 
                 369/509 
                 468/509 
                 372/509 
                 371/509 
               
               
                 51 
                   Serratia marcescens  2880STDY5682884 
                 482/509 
                 479/509 
                 371/509 
                 369/509 
                 467/509 
                 372/509 
                 372/509 
               
               
                 52 
                   Serratia marcescens  D-3 
                 484/509 
                 481/509 
                 372/509 
                 370/509 
                 467/509 
                 373/509 
                 372/509 
               
               
                 53 
                   Serratia marcescens  2880STDY5682957 
                 483/509 
                 480/509 
                 371/509 
                 370/509 
                 466/509 
                 373/509 
                 371/509 
               
               
                 54 
                   Serratia marcescens  YDC563 
                 482/509 
                 479/509 
                 371/509 
                 370/509 
                 465/509 
                 373/509 
                 371/509 
               
               
                 55 
                   Serratia marcescens  2880STDY5683035 
                 483/509 
                 480/509 
                 372/509 
                 370/509 
                 466/509 
                 373/509 
                 372/509 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 3-3 
               
               
                   
               
             
            
               
                 56 
                   Serratia marcescens  2880STDY5682930 
                 482/509 
                 479/509 
                 371/509 
                 369/509 
                 465/509 
                 372/509 
                 371/509 
               
               
                 57 
                   Serratia marcescens  790 
                 481/509 
                 480/509 
                 373/509 
                 371/509 
                 465/509 
                 374/509 
                 373/509 
               
               
                 58 
                   Serratia marcescens  UMH5 
                 476/509 
                 473/509 
                 370/509 
                 371/509 
                 460/509 
                 370/509 
                 369/509 
               
               
                 59 
                   Serratia marcescens  2880STDY5682988 
                 475/509 
                 472/509 
                 370/509 
                 371/509 
                 459/509 
                 370/509 
                 389/509 
               
               
                 60 
                   Serratia marcescens  945154301 
                 483/509 
                 480/509 
                 373/509 
                 373/509 
                 465/509 
                 375/509 
                 374/509 
               
               
                 61 
                   Serratia marcescens  at 10508 
                 482/509 
                 479/509 
                 374/509 
                 374/509 
                 464/509 
                 375/509 
                 375/509 
               
               
                 62 
                   Serratia marcescens  ML2637 
                 481/509 
                 478/509 
                 373/509 
                 374/509 
                 463/509 
                 375/509 
                 375/509 
               
               
                 63 
                   Serratia marcescens  SM1978 
                 480/509 
                 477/509 
                 373/509 
                 373/509 
                 462/509 
                 375/509 
                 375/509 
               
               
                 64 
                   Serratia marcescens  PWN146 
                 479/509 
                 476/509 
                 371/509 
                 371/509 
                 463/509 
                 371/509 
                 373/509 
               
               
                 65 
                   Serratia marcescens  H1q 
                 471/509 
                 471/509 
                 369/509 
                 369/509 
                 476/509 
                 370/509 
                 372/509 
               
               
                 66 
                   Serratia marcescens  UMH6 
                 464/509 
                 464/509 
                 370/509 
                 372/539 
                 507/509 
                 374/509 
                 373/509 
               
               
                 67 
                   Serratia nematodiphila  WCU338 
                 464/509 
                 464/509 
                 370/509 
                 372/509 
                 506/509 
                 374/509 
                 373/509 
               
               
                 68 
                   Serratia  sp. OLEL1 
                 463/509 
                 463/509 
                 371/509 
                 373/509 
                 508/509 
                 375/509 
                 374/509 
               
               
                 69 
                   Serratia marcescens  7209 
                 463/509 
                 463/509 
                 369/509 
                 371/509 
                 506/509 
                 373/509 
                 372/509 
               
               
                 70 
                   Serratia marcescens  sicaria (Ss1) 
                 463/509 
                 463/509 
                 370/509 
                 372/509 
                 506/509 
                 373/509 
                 373/509 
               
               
                 71 
                   Serratia  sp. OLFL2 
                 462/509 
                 462/509 
                 370/509 
                 372/509 
                 507/509 
                 374/509 
                 373/509 
               
               
                 72 
                   Serratia marcescens  BIDMC 81 
                 462/509 
                 462/509 
                 371/509 
                 373/509 
                 507/509 
                 375/509 
                 374/509 
               
               
                 73 
                   Serratia marcescens  BIDMC 50 
                 462/509 
                 462/509 
                 370/509 
                 372/509 
                 505/509 
                 374/509 
                 373/509 
               
               
                 74 
                   Serratia marcescens  UMH7 
                 461/509 
                 461/509 
                 371/509 
                 373/509 
                 508/509 
                 375/509 
                 374/509 
               
               
                 75 
                   Serratia marcescens  RSC-14 
                 461/509 
                 461/509 
                 371/509 
                 374/509 
                 505/509 
                 376/509 
                 375/509 
               
               
                 76 
                   Serratia marcescens  SMO3 
                 470/509 
                 470/509 
                 368/509 
                 368/509 
                 476/509 
                 369/509 
                 371/509 
               
               
                 77 
                   Serratia marcescens  90-166 
                 459/509 
                 457/509 
                 369/509 
                 374/509 
                 492/509 
                 375/509 
                 372/509 
               
               
                 78 
                   Serratia marcescens  UMH2 
                 462/509 
                 462/509 
                 371/509 
                 373/509 
                 505/509 
                 375/509 
                 374/509 
               
               
                 79 
                   Serratia plymuthica  AS9 
                 369/509 
                 366/509 
                 492/509 
                 433/509 
                 374/509 
                 438/509 
                 426/509 
               
               
                 80 
                   Serratia plymuthica  tumat 205 
                 370/509 
                 367/509 
                 499/509 
                 439/509 
                 374/509 
                 441/509 
                 429/509 
               
               
                 81 
                   Serratia plymuthica  A30 
                 368/509 
                 365/509 
                 503/509 
                 436/509 
                 371/509 
                 440/509 
                 428/509 
               
               
                 82 
                   Serratia plymuthica  4Rx13 
                 368/509 
                 365/509 
                 498/509 
                 437/509 
                 372/509 
                 440/509 
                 429/509 
               
               
                 83 
                   Serratia plymuthica  V4 
                 368/509 
                 365/509 
                 501/509 
                 437/509 
                 372/509 
                 440/509 
                 429/509 
               
               
                 84 
                   Serratia plymuthica  3Rp8 
                 368/509 
                 365/509 
                 502/509 
                 438/509 
                 372/509 
                 441/509 
                 428/509 
               
               
                 85 
                   Serratia proteamaculans  MFPA44A14 
                 368/509 
                 366/509 
                 443/509 
                 471/509 
                 373/509 
                 503/509 
                 444/509 
               
               
                 86 
                   Serratia plymuthica  A153 
                 367/509 
                 364/509 
                 505/509 
                 438/509 
                 371/509 
                 441/509 
                 430/509 
               
               
                   
               
            
           
         
       
     
     The nucleic acids encoding the polypeptides described in (a) to (c) according to the present invention may contain an additional sequence that encodes a peptide or protein added to the original polypeptides at the N terminus andor the C terminus. Examples of such a peptide or protein can include secretory signal sequences, translocation proteins, binding proteins, peptide tags for purification, and fluorescent proteins. Among those peptides or proteins, a peptide or protein with a desired function can be selected depending on the purpose and can be added to the polypeptides of the present invention by those skilled in the art. It should be noted that the amino acid sequence of such a peptide or protein is excluded from the calculation of sequence identity. 
     The nucleic acids encoding the polypeptides represented by SEQ ID NOs: 1 to 86 are not specifically limited, provided that the nucleic acids have nucleotide sequences that can be translated to the amino acid sequences represented by SEQ ID NOs: 1 to 86, and the nucleotide sequences can be deteunined considering the set of codons (standard genetic code) corresponding to each amino acid. In this respect, the nucleotide sequences may be redesigned using codons that are frequently used by a host microorganism used in the present invention. 
     Specific examples of the nucleotide sequences of the nucleic acids that encode the polypeptides with the amino acid sequences represented by SEQ ID NOs: 1 to 86 include the nucleotide sequences represented by SEQ ID NOs: 87 to 172. 
     In the present invention, whether or not a polypeptide encoded by a certain nucleic acid has 3-oxoadipyl-CoA reductase activity is determined as follows: transformants A and B below are produced and grown in a culture test; if 3-hydroxyadipic acid or α-hydromuconic acid is confirmed in the resulting culture medium, it is judged that the nucleic acid encodes a polypeptide having 3-oxoadipyl-CoA reductase activity. The determination method will be described using the scheme 1 below which shows a biosynthesis pathway. 
     
       
         
         
             
             
         
       
     
     The above scheme 1 shows an exemplary reaction pathway required for the production of 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid. In this scheme, the reaction A represents a reaction that generates 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA. The reaction B represents a reaction that generates 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA. The reaction C represents a reaction that generates 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. The reaction D represents a reaction that generates adipyl-CoA from 2,3-dehydroadipyl-CoA. The reaction E represents a reaction that generates 3-hydroxyadipic acid from 3-hydroxyadipyl-CoA. The reaction F represents a reaction that generates α-hydromuconic acid from 2,3-dehydroadipyl-CoA. The reaction G represents a reaction that generates adipic acid from adipyl-CoA. 
     The transformant A has enzymes that catalyze the reactions A, E, and F. The transformant B has enzymes that catalyze the reactions A, C, E, and F. 
     The transformant A is first produced. Plasmids fbr the expression of the enzymes that catalyze the reactions A, E, and F, respectively, are produced. The reactions E and F can be catalyzed by an identical enzyme. The plasmids are introduced into  Escherichia coli  strain BL21 (DE3), which is a microorganism strain lacking abilities to produce all of 3-hydroxyadipic acid, α-hydromuconic acid, and adipic acid. Into the obtained transformant, an expression plasmid carrying a nucleic acid that encodes a polypeptide to be analyzed for the presence of the enzymatic activity of interest and is integrated downstream of an appropriate promoter is introduced to obtain the transformant A. The transformant A is cultured, and the post-culture fluid is examined for the presence of 3-hydroxyadipic acid. Once the presence of 3-hydroxyadipic acid in the culture fluid is confirmed, the transformant B is then produced. The transformant B is obtained by producing a plasmid for the expression of an enzyme that catalyzes the reaction C and introducing the resulting plasmid into the transformant A. The transformant B is cultured, and the post-culture fluid is examined for the presence of α-hydromuconic acid. When the presence of α-hydromuconic acid in the post-culture fluid is confirmed, it indicates that 3-hydroxyadipic acid produced in the transformant A and α-hydromuconic acid produced in the transformant B are generated via production of 3-hydroxyadipyl-CoA, and that the polypeptide of interest has 3-oxoadipyl-CoA reductase activity. 
     As the gene encoding the enzyme that catalyzes the reaction A, pcaF from  Pseudomonas putida  strain KT2440 (NCBI Gene ID: 1041755; SEQ ID NO: 174) is used. 
     As the genes encoding the enzyme that catalyzes the reactions E and F, a continuous sequence including the full lengths of peal and peal from  Pseudomonas putida  strain KT2440 (NCBI Gene IDs: 1046613 and 1046612; SEQ ID NOs: 175 and 176) is used. The polypeptides encoded by peal and peal forms a complex and then catalyze the reactions E and F. 
     As the nucleic acid encoding the enzyme that catalyzes the reaction C, the paaF gene from Pseudomonas putida strain KT2440 (NCBI Gene ID: 1046932, SEQ ID NO: 177) is used. 
     The method of culturing the transformant A and the transformant B is as follows. Antibiotics for stable maintenance of the plasmids and inducer substances for induction of expression of the polypeptides encoded by the incorporated nucleic acids may be added as appropriate to the culture. A loopful of either the transformant A or B is inoculated into 5 mL of the culture medium I (10 gL Bacto Tryptone (manufactured by Difco Laboratories), 5 gL Bacto Yeast Extract (manufactured by Difco Laboratories), 5 gL sodium chloride) adjusted at pH 7 and is cultured at 30° C. with shaking at 120 min −1  for 18 hours to prepare a preculture fluid. Subsequently, 0.25 mL of the preculture fluid is added to 5 mL of the culture medium II (10 gL succinic acid, 10 gL glucose, 1 gL ammonium sulfate, 50 mM potassium phosphate, 0.025 gL magnesium sulfate, 0.0625 mgL iron sulfate, 2.7 mgL manganese sulfate, 0.33 mgL calcium chloride, 1.25 gL sodium chloride, 2.5 gL Bacto Tryptone, 1.25 gL Bacto Yeast Extract) adjusted to pH 6.5 and is cultured at 30° C. with shaking at 120 min −1  for 24 hours. The obtained culture fluid is examined for the presence of 3-hydroxyadipic acid or α-hydromuconic acid. 
     The presence of 3-hydroxyadipic acid or α-hydromuconic acid in the culture fluid can be confirmed by centrifuging the culture fluid and analyzing the supernatant with LC-MSMS. The analysis conditions are as described below:
     HPLC: 1290 Infinity (manufactured by Agilent Technologies, Inc.)   Column: Synergi hydro-RP (manufactured by Phenomenex Inc.), length: 100 mm,   internal diameter: 3 mm, particle size: 2.5 μm   Mobile phase: 0.1% aqueous formic acid solution methanol =7030   Flow rate: 0.3 mLmin   Column temperature: 40° C.   LC detector: DAD (210 nm)   MSMS: Triple-Quad LCMS (manufactured by Agilent Technologies, Inc.) Ionization method: ESI in negative mode.   

     The 3-oxoadipyl-CoA reductase activity value can be calculated by quantifying 3-hydroxyadipyl-CoA generated from 3-oxoadipyl-CoA used as a substrate by using purified 3-oxoadipyl-CoA reductase, wherein the 3-oxoadipyl-CoA is prepared from 3-oxoadipic acid by an enzymatic reaction. The specific method is as follows. 
     3-Oxoadipic acid can be prepared by a known method (for example, a method described in Reference Example 1 of WO 2017099209). 
     Preparation of 3-oxoadipyl-CoA solution: A PCR using the genomic DNA of  Pseudomonas putida  strain KT2440 as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase (peal and pcaJ; NCBI-GenelDs: 1046613 and 1046612) in the full-length form. The nucleotide sequences of primers used in this PCR are, for example, those represented by SEQ ID NOs: 194 and 195. The amplified fragment is inserted into the Kpnl site of pRSF-1b (manufactured by Novagen), an expression vector for  E. coli,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and the enzyme is expressed from the plasmid under isopropyl-β-thiogalactopyranoside (IPTG) induction and is then purified using the histidine tag from the culture fluid in accordance with routine procedures to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution for 3-oxoadipyl-CoA preparation with the following composition, and the enzymatic reaction solution is kept at 25° C. for 3 minutes to allow the reaction to proceed and is then filtered through a UF membrane (Amicon Ultra-0.5mL 10K; manufactured by Merck Millipore) to remove the enzyme, and the obtained filtrate is designated as 3-oxoadipyl-CoA solution. 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pH 8.2) 
     10 mM MgCl 2    
     0.5 mM succinyl-CoA 
     5 mM 3-oxoadipic acid sodium salt 
     2 μM CoA transferase. 
     Identification of 3-oxoadipyl-CoA reductase activity: A PCR using the genomic DNA of a microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding 3-oxoadipyl-CoA reductase in the full-length form. The nucleotide sequences of primers used in this PCR are, for example, those represented by SEQ ID NOs: 196 and 197. The amplified fragment is inserted into the Ba.mHI site of pACYCDuet-1 (manufactured by Novagen), an expression vector for  E. coli,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and the enzyme is expressed from the plasmid under isopropyl(3-thiogalactopyranoside (IPTG) induction and is then purified using the histidine tag from the culture fluid in accordance with routine procedures to obtain a 3-oxoadipyl-CoA reductase solution. The 3-oxoadipyl-CoA reductase activity can be determined by using the enzyme solution to prepare an enzymatic reaction solution with the following composition and quantifying 3-hydroxyadipyl-CoA generated at 25° C. 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pH 8.2) 
     10 mM MgCl 2    
     150 μLmL 3-oxoadipyl-CoA solution 
     0.5 mM NADH 
     1 mM dithiothreitol 
     10 μM 3-oxoadipyl-CoA reductase. 
     In the present invention, the genetically modi lied microorganism in which the expression of any one of the polypeptides described in (a) to (c) is enhanced is a microorganism as a host which originally has a nucleic acid encoding any one of the polypeptides described in (a) to (c) and is genetically modified for increased expression of any one of the polypeptides described in (a) to (c) which are owned by the host microorganism. 
     Specific examples of the microorganism which originally has a nucleic acid encoding any one of the polypeptides described in (a) to (c) include the following microorganisms of the genus  Serratia,  including  Serratia marcescens  (a microorganism having the sequences represented by SEQ ID NOs: 1, 18 to 28, 30 to 33, 35 to 66, 69, 70, 72 to 78, and 79),  Serratia nematodiphila  (a microorganism having the sequences represented by SEQ ID NOs: 2, 29, and 67),  Serratia plymuthica  (a microorganism having the sequences represented by SEQ ID NOs: 3, 79 to 84, and 86),  Serratia proteamaculans  (a microorganism having the sequences represented by SEQ ID NOs: 4 and 85),  Serratia ureilytica  (a microorganism having the sequence represented by SEQ ID NO: 5),  Serratia  sp. BW106 (a microorganism having the sequence represented by SEQ ID NO: 6),  Serratia liquefaciens  (a microorganism having the sequence represented by SEQ ID NO: 7),  Serratia  sp. S119 (a microorganism having the sequence represented by SEQ ID NO: 8),  Serratia  sp. YD25 (a microorganism having the sequence represented by SEQ ID NO: 9),  Serratia  sp. FS14 (a microorganism having the sequence represented by SEQ ID NO: 10).  Serratia  sp. HMSC15F11 (a microorganism having the sequence represented by SEQ ID NO: I1),  Serratia  sp. JKS000199 (a microorganism having the sequence represented by SEQ ID NO: 12),  Serratia  sp. TEL (a microorganism having the sequence represented by SEQ ID NO: 13),  Serratia  sp. ISTD04 (a microorganism having the sequence represented by SEQ ID NO: 14),  Serratia  sp. SCBI (a microorganism having the sequence represented by SEQ ID NO: 15),  Serratia  sp. S4 (a microorganism having the sequence represented by SEQ ID NO: 16),  Serratia  sp. C-1 (a microorganism having the sequence represented by SEQ ID NO: 17),  Serratia  sp. OMLW3 (a microorganism having the sequence represented by SEQ ID NO: 34),  Serratia  sp. OLEL1 (a microorganism having the sequence represented by SEQ ID NO: 68),  Serratia  sp. OLEL2 (a microorganism having the sequence represented by SEQ ID NO: 71), and the like. 
     Each of the polypeptides as described above in (a), (b), and (c) also has 3-hydroxybutyryl-CoA dehydrogenase activity, and the 3-hydroxybutyryl-CoA dehydrogenase is encoded by a 3-hydroxybutyryl-CoA dehydrogenase gene, which forms a gene cluster with the 5-aminolevulinic acid synthase gene in the microorganisms of the genus  Serratia.    
     As used herein, the term “gene cluster” in the phrase “the 3-hydroxybutyryl-CoA dehydrogenase gene, which forms a gene cluster with 5-aminolevulinic acid synthase gene in the microorganisms of the genus  Serratia ” refers to a region in which a set of nucleic acids encoding proteins with related functions are located in close proximity to each other. Specific components in a gene cluster include, for example, nucleic acids which are transcribed under the control of a single transcription regulator, and those in an operon which are transcribed under the control of a single transcription promoter. Whether or not a certain nucleic acid is a nucleic acid component of a gene cluster can also be investigated using an online gene cluster search program, such as antiSMASH. Additionally, whether or not a certain polypeptide is classified as a 3-hydroxybutyryl-CoA dehydrogenase or a 5-aminolevulinic acid synthase can be determined by BLAST (Basic Local Alignment Search Tool) searching on a website, such as that of NCR! (National Center for Biotechnology Information) or KEGG (Kyoto Encyclopedia of Genes and Genomes), to find any enzyme with a high degree of homology to the polypeptide in amino acid sequence. For example, the amino acid sequence represented by SEQ ID NO: 4 is registered in an NCBI database under Protein ID: ABV40935.1, which is annotated as a putative protein with 3-hydroxybutyryl-CoA dehydrogenase activity, as judged from the amino acid sequence. A gene encoding the amino acid sequence represented by SEQ ID NO: 4 is registered in an NCBI database under Gene ID: CP000826.1 and can be identified through a database search as conserved in the genome of  Serratia proteamaculans  strain 568 or as conserved in the region from 2015313 to 2016842 bp on the sequence of Gene ID: CP000826.1. Furthermore, the positional information of the gene can lead to identification of the sequences of flanking genes, from which the gene can be found to form a gene cluster with the 5-aminolevulinic acid synthase gene (Protein ID: ABV40933.1), as shown in  FIG. 1 . Similarly, for the amino acid sequences represented by SEQ ID NOs: 1 to 3, 6 to 20, 22 to 30,32 to 35,37,38,40,42 to 48,51 to 56, 59 to 63, 65, 66, 68 to 73, 75 to 81, and 83 to 85, the information can be checked on the NCBI site with the Protein IDs and Gene IDs presented in Tables 3-4 and 3-5. 
     
       
         
           
               
               
               
             
               
                 TABLE 3-4 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Gene ID:position (from . . . to) 
                 Protein ID 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 JMPQ01000047.1:133194 . . . 134723 
                 KFD11732.1 
               
               
                 2 
                 JPUX00000000.1:4202615 . . . 4204144 
                 WP_033633399.1 
               
               
                 3 
                 BCTU01000013.1:85647 . . . 87176 
                 WP_063199278.1 
               
               
                 4 
                 CP000826.1:2015313 . . . 2016842 
                 ABV40935.1 
               
               
                 6 
                 MCGS01000002.1:43811 . . . 45340 
                 WP_099061672.1 
               
               
                 7 
                 CP006252.1:1825868 . . . 1827397 
                 AGQ30498.1 
               
               
                 8 
                 MSFH01000022.1:147976 . . . 149505 
                 ONK16968.1 
               
               
                 9 
                 CP016948.1:1213474 . . . 1215003 
                 AOE98783.1 
               
               
                 10 
                 CP005927.1:4244665 . . . 4246194 
                 WP_044031504.1 
               
               
                 11 
                 LWNG01000196.1:83086 . . . 84615 
                 OFS85208.1 
               
               
                 12 
                 LT907843.1:1172733 . . . 1174262 
                 SNY82966.1 
               
               
                 13 
                 LDEG01000005.1:19627 . . . 21156 
                 KLE40298.1 
               
               
                 14 
                 MBDW01000089.1:53478 . . . 55007 
                 ODJ15373.1 
               
               
                 15 
                 CP003424.1:1869825 . . . 1871300 
                 AIM21329.1 
               
               
                 16 
                 APLA01000003.1:1964823 . . . 1966352 
                 WP_017892361.1 
               
               
                 17 
                 CAQO01000118.1:101692 . . . 103221 
                 WP_062792820.1 
               
               
                 18 
                 JVDI01000070.1:19399 . . . 20928 
                 WP_049300487.1 
               
               
                 19 
                 FCGF01000001.1:938090 . . . 939619 
                 WP_060444298.1 
               
               
                 20 
                 NC_020211.1:1963542 . . . 1965071 
                 WP_015377392.1 
               
               
                 22 
                 JVNC01000043.1:47711 . . . 49240 
                 WP_049187553.1 
               
               
                 23 
                 MCNK01000010.1:591271 . . . 592800 
                 WP_076740355.1 
               
               
                 24 
                 JVZV01000138.1:53080 . . . 54609 
                 WP_049277247.1 
               
               
                 25 
                 CP021984.1:1963542 . . . 1965071 
                 WP_088381461.1 
               
               
                 26 
                 NERL01000025.1:86571 . . . 88100 
                 WP_060559176.1 
               
               
                 27 
                 MTEH01000001.1:215863 . . . 217392 
                 WP_085336366.1 
               
               
                 28 
                 JVCS01000001.1:19397 . . . 20926 
                 WP_049239700.1 
               
               
                 29 
                 MTBJ01000002.1:216232 . . . 217761 
                 WP_082996863.1 
               
               
                 30 
                 AORJ01000010.1:70272 . . . 71801 
                 WP_033645451.1 
               
               
                 32 
                 LFJS01000012.1:944087 . . . 945616 
                 WP_025302345.1 
               
               
                 33 
                 CP018930.1:1161338 . . . 1162867 
                 WP_060447438.1 
               
               
                 34 
                 MSTK01000013.1:54046 . . . 55575 
                 WP_099817374.1 
               
               
                 35 
                 CP018929.1:1167577 . . . 1170106 
                 WP_089180755.1 
               
               
                 37 
                 FCGS01000006.1:98915 . . . 100444 
                 WP_060438851.1 
               
               
                 38 
                 MQRI01000002.1:585500 . . . 587029 
                 WP_060387554.1 
               
               
                 40 
                 FCFE01000001.1:962839 . . . 964368 
                 WP_060435888.1 
               
               
                 42 
                 FCIO01000002.1:145369146898 . . . 
                 WP_033637938.1 
               
               
                 43 
                 AP013063.1:1329259 . . . 1330788 
                 WP_041034581.1 
               
               
                 44 
                 MQRJ01000004.1:178926 . . . 180455 
                 WP_074026553.1 
               
               
                 45 
                 HG738868.1:1928329 . . . 1929858 
                 WP_060437960.1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 3-5 
               
               
                   
               
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO: 
                 Gene ID:position (from . . . to) 
                 Protein ID 
               
               
                   
               
             
            
               
                 46 
                 FCHQ01000006.1:51377 . . . 52906 
                 WP_060420535.1 
               
               
                 47 
                 NPGG01000001.1:301231 . . . 302760 
                 WP_047568134.1 
               
               
                 48 
                 FCME01000002.1:205632 . . . 207161 
                 WP_060443161.1 
               
               
                 51 
                 FCIH01000014.1:52403 . . . 53932 
                 WP_060429049.1 
               
               
                 52 
                 NBWV01000007.1:110621 . . . 112150 
                 WP_039566649.1 
               
               
                 53 
                 FCKI01000001.1:594106 . . . 595635 
                 WP_060429902.1 
               
               
                 54 
                 JPOB01000010.1:81351 . . . 82880 
                 WP_033654196.1 
               
               
                 55 
                 FCFI01000001.1:582222 . . . 583751 
                 WP_060443342.1 
               
               
                 56 
                 FCML01000001.1:1005802 . . . 1007331 
                 WP_060456892.1 
               
               
                 59 
                 FCMR01000001.1:1873566 . . . 1875095 
                 WP_060440240.1 
               
               
                 60 
                 LJEV02000002.1:115432 . . . 116961 
                 WP_047727865.1 
               
               
                 61 
                 NPIX01000027.1:38249 . . . 39778 
                 WP_094461128.1 
               
               
                 62 
                 NDXU01000091.1:70343 . . . 71872 
                 WP_048233299.1 
               
               
                 63 
                 FNXW01000055.1:13619 . . . 15148 
                 WP_080490898.1 
               
               
                 65 
                 AYMO01000023.1:23978 . . . 25507 
                 WP_025160335.1 
               
               
                 66 
                 CP018926.1:1215941 . . . 1217470 
                 WP_089191486.1 
               
               
                 68 
                 MORG01000026.1:13723 . . . 15252 
                 WP_099782744.1 
               
               
                 69 
                 PEHC01000008.1:57274 . . . 58803 
                 PHY81681.1 
               
               
                 70 
                 MEDA01000063.1:13491 . . . 15020 
                 WP_072627918.1 
               
               
                 71 
                 MORH01000030.1:13633 . . . 15162 
                 WP_099789708.1 
               
               
                 72 
                 KK214286.1:392757 . . . 394286 
                 WP_033650708.1 
               
               
                 73 
                 KI929259.1:1574567 . . . 1576096 
                 WP_033642621.1 
               
               
                 75 
                 CP012639.1:230596 . . . 232125 
                 WP_060659686.1 
               
               
                 76 
                 LZOB01000011.1:1613417 . . . 1614946 
                 WP_074054551.1 
               
               
                 77 
                 LCWI01000024.1:46336 . . . 47865 
                 WP_046899223.1 
               
               
                 78 
                 CP018924.1:1213305 . . . 1214834 
                 WP_089194521.1 
               
               
                 79 
                 NC_015567.1:1930552 . . . 1932081 
                 WP_013812379.1 
               
               
                 80 
                 MQML01000205.1:9362 . . . 10891 
                 WP_073439751.1 
               
               
                 81 
                 AMSV01000032.1:251478 . . . 253007 
                 WP_006324610.1 
               
               
                 83 
                 CP007439.1:1991332 . . . 1992861 
                 AHY06789.1 
               
               
                 84 
                 CP012096.1:319897 . . . 321426 
                 WP_037432641.1 
               
               
                 85 
                 FWWG01000018.1:38528 . . . 40057 
                 WP_085116175.1 
               
               
                   
               
            
           
         
       
     
     A nucleic acid encoding a polypeptide encoded by the 3-hydroxybutyryl-CoA dehydrogenase gene of a microorganism of the genus  Serratia  , which forms a gene cluster with the 5-aminolevulinic acid synthase gene, is hereinafter referred to as “the 3-hydroxybutyryl-CoA dehydrogenase gene used in the present invention,” and the polypeptide encoded by the 3-hydroxybutyryl-CoA dehydrogenase gene is referred as “the 3-hydroxybutyryl-CoA dehydrogenase used in the present invention.” 
     A gene cluster including the 3-hydroxybutyryl-CoA dehydrogenase gene used in the present invention may include other nucleic acids, provided that the gene cluster includes at least the 3-hydroxybutyryl-CoA dehydrogenase gene and the 5-aminolevulinic acid synthase gene.  FIG. 1  shows a specific example of the gene cluster including the 3-hydroxybutyryl-CoA dehydrogenase gene used in the present invention. 
     Specific examples of the microorganisms of the genus  Serratia  that contain the above gene cluster include  S. marcescens, S. nematodiphila, S. plymuthica, S. proteamaculans, S. ureilytica, S. liquelaciens. Serratia  sp. BW106,  Serratia  sp. S119,  Serratia  sp. YD25,  Serratia  sp. FS14.  Serratia  sp. HMSC15F11,  Serratia  sp. JKS000199,  Serratia  sp. TEL,  Serratia  sp. ISTD04,  Serratia  sp. SCBI,  Serratia  sp. S4,  Serratia  sp. C-1,  Serratia  sp. OMLW3.  Serratia  sp. OLEL1,  Serratia  sp. OLEL2, and  S. liquefaciens.    
     The 3-hydroxybutyryl-CoA dehydrogenase used in the present invention has an excellent 3-oxoadipyl-CoA reductase activity. Whether or not a 3-hydroxybutyryl-CoA dehydrogenase-encoding nucleic acid has a 3-oxoadipyl-CoA reductase activity can be determined by the same method as described above. 
     The polypeptide encoded by the 3-hydroxybutyryl-CoA dehydrogenase gene used in the present invention is characterized by containing the common sequence 1. Specific examples of amino acid sequences of such polypeptides include the amino acid sequences represented by SEQ ID NOs: 1 to 86. 
     In the present invention, a nucleic acid encoding a polypeptide composed of the same amino acid sequence as that represented by any one of SEQ ID NOs: 8 to 86, except that one or several amino acids are substituted, deleted, inserted, andor added, and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA can also be suitable for use, provided that the common sequence 1 is contained in the polypeptide. In this respect, the range represented by the phrase “gone or several” is preferably 10 or less, more preferably 5 or less, especially preferably 4 or less, and most preferably one or two. In the case of amino acid substitution, the activity of the original polypeptide is more likely to be maintained when an amino acid(s) isare replaced by an amino acid(s) with similar properties (i.e., conservative substitution as described above). A nucleic acid encoding a polypeptide composed of an amino acid sequence with a sequence identity to not less than 70%, preferably not less than 80%, more preferably not less than 85%, further preferably not less than 90%, still further preferably not less than 95%, yet further preferably not less than 97%, even further preferably not less than 99%, to the sequence represented by any one of SEQ ID NOs: 8 to 86 and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA can also be suitably used. 
     On the other hand, examples of a polypeptide that is not the 3-hydroxybutyryl-CoA dehydrogenase used in the present invention but has 3-oxoadipyl-CoA reductase activity include PaaH from  Pseudomonas putida  strain KT2440 (SEQ ID NO: 178), PaaH from  Escherichia coli  strain K-12 substrain MG1655 (SEQ ID NO: 179). DcaH from  Acinetobacter haylyi  strain ADP1 (SEQ ID NO: 180), and PaaH from  Serratia plymuthica  strain NBRC102599 (SEQ ID NO: 181). As shown in Tables 4 and 5, these polypeptides are found not to contain the common sequence I. It should be noted that those polypeptides are neither (b) polypeptides composed of the same amino acid sequence as that represented by any one of SEQ ID NOs: 1 to 7, except that one or several amino acids are substituted, deleted, inserted, andor added, and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, nor (c) polypeptides having an amino acid sequence with a sequence identity of not less than 70% to the sequence represented by any one of SEQ ID NOs: 1 to 7 and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. 
     
       
         
           
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   5  10  15  20  25  30  35  40 
               
               
                   
                 Consensus sequence1 
               
               
                   
                     GAGTMGRG|AYLXAXXX|XTXLYN 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 1. 
                 1 
                   Serratia marcescens  ATCC13880 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 2. 
                 2 
                   Serratia nematodiphila  DSM21420 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 3. 
                 3 
                   Serratia plymuthica  NBRC102599 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 4. 
                 4 
                   Serratia proteamaculans  568 
                 MAENNSA|HSVAV|GAGTMGRG|AYLLAQNG|RTLLYNRS 
               
               
                 5. 
                 5 
                   Serratia ureilytica  Lr5/4 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 6. 
                 6 
                   Serratia  sp. BW106 
                 MAENNSA|HSVAV|GAGTMGRG|AYLLAQNG|RTLLYNRS 
               
               
                 7. 
                 7 
                   Serratia liquefaciens  FK01 
                 MAENNTA|DSVAV|GAGTMGRG|AYLLALNG|RTLLYNRN 
               
               
                 8. 
                 8 
                   Serratia  sp. S119 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 9. 
                 9 
                   Serratia  sp. YD25 
                 MAERNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 10. 
                 10 
                   Serratia  sp. FS14 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 11. 
                 11 
                   Serratia  sp. HMSC15F11 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 12. 
                 12 
                   Serratia  sp. JKS000199 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 13. 
                 13 
                   Serratia  sp. TEL 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 14. 
                 14 
                   Serratia  sp. ISTD04 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 15. 
                 15 
                   Serratia  sp. SCBI 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 16. 
                 16 
                   Serratia  sp. S4 
                 MAENNSA|HSVAV|GAGTMGRG|AYLLAQNG|RTLLYNRS 
               
               
                 17. 
                 17 
                   Serratia  sp. C-1 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 18. 
                 18 
                   Serratia marcescens  532 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 19. 
                 19 
                   Serratia marcescens  2880STDY5683033 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 20. 
                 20 
                   Serratia marcescens  WW4 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 21. 
                 21 
                   Serratia marcescens  K27 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 22. 
                 22 
                   Serratia marcescens  280 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 23. 
                 23 
                   Serratia marcescens  19F 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 24. 
                 24 
                   Serratia marcescens  1185 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 25. 
                 25 
                   Serratia marcescens  S217 
                 MAESNAA|QSAAI|GAGTMGRG|ATLFAQKG|PTMLYNRN 
               
               
                 26. 
                 26 
                   Serratia marcescens  KHCo-24B 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 27. 
                 27 
                   Serratia marcescens  Z6 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 28. 
                 28 
                   Serratia marcescens  546 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 29. 
                 29 
                   Serratia nematodiphila  MB307 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 30. 
                 30 
                   Serratia marcescens  VGH107 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|PTMLYNRN 
               
               
                 31. 
                 31 
                   Serratia marcescens  MCB 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 32. 
                 32 
                   Serratia marcescens  AH0650 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 33. 
                 33 
                   Serratia marcescens  UMH12 
                 MAESNAE|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 34. 
                 34 
                   Serratia  sp. OMLW3 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 35. 
                 35 
                   Serratia marcescens  UMH11 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRS 
               
               
                 36. 
                 36 
                   Serratia marcescens  UMH1 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 37. 
                 37 
                   Serratia marcescens  2880STDY5683020 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 38. 
                 38 
                   Serratia marcescens  99 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 39. 
                 39 
                   Serratia marcescens  374 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 40. 
                 40 
                   Serratia marcescens  2880STDY5683036 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 41. 
                 41 
                   Serratia marcescens  2880STDY5683034 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 42. 
                 42 
                   Serratia marcescens  2880STDY5682892 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 43. 
                 43 
                   Serratia marcescens  SM39 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 5  10  15  20  25  30  35  40 
               
               
                   
                 Consensus sequence1 
               
               
                   
                     GAGTMGRG|AYLXAXXX|XTXLYN 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 44. 
                 44 
                   Serratia marcescens  189 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 45. 
                 45 
                   Serratia marcescens  SMB2099 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 46. 
                 46 
                   Serratia marcescens  2880STDY5682862 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 47. 
                 47 
                   Serratia marcescens  SE4145 
                 MAESNAE|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 48. 
                 48 
                   Serratia marcescens  2880STDY5682876 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 49. 
                 49 
                   Serratia marcescens  709 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 50. 
                 50 
                   Serratia marcescens  MGH136 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 51. 
                 51 
                   Serratia marcescens  2880STDY5682884 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 52. 
                 52 
                   Serratia marcescens  D-3 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 53. 
                 53 
                   Serratia marcescens  2880STDY5682957 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 54. 
                 54 
                   Serratia marcescens  YDC563 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 55. 
                 55 
                   Serratia marcescens  2880STDY5683035 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 56. 
                 56 
                   Serratia marcescens  2880STDY5682930 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 57. 
                 57 
                   Serratia marcescens  790 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 58. 
                 58 
                   Serratia marcescens  UMH5 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 59. 
                 59 
                   Serratia marcescens  2880STDY5682988 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 60. 
                 60 
                   Serratia marcescens  945154301 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 61. 
                 61 
                   Serratia marcescens  at10508 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 62. 
                 62 
                   Serratia marcescens  ML2637 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 63. 
                 63 
                   Serratia marcescens  SM1978 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 64. 
                 64 
                   Serratia marcescens  PWN146 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 65. 
                 65 
                   Serratia marcescens  H1q 
                 MAERNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 66. 
                 66 
                   Serratia marcescens  UMH6 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKS|RTVLYNRN 
               
               
                 67. 
                 67 
                   Serratia nematodiphila  WCU338 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKS|RTVLYNRN 
               
               
                 68. 
                 68 
                   Serratia  sp. OLEL1 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 69. 
                 69 
                   Serratia marcescens  7209 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKS|RTVLYNRN 
               
               
                 70. 
                 70 
                   Serratia marcescens  sicaria (Ss1) 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKS|RTVLYNRN 
               
               
                 71. 
                 71 
                   Serratia  sp. OLFL2 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 72. 
                 72 
                   Serratia marcescens  BIDMC 81 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 73. 
                 73 
                   Serratia marcescens  BIDMC 50 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKS|RTVLYNRN 
               
               
                 74. 
                 74 
                   Serratia marcescens  UMH7 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 75. 
                 75 
                   Serratia marcescens  RSC-14 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 76. 
                 76 
                   Serratia marcescens  SMO3 
                 MAERNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 77. 
                 77 
                   Serratia marcescens  90-166 
                 MAESNAA|QSAAI|GAGTMGRG|AYLFAQKG|RTVLYNRN 
               
               
                 78. 
                 78 
                   Serratia marcescens  UMH2 
                 MAESNAA|QSAAI|GAGTMGRG|AYLLAQKS|RTVLYNRN 
               
               
                 79. 
                 79 
                   Serratia plymuthica  AS9 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 80. 
                 80 
                   Serratia plymuthica  tumat 205 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 81. 
                 81 
                   Serratia plymuthica  A30 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 82. 
                 82 
                   Serratia plymuthica  4Rx13 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 83. 
                 83 
                   Serratia plymuthica  V4 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 84. 
                 84 
                   Serratia plymuthica  3Rp8 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                 85. 
                 85 
                   Serratia proteamaculans  MFPA44A14 
                 MAENNSA|HSVAV|GAGTMGRG|AYLLAQNG|RTLLYNRS 
               
               
                 86. 
                 86 
                   Serratia plymuthica  A153 
                 MAENNSA|RSAAV|GAGTMGRG|AYLLALNG|RTVLYNRN 
               
               
                   
               
            
           
         
       
     
     In the present invention, impairing the function of pyruvate kinase or a phosphotransferase system enzyme means impairing the enzymatic activity of the enzyme. The method of impairment of the function is not limited to a particular method, but the function can be impaired, for example, by disrupting a gene that encodes the enzyme, such as via partial or complete deletion of the gene by mutagenesis with a chemical mutagen, ultraviolet irradiation, or the like, or by site-directed mutagenesis or the like, or via introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene. Alternatively, recombinant DNA technologies can be used to disrupt the gene by partial or complete deletion of the nucleotide sequence or by partial or complete substitution of the nucleotide sequence with another nucleotide sequence. Among those, the methods for partial or complete deletion of the nucleotide sequence are preferred. 
     Pyruvate kinase is classified as EC 2.7.1.40 and is an enzyme that catalyzes a reaction to dephosphorylate phosphoenolpyruvic acid (in this specification, also referred to as PEP) to pyruvic acid and ATP. Specific examples of pyruvate kinase include pykF (NCBI-Protein ID: NP 416191, SEQ ID NO: 182) and pykA (NCBI-Protein ID: NP 416368, SEQ ID NO: 183) from  Escherichia coli  strain K-12 substrain MG1655, and pykF (SEQ ID NO: 184) and pykA (SEQ ID NO: 185) from  Serratia grimesii  strain NBRC13537. 
     In cases where a microorganism used in the present invention has two or more genes that each encode a pyruvate kinase, as illustrated in the metabolic pathway shown in the scheme 2 below, it is desirable to impair the function of all the pyruvate kinases. Whether or not a polypeptide encoded by a certain gene of a microorganism used in the present invention is a pyruvate kinase may be determined by BLAST (Basic Local Alignment Search Tool) searching on a website, such as that of NCBI (National Center for Biotechnology Information) or KEGG (Kyoto Encyclopedia of Genes and Genomes). 
     In the genetically modified microorganism of the present invention, it is desirable to further impair the function of a phosphotransferase system enzyme. The phosphotransferase system enzyme is relevant to the phosphoenolpyruvate 
     (PEP)-dependent phosphotransferase system (PTS) (in this specification, also referred to as a PTS enzyme). PTS is a major mechanism for the uptake of carbohydrates such as hexose, hexitol, and disaccharide into a cell, as illustrated in the metabolic pathway shown in the scheme 2 below. PTS involves uptake of carbohydrates into a cell and simultaneous conversion of the carbohydrates to a phosphate ester, while converting a phosphate donor, PEP, to pyruvic acid. Therefore, the conversion reaction from PEP to pyruvic acid is inhibited in a mutant microorganism with a disrupted PTS enzyme gene. 
     
       
         
         
             
             
         
       
     
     PTS enzymes are composed of two common enzymes that exert their functions on any type of carbohydrate, phosphoenolpyruvate sugar phosphotransferase enzyme I and phospho carrier protein HPr, and membrane-bound sugar specific permeases (enzymes II) that are specific for particular carbohydrates. The enzymes II are further composed of sugar-specific components IIA, IIB, and IIC. The enzymes II exist as independent proteins or as fused domains in a single protein, and this depends on the organism which those enzymes are originated from. In microorganisms, phosphoenolpyruvate sugar phosphotransferase enzyme I is encoded by the pts gene, and phospho carrier protein 1-1Pr is encoded by the ptsH gene, and glucose-specific enzyme HA is encoded by the crr gene, and glucose-specific enzymes IIB and HC are encoded by the ptsG gene. The enzyme encoded by the ptsG gene is classified as EC 2.7.1.199 and is called protein-Npi-phosphohistidinc-D-glucose phosphotransferase. 
     In the present invention, one or more of the above PTS enzyme genes may be disrupted. Although any of the above PTS enzyme genes may be disrupted, it is desirable to impair an enzyme gene that is involved in glucose uptake, particularly the ptsG gene. Specific examples of the ptsG gene include ptsG from  Escherichia coli  strain K-12 substrain MG1655 (NCBI-Gene ID: 945651) andptsG from  Serratia grimesii  strain NBRC13537 (SEQ ID NO: 238). 
     Whether or not a polypeptide encoded by a certain gene of a microorganism used in the present invention is a protein-Npi-phosphohistidine-D-glucose phosphotransferase may be determined by BLAST searching on a website, such as that of NCBI or KEGG. 
     As described below,  E. coli  is a microorganism that has an ability to produce 3-hydroxyadipic acid and α-hydromuconic acid, and JP 2008-527991 A describes production of a genetically modified E. coli strain with defects in the pykF and pykA genes, which each encode a pyruvate kinase, and in the ptsG gene, which encodes a phosphotransferase system enzyme, wherein the yield of succinic acid is increased, and the yields of acetic acid and ethanol are decreased, by culturing the genetically modified strain under anaerobic conditions. In this respect, acetic acid and ethanol are compounds generated from the metabolism of acetyl-CoA, as illustrated in the metabolic pathway shown in the above scheme 2. That is, in JP 2008-527991 A. it is presumed that the defects of the ptsG, pykF, and pykA genes in  E. coli  resulted in a reduced supply of acetyl-CoA and in turn a lower yield of acetic acid and ethanol. 
     The 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid produced by the method of the present invention are compounds generated through reactions in the metabolism of 3-oxoadipyl-CoA, which is produced from acetyl-CoA and succinyl-CoA by the reaction A, as described above. Accordingly, from the description in JP 2008-527991 A, it is expected that disruption of genes encoding pyruvate kinase and a phosphotransferase system enzyme also results in a decreased yields of 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid due to the reduced supply of acetyl-CoA. However, in the present invention, disruption of genes encoding pyruvate kinase and a phosphotransferase system enzyme increases the yields of 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid and also the yields of acetic acid and ethanol in a genetically modified microorganism expressing an enzyme that exhibits excellent activity in a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, which is contrary to the above expectation. 
     In the present invention, examples of the microorganism that can be used as a host to obtain the genetically modified microorganism include microorganisms belonging to the genera  Escherichia, Serratia, Hafnia, Pseudomonas, Corynebacterium, Bacillus. Streptomyces, Cupriavidus, Acinetobacter, Alcaligenes, Brevibacterium, Delftia, Aerobacter, Rhizobium, ThermoNfida, Clostridium, Schizosaccharomyces, Kluyveromyees, Pichia,  and  Candida.  Among those, microorganisms belonging to the genera  Escherichia, Serratia, Hafnia,  and  Pseudomonas  are preferred. 
     The method of producing 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid by using a genetically modified microorganism of the present invention will be described. 
     As a microorganism that has an ability to produce 3-hydroxyadipic acid, a microorganism that has an ability to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (the reaction A), and an ability to generate 3-hydroxyadipic acid from 3-hydroxyadipyl-CoA (the reaction E) is used. The microorganism with these production abilities can be used as a host microorganism to obtain a genetically modified microorganism according to the present invention with an ability to abundantly produce 3-hydroxyadipic acid. 
     Microorganisms that are speculated to originally have abilities to catalyze the above reactions A and E include microorganisms belonging to the following species: 
     species of the genus  Escherichia,  such as  Escherichia fergusonii  and  Escherichia coli;    
     species of the genus  Pseudomonas,  such as  Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas azotoformans,  and  Pseudomonas chlororaphis  subsp.  aureofaciens;    
     species of the genus  Hafnia,  such as  Hafnia alvei;    
     species of the genus  Corynebacterium,  such as  Corynebacterium acetoacidophilum, Corynebacterium acetoglulamicum, Corynebacterium ammoniagenes,  and  Corynebacterium glutamicum;    
     species of the genus  Bacillus,  such as  Bacillus badius, Bacillus magaterium,  and  Bacillus roseus;    
     species of the genus  Streptomyces,  such as  Streptomyces vinaceus, Streptomyces karnatakensis,  and  Streptomyces olivaceus;    
     species of the genus  Cupriavidus,  such as  Cupriavidus metallidurans, Cupriavidus necator,  and  Cupriavidus oxalaticus;    
     species of the genus  Acinetobacter,  such as  Acinetobacter baylyi  and  Acinetobacter radioresistens;    
     species of the genus  Alcaligenes,  such as  Alcaligenes faecalis;    
     species of the genus  Nocardioides,  such as  Nocardioides albus;    
     species of the genus  Brevibacterium,  such as  Brevibacterium iodinum;    
     species of the genus  Delfila,  such as  Delftia acidovorans;    
     species of the genus  Shimwellia,  such as  Shimwellia blattae;    
     species of the genus  Aerobacter,  such as  Aerobacter cloacae;    
     species of the genus  Rhizobium,  such as  Rhizobium radiobacter;    
     species of the genus  Serratia,  such as  Serratia grimesii, Serratia ficaria, Serratia fonticokt, Serratia odorfera, Serratia plymuthica, Serratia entomophila,  and  Serratia nematodiphila.    
     Even a microorganism that originally has no abilities to catalyze the reactions A andor E can also be used as the aforementioned host microorganism when an appropriate combination of nucleic acids that encode enzymes catalyzing the reactions A and E is introduced into the microorganism to impart those production abilities. 
     As a microorganism that has an ability to produce α-hydromuconic acid, a microorganism that has an ability to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (the reaction A), an ability to generate 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA through dehydration (the reaction C), and an ability to generate α-hydromuconic acid from 2.3-dehydroadipyl-CoA (the reaction F) is used. The microorganism with these production abilities can be used as a host microorganism to obtain a genetically modified microorganism according to the present invention with an ability to abundantly produce α-hydromuconic acid. 
     Microorganisms that are speculated to originally have abilities to catalyze the above reactions A, C, and F include microorganisms belonging to the following species: 
     species of the genus  Escherichia,  such as  Escherichia jergusonii  and  Escherichia coli;    
     species of the genus  Pseudomonas,  such as  Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans,  and  Pseudomonas chlororaphis  subsp.  aureofaciens;    
     species of the genus  Hafnia,  such as  Hafnia alvei;    
     species of the genus  Bacillus,  such as  Bacillus badius;    
     species of the genus  Cupriavidus,  such as  Cupriavidus metallidurans, Cupriavidus numazuensis,  and  Cupriavidus oxalaticus;    
     species of the genus  Acinetobacter,  such as  Acinetobacter haylyi  and  Acinetobacter radioresistens;    
     species of the genus  Alcaligenes,  such as  Alcaligenes faecalis;    
     species of the genus  Delftia,  such as  Delftict acidovorans;    
     species of the genus  Shimwellia,  such as  Shimwellia blattae;    
     species of the genus  Serratia,  such as  Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila,  and  Serratia nematodiphila.    
     Even a microorganism that originally has no abilities to catalyze the reactions A, C. andor F can also be used as the aforementioned host microorganism when an appropriate combination of nucleic acids that encode enzymes catalyzing the reactions A, C, and F is introduced into the microorganism to impart those production abilities. 
     As a microorganism that has an ability to produce adipic acid, a microorganism that has an ability to generate 3-oxoadipyl-CoA and coenzyme A from succinyl-CoA (the reaction A), an ability to generate 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA through dehydration (the reaction C), an ability to reduce 2,3-dehydroadipyl-CoA to adipyl-CoA (the reaction D), and an ability to generate adipic acid from adipyl-CoA (the reaction G) is used. The microorganism with these production abilities can be used as a host microorganism to obtain a genetically modified microorganism with an ability to abundantly produce adipic acid. 
     Microorganisms that are speculated to originally have abilities to catalyze the above reactions A, C, D, and G include microorganisms of the genus  Thermobifida,  such as  Thermobifida fusca.    
     Even a microorganism that originally has no abilities to catalyze the reactions A, C, D, and G can also be used as the aforementioned host microorganism when an appropriate combination of nucleic acids that encode enzymes catalyzing the reactions A, C, D, and G is introduced into the microorganism to impart those production abilities. 
     Specific examples of the enzymes that catalyze the reactions A and C to G are presented below. 
     As an enzyme that catalyzes the reaction A to generate 3-oxoadipyl-CoA, for example, an acyl transferase (P-ketothiolase) can be used. The acyl transferase is not limited to a particular number in the EC classification but is preferably an acyl transferase classified into EC 2.3.1.-, specifically including an enzyme classified as 3-oxoadipyl-CoA thiolase and classified into EC number 2.3.1.174, an enzyme classified as acetyl-CoA C-acetyltransferase and classified into EC number 2.3.1.9, and an enzyme classified as acetyl-CoA C-acyl transferase and classified into EC number 2.3.1.16. Among these, PaaJ from Escherichia coli strain MG1655 (NCBI-Protein ID: NP 415915), PcaF from Pseudomonas putida strain KT2440 (NCBI-Protein ID: NP 743536), and the like can be suitably used. 
     Whether or not the above acyl transferases can generate 3-oxoadipyl-CoA from succinyl-CoA and acetyl-CoA as substrates can be determined by measuring a decrease in NADH coupled with reduction of 3-oxoadipyl-CoA in a combination of the reaction catalyzed by purified acyl transferase to generate 3-oxoadipyl-CoA and a reaction catalyzed by purified 3-oxoadipyl-CoA reductase to reduce 3-oxoadipyl-CoA as a substrate. The specific measurement method is, for example, as follows. 
     Identification of acyl transferase activity: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding an acyl transferase in the full-length form. The amplified fragment is inserted into the Suet site of pACYCDuet-1 (manufactured by Novagen), an expression vector for E. colt, in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and expression of the enzyme is induced with isopropyl-P-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain an acyl transferase solution. The acyl transferase activity can be determined by using the enzyme solution to prepare an enzymatic reaction solution with the following composition and measuring a decrease in absorbance at 340 ran coupled with oxidation of NADH at 30° C. 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pH 8.0) 
     10 mM MgCl 2    
     0.1 mM succinyl-CoA 
     0.2 mM acetyl-CoA 
     0.2 mM NADH 
     1 mM dithiothreitol 
     10 μgmL 3-oxoadipyl-CoA reductase 
     5 μgmL acyl transferase. 
     Whether or not an enzyme originally expressed in a host microorganism used in the present invention has acyl transferase activity can be determined by performing the above-described measurement using cell homogenate (cell free extract: CFE) instead of purified acyl transferase. The specific measurement method targeted to  E. coli  is, for example, as follows. 
     Preparation of CFE: A loopful of  E. coli  strain MG1655 to be subjected to the measurement of the activity is inoculated into 5 mL of a culture medium (culture medium composition: 10 gL tryptone, 5 gL yeast extract, 5 gL sodium chloride) adjusted to pH 7, and incubated at 30° C. with shaking for 18 hours. The obtained culture fluid is added to 5 mL of a culture medium (culture medium composition: 10 gL tryptone, 5 gL yeast extract, 5 gL sodium chloride, 2.5 mM ferulic acid, 2.5 mMp-coumaric acid, 2.5 mM benzoic acid, 2.5 mM cis,cis-muconic acid, 2.5 mM protocatechuic acid, 2.5 m1\4 catechol. 2.5 mM 30A, 2.5 mM 3-hydroxyadipic acid, 2.5 mM cc-hydromuconic acid. 2.5 mM adipic acid, 2.5 mM phenylethylamine) adjusted to pH 7. and incubated at 30° C. with shaking for 3 hours. 
     The obtained culture fluid is supplemented with 10 mL of 0.9% sodium chloride and then centrifuged to remove the supernatant from bacterial cells, and this operation is repeated three times in total to wash the bacterial cells. The washed bacterial cells are suspended in 1 mL of a Tris-HCl buffer composed of 100 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol, and glass beads (with a diameter of 0.1 mm) are added to the resulting suspension to disrupt the bacterial cells at 4° C. with an ultrasonic disruptor. The resulting bacterial homogenate is centrifuged to obtain the supernatant, and 0.5 mL of the supernatant is filtered through a UF membrane (Amicon Ultra-0.5mL 10K; manufactured by Merck Millipore) to remove the resulting filtrate, followed by application of 0.4 mL of the Tris-HCl buffer to the UF membrane, and this operation is repeated three times in total to remove low-molecular-weight impurities, and the resulting supernatant is then resuspended in the Tris-HCl buffer to a final volume of 0.1 mL, which is designated as CFE. Instead of purified enzyme, 0.05 mL of the CFE is added to a total of 0.1 mL of the enzymatic reaction solution to determine the enzymatic activity. 
     As an enzyme that catalyzes the reaction C to generate 2,3-dehydroadipyl-CoA, for example, an enoyl-CoA hydratase can be used. The enoyl-CoA hydratase is not limited by a particular number in the EC classification, and is preferably an enoyl-CoA hydratase classified into EC 4.2.1.-, specifically including an enzyme classified as enoyl-CoA hydratase or 2,3-dehydroadipyl-CoA hydratase and classified into EC 4.2.1.17. Among them, PaaF from  Escherichia coli  strain MG1655 (NCBI-ProteinlD: NP_415911), PaaF from  Pseudomonas puhda  strain KT2440 (NCBI-ProteinlD: NP_745427), and the like can be suitably used. 
     Since the reaction catalyzed by enoyl-CoA hydratase is generally reversible, whether or not an enoyl-CoA hydratase has an activity to catalyze a reaction that generates 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA used as a substrate can be determined by detecting 3-hydroxyadipyl-CoA generated using purified enoyl-CoA hydratase with 2.3-dehydroadipyl-CoA used as a substrate thereof, which is prepared from α-hydromuconic acid through an enzymatic reaction. The specific measurement method is, for example, as follows. 
     The α-hydromuconic acid used in the above reaction can be prepared by a known method (for example, a method described in Reference Example 1 of WO 2016199858 A1). 
     Preparation of 2,3-dehydroadipyl-CoA solution: A PCR using the genomic DNA of Pseudomonas putida strain KT2440 as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase (including peal and peal; NCBI-GenelDs: 1046613 and 1046612) in the full-length form. The amplified fragment is inserted into the Kpnl site of pRSF- − lb (manufactured by Novagen), an expression vector for  E. coli,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and expression of the enzyme is induced with isopropyl-13-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution for 2,3-dehydroadipyl-CoA preparation with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5mL 10K; manufactured by Merck Millipore) to remove the enzyme, and the obtained filtrate is designated as 2,3-dehydroadipyl-CoA solution. 
     (Enzymatic Reaction Solution) 
     100 mM Tris-IICl (pH 8.0) 
     10 mM MgCI, 
     0.4 mM succinyl-CoA 
     2 mM ci-hydromuconic acid sodium salt 
     20 μgmL CoA transferase. 
     Identification of enoyl-CoA hydratase activity: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding an enoyl-CoA hydratase in the lull-length form. The amplified fragment is inserted into the Ndel site of pET-16b (manufactured by Novagen), an expression vector for  E. coil,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain an enoyl-CoA hydratase solution. The solution is used to prepare an enzymatic reaction solution with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5mL 10K; manufactured by Merck Millipore) to remove the enzyme. The enoyl-CoA hydratase activity can be confirmed by detecting 3-hydroxyadipyl-CoA in the resulting filtrate on high-performance liquid chromatograph-tandem mass spectrometer (LC-MSMS) (Agilent Technologies, Inc.). 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pH 8.0) 
     10 mM MgCl, 
     300 μLmL 2,3-dehydroadipyl-CoA solution 
     1 mM dithiothreitol 
     20 ugmL enoyl-CoA hydratase. 
     Whether or not an enzyme originally expressed in a host microorganism used in the present invention has enoyl-CoA hydratase activity can be determined by adding 0.05 mL of the CFE, instead of purified enoyl-CoA hydratase, to a total of 0.1 mL of the enzymatic reaction solution and performing the above-described measurement. The specific CFE preparation method targeted to  E. coli  is as described for that used in determination of acyl transferase activity. 
     As an enzyme that catalyzes the reaction D to generate adipyl-CoA, for example, an enoyl-CoA reductase can be used. The enoyl-CoA reductase is not limited by a particular number in the EC classification, and is preferably an enoyl-CoA reductase classified into EC 1.3.-.-, specifically including an enzyme classified as trans-2-enoyl-CoA reductase and classified into EC 1.3.1.44, and an enzyme classified as acyl-CoA dehydrogenase and classified into EC 1.3.8.7. These specific examples are disclosed in, for example JP 2011-515111 A, J Appl Microbiol. 2015 Oct; 119 (4): 1057-63., and the like; among them, TER from  Euglena gracilis  strain Z (UniProtKB: Q5E − 1590), Tfu 1647 from  Thermobilida fitsca  strain YX (NCBI-ProteinID: AAZ55682), DcaA from  Acinetobacter baylyi  strain ADPI (NCBI-ProteinID: AAL09094.1), and the like can be suitably used. 
     Whether or not an enoyl-CoA reductase has an activity to generate adipyl-CoA from 2,3-dehydroadipyl-CoA used as a substrate can be determined by measuring a decrease in NADH coupled with reduction of 2,3-dehydroadipyl-CoA in a reaction using purified enoyl-CoA reductase with 2,3-dehydroadipyl-CoA used as a substrate thereof, which is prepared from α-hydromuconic acid through another enzymatic reaction. 
     Preparation of α-hydromuconic acid and of 2,3-dehydroadipyl-CoA solution can be performed in the same manner as described above. 
     Identification of enoyl-CoA reductase activity: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding an enoyl-CoA reductase in the full-length form. The amplified fragment is inserted into the NdeI site of pET-16b (manufactured by Novagen), an expression vector for  E. coli,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and expression of the enzyme is induced with isopropyl-13-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain an enoyl-CoA reductase solution. The enoyl-CoA reductase activity can be determined by using the enzyme solution to prepare an enzymatic reaction solution with the following composition and measuring a decrease in absorbance at 340 nm coupled with oxidation of NADH at 30° C. 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pH 8.0) 
     10 mM MgCl 2    
     300 μLmL 2,3-dehydroadipyl-CoA solution 
     0.2 mM NADH 
     1 mM dithiothreitol 
     20 μgmI. enoyl-CoA reductase. 
     Whether or not an enzyme originally expressed in a host microorganism used in the present invention has enoyl-CoA reductase activity can be determined by adding 0.05 mL of the CFE, instead of purified enoyl-CoA reductase, to a total of 0.1 mL of the enzymatic reaction solution and performing the above-described measurement. The specific CFE preparation method targeted to E. coli is as described for that used in determination of acyl transferase activity. 
     As an enzyme that catalyzes the reaction E to generate 3-hydroxyadipic acid, the reaction F to generate cc-hydromuconic acid, and the reaction G to generate adipic acid, for example, a CoA transferase or an acyl-CoA hydrolase, preferably a CoA transferase, can be used. 
     The CoA transferase is not limited by a particular number in the EC classification, and is preferably a CoA transferase classified into EC 2.8.3.-, specifically including an enzyme classified as CoA transferase or acyl-CoA transferase and classified into EC 2.8.3.6, and the like. 
     In the present invention, the term “CoA transferase” refers to an enzyme with activity (CoA transferase activity) to catalyze a reaction that generates carboxylic acid and succinyl-CoA from acyl-CoA and succinic acid used as substrates. 
     As an enzyme that catalyzes the reaction E to generate 3-hydroxyadipic acid and the reaction F to generate ct-hydromuconic acid, Peal and PcaJ from  Pseudomonas putido  strain KT2440 (NCBI-ProteinlDs: NP 746081 and NP 746082), and the like can be suitably used, among others. 
     As an enzyme that catalyzes the reaction G to generate adipic acid, Deal and DcaJ from  Acinetobacter haylyi  strain ADP1 (NCBI-ProteinlDs: CAG68538 and CAG68539), and the like can be suitably used. 
     Since the above enzymatic reactions are reversible, the CoA transferase activity against 3-hydroxyadipyl-CoA, 2,3-dehydroadipyl-CoA, or adipyl-CoA used as a substrate can be determined by detecting 3-hydroxyadipyl-CoA, 2,3-dehydroadipyl-CoA, or adipyl-CoA generated respectively using purified CoA transferase with 3-hydroxyadipic acid and succinyl-CoA, α-hydromuconic acid and succinyl-CoA, or adipic acid and succinyl-CoA used as substrates thereof. The specific measurement method is, for example, as follows. 
     Preparation of 3-hydroxyadipic acid: Preparation of 3-hydroxyadipic acid is performed according to the method described in Reference Example 1 of WO 2016199856 A1. 
     Identification of CoA transferase activity using 3-hydroxyadipic acid as a substrate: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase in the full-length form. The amplified fragment is inserted into the Kpnl site of pRSF-1 b (manufactured by Novagen), an expression vector for  E. coli,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and expression of the enzyme is induced with isopropyl-1-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5mL 10K; manufactured by Merck Millipore) to remove the enzyme. The CoA transferase activity can be confirmed by detecting 3-hydroxyadipyl-CoA in the resulting filtrate on high-performance liquid chromatograph-tandem mass spectrometer (LC-MSMS) (Agilent Technologies, Inc.). 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pII 8.0) 
     10 mM MgCl, 
     0.4 mM succinyl-CoA 
     2 mM 3-hydroxyadipic acid sodium salt 
     20 μgmL CoA transferase. 
     Preparation of α-hydromuconic acid: Preparation of α-hydromuconic acid is performed according to the method described in Reference Example 1 of WO 2016199858 A1. 
     Identification of CoA transferase activity using α-hydromuconic acid as a substrate: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase in the full-length lot III. The amplified fragment is inserted into the Kpnl site of pRSF-lb (manufactured by Novagen), an expression vector for  E. coli,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5mL 10K; manufactured by Merck Millipore) to remove the enzyme. The CoA transferase activity can be confirmed by detecting 2,3-dehydroadipyl-CoA in the resulting filtrate on high-performance liquid chromatograph-tandem mass spectrometer (LC-MSMS) (Agilent Technologies, Inc.). 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pH 8.0) 
     10 niM MgCl 2    
     0.4 mM succinyl-CoA 
     2 mM α-hydromuconic acid sodium salt 
     20 μgmL CoA transferase. 
     Identification of CoA transferase activity using adipic acid as a substrate: A PCR using the genomic DNA of a subject microorganism strain as a template is performed in accordance with routine procedures, to amplify a nucleic acid encoding a CoA transferase in the full-length form. The amplified fragment is inserted into the Kpnl site of pRSF-1 b (manufactured by Novagen), an expression vector for  E. coli,  in-frame with the histidine-tag sequence. The plasmid is introduced into  E. coli  BL21 (DE3), and expression of the enzyme is induced with isopropyl-β-thiogalactopyranoside (IPTG) in accordance with routine procedures and the enzyme is purified using the histidine tag from the culture fluid to obtain a CoA transferase solution. The solution is used to prepare an enzymatic reaction solution with the following composition, which is allowed to react at 30° C. for 10 minutes and then filtered through a UF membrane (Amicon Ultra-0.5mL 10K; manufactured by Merck Millipore) to remove the enzyme. The CoA transferase activity can be confirmed by detecting adipyl-CoA in the resulting filtrate on high-performance liquid chromatograph-tandem mass spectrometer (LC-MSMS) (Agilent Technologies, Inc.). 
     (Enzymatic Reaction Solution) 
     100 mM Tris-HCl (pH 8.0) 
     10 mM MgCl 2    
     0.4 mM succinyl-CoA 
     2 mM adipic acid sodium salt 
     20 μgmL CoA-transferase. 
     Whether or not an enzyme originally expressed in a host microorganism used in the present invention has CoA transferase activity can be determined by adding 0.05 mL of the CFE, instead of purified CoA transferase, to a total of 0.1 mL of the enzymatic reaction solution and performing the above-described measurement. The specific CFE preparation method targeted to  E. coli  is as described for that used in determination of acyl transferase activity. 
     Either the polypeptides described in (a) to (c) or the 3-hydroxybutyryl-CoA dehydrogenase in the present invention is characterized by having higher activity than 3-oxoadipyl-CoA reductases used in conventional techniques. In this respect, the phrase “higher activity” refers to production of 3-hydroxyadipic acid, α-hydromuconic acid, or adipic acid with a higher yield in a genetically modified microorganism expressing any one of the polypeptides than in a genetically modified microorganism expressing a conventional 3-oxoadipyl-CoA reductase when those microorganisms are derived from the same host microorganism species and are cultured under the same expression conditions in a culture medium containing a carbon source as a material for fermentation. In this respect, the yield of 3-hydroxyadipic acid is calculated according to the formula (2). The yield of u-hydromuconic acid or adipic acid is calculated according to the formula (2), where 3-hydroxyadipic acid is replaced by u-hydromuconic acid or adipic acid, respectively. 
     
       
         
           
             
                 
             
             ⁢ 
             
               Formula 
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               ⁢ 
               
                 ( 
                 2 
                 ) 
               
             
           
         
       
       
         
           
             
               Yield 
               ⁢ 
               
                   
               
               ⁢ 
               
                 ( 
                 % 
                 ) 
               
             
             = 
             
               amount 
               ⁢ 
               
                   
               
               ⁢ 
               of 
               ⁢ 
               
                   
               
               ⁢ 
               generated 
               ⁢ 
               
                   
               
               ⁢ 
               3 
               ⁢ 
               
                 - 
               
               ⁢ 
               hydroxyadipic 
               ⁢ 
               
                   
               
               ⁢ 
               acid 
               ⁢ 
               
                   
               
               ⁢ 
               
                 ( 
                 mol 
                 ) 
               
               ⁢ 
               
                 / 
               
               ⁢ 
               amount 
               ⁢ 
               
                   
               
               ⁢ 
               of 
               ⁢ 
               
                   
               
               ⁢ 
               consumed 
               ⁢ 
               
                   
               
               ⁢ 
               carbon 
               ⁢ 
               
                   
               
               ⁢ 
               source 
               ⁢ 
               
                   
               
               ⁢ 
               
                 ( 
                 mol 
                 ) 
               
               × 
               100 
             
           
         
       
     
     The specific method to confirm the higher activity of either the polypeptides described in (a) to (c) or the 3-hydroxybutyryl-CoA dehydrogenase in the present invention compared to the activity of 3-oxoadipyl-CoA reductases used in conventional techniques is as follows. The pBBR1 MCS-2 vector, which is able to self-replicate in  E. coli  (ME Kovach, (1995), Gene 166: 175-176), is cleaved with Xhol to obtain pBBR1MCS-2Xhol. To integrate a constitutive expression promoter into the vector, an upstream 200-b region (SEQ ID NO: 186) of gapA (NCBI Gene ID: NC 000913.3) is amplified by PCR using the genomic DNA of  Escherichia coli  K-12 MG1655 as a template in accordance with routine procedures (for example, primers represented by SEQ ID NOs: 187 and 188 are used), and the obtained fragment and the pBBR1MCS-2Xhol are ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain the plasmid pBBR1MCS-2::Pgap. The pBBR1MCS-2::Pgap is cleaved with Scol to obtain pBBR1MCS-2::PgapScaI. A nucleic acid encoding an acyl transferase in the full length form is amplified by PCR in accordance with routine procedures (for example, primers represented by SEQ ID NOs: 190 and 191 are used), and the obtained fragment and pBBR1MCS-2::PgapScaI are ligated together using the In-Fusion HD Cloning Kit to obtain the plasmid pBBR1MCS-2::AT. The pBBR − 1MCS-2::AT is cleaved with Hpal to obtain pBBR1MCS-2::ATIIpal. A nucleic acid encoding a CoA transferase in the full length form is amplified by PCR in accordance with routine procedures (for example, primers represented by SEQ ID NOs: 194 and 195 are used), and the obtained fragment and pBBR1MCS-2::ATHpaI are ligated together using “the In-Fusion HD Cloning Kit” to obtain the plasmid pBBR1MCS-2::ATCT. 
     On the other hand, the pACYCDuet-1 expression vector (manufactured by Novagen), which is able to self-replicate in  E. coli,  is cleaved with BaivHl to obtain pACYCDuet-1BamHI. A nucleic acid encoding a polypeptide represented by any one of SEQ ID NOs: 1 to 86 or encoding a conventionally used 3-oxoadipyl-CoA reductase, is amplified by PCR in accordance with routine procedures (for example, primers represented by SEQ ID NOs: 196 and 197 are used), and the obtained fragment and pACYCDuet-1BamIII are ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) to obtain a plasmid that expresses the polypeptide represented by any one of SEQ ID NOs: 1 to 86 or expresses the conventionally used 3-oxoadipyl-CoA reductase. 
     The obtained plasmid and the pBBR1MCS-2::ATCT are introduced into  E. coli  strain BL21 (DE3) by electroporation (NM Calvin. PC Hanawalt. J. Bacteriol, 170 (1988), pp. 2796-2801). A loopful of the strain after the introduction is inoculated into 5 mL of the culture medium I (10 gL Bacto Tryptone (manufactured by Difco Laboratories), 5 gL Bacto Yeast Extract (manufactured by Difco Laboratories), 5 gL sodium chloride, 25 ugmL kanamycin, and 15 μgmL chloramphenicol) adjusted to pH 7, and incubated at 30° C. with shaking at 120 min −1  :for 18 hours. Subsequently, 0.25 mL of the culture fluid is added to 5 mL of the culture medium II (10 gL succinic acid, 10 gL glucose, 1 gl, ammonium sulfate, 50 mM potassium phosphate,0.025 gL magnesium sulfate,0.0625 mgL iron sulfate, 2.7 mgL manganese sulfate, 0.33 mgt, calcium chloride, 1.25 gL sodium chloride, 2.5 gL Bacto Tryptone. 1.25 gL Bacto Yeast Extract. 25 μgmL kanamycin, 15 μgmL chloramphenicol, and 0.01 mM IPTG) adjusted to plI 6.5. and incubated at 30° C. with shaking at 120 min −I  for 24 hours. The supernatant separated from bacterial cells by centrifugation of the culture fluid is processed by membrane treatment using Millex-GV (0.22 μm; PVDF; manufactured by Merck KGaA), and the resulting filtrate is analyzed to measure the 3-hydroxyadipic acid and carbon source concentrations in the culture supernatant. Quantitative analysis of 3-hydroxyadipic acid on LC-MSMS is performed under the following conditions.
     HPLC: 1290 Infinity (manufactured by Agilent Technologies, Inc.)   Column: Synergi hydro-RP (manufactured by Phenomenex Inc.), length: 100 mm,   internal diameter: 3 mm, particle size: 2.5 um   

     Mobile phase: 0.1% aqueous formic acid solution methanol =7030
     Flow rate: 0.3 mLmin   Column temperature: 40° C.   LC detector: DAD (210 nm)   MSMS: Triple-Quad LCMS (manufactured by Agilent Technologies, Inc.)   Ionization method: ESI in negative mode.   

     Quantitative analysis of carbon sources, such as sugars and succinic acid, on HPLC is performed under the following conditions.
     HPLC: Shimazu Prominence (manufactured by Shimadzu Corporation)   Column: Shodex Sugar SH1011 (manufactured by Showa Denko K.K.), length: 300 mm, internal diameter: 8 mm, particle size: 6 μm   Mobile phase: 0.05M aqueous sulfuric acid solution   Flow rate: 0.6 mLmin   Column temperature: 65° C.   Detector: RI.   

     When a nucleic acid encoding any one selected from the group of the acyl transferase, the CoA transferase, the enoyl-CoA hydratase, and the enoyl-CoA reductase is introduced into a host microorganism in the present invention, the nucleic acid may be artificially synthesized based on the amino acid sequence information of the enzyme in a database, or isolated from the natural environment. In cases where the nucleic acid is artificially synthesized, the usage frequency of codons corresponding to each amino acid in the nucleic acid sequence may be changed depending on the host microorganism into which the nucleic acid is introduced. 
     In the present invention, the method of introducing a nucleic acid encoding any one selected from the group of the acyl transferase, the CoA transferase, the enoyl-CoA hydratase, and the enoyl-CoA reductase into the host microorganism method is not limited to a particular method; for example, a method in which the nucleic acid is integrated into an expression vector capable of autonomous replication in the host microorganism and then introduced into the host microorganism, a method in which the nucleic acid is integrated into the genome of the host microorganism, and the like can he used. 
     In cases where a nucleic acid encoding any one of the enzymes is isolated from the natural environment, the sources of the genes are not limited to particular organisms, and examples of the organisms include those of the genus  Acinetobacter,  such as  Acinetobacter baylyi  and  Acinetobacter radioresistens;  the genus  Aerobacter,  such as  Aerobacter cloacae;  the genus  Alcaligenes,  such as  Alcaligenesfaecalis;  the genus  Bacillus,  such as  Bacillus badius, Bacillus magaterium,  and  Bacillus roseus;  the genus  Brevibacterium.  such as  Brevibacterium todinum;  the genus  Corynebacterium,  such as  Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,  and  Corynebacterium glutamicum;  the genus  Cupriavidus,  such as  Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus numazuensis,  and  Cupriavidus oxalaticus;  the genus  Delflia,  such as  Delftia acidovorans;  the genus  Escherichia,  such as  Escherichia coli  and  Escherichia lergusonii;  the genus  Hafnia,  such as  Hafnia alvei;  the genus  Microbacterium,  such as  Microbacterium ammoniaphilum;  the genus  Nocardioides,  such as  Nocardioides alhus;  the genus  Planomicrohium,  such as  Planomicrobium okeanokoites;  the genus  Pseudomonas,  such as  Pseudomonas azotoformans, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas Pseudomonas putida,  and  Pseudomonas reptilivora;  the genus  Rhizobium,  such as  Rhizobium radiohacter;  the genus  Rhodosporidium,  such as  Rhodosporidium toruloides;  the genus  Saccharomyces,  such as  Saccharomyces cerevisiae;  the genus  Serratia,  such as  Serratia entoinophila, Serratialicaria, Serratia fonticola, Serratia grimesii, Serratia nematodiphila, Serratia odorilera,  and  Serratia plymuthica;  the genus  Shimwellia,  such as  Shimwellia blattae;  the genus  Streptomyces,  such as  Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus,  and  Streptomyces vinaceus;  the genus  Yarrowia,  such as  Yarrowia lipolytica;  the genus  Yersinia,  such as  Yersinia ruckeri;  the genus  Euglena,  such as  Euglena gracilis;  and the genus  Thermobifida,  such as  Thermobifidalitsca.  Preferably, the organisms are those of the genera  Acinetobacter, Corynebacterium, Escherichia, Pseudomonas, Serratia, Euglena,  and  Thermobifida.    
     When a nucleic acid encoding a polypeptide expressed in the present invention is integrated into an expression vector or the genome of a host microorganism, the nucleic acid being integrated into the expression vector or the genome is preferably composed of a promoter, a ribosome-binding sequence, a nucleic acid encoding the polypeptide to be expressed, and a transcription termination sequence, and may additionally contain a gene that controls the activity of the promoter. 
     The promoter used in the present invention is not limited to a particular promoter, provided that the promoter drives expression of the enzyme in the host microorganism; examples of the promoter include gap promoter, trp promoter, lac promoter, toe promoter, and T7 promoter. 
     In cases where an expression vector is used in the present invention to introduce the nucleic acid or to enhance the expression of the polypeptide, the expression vector is not limited to a particular vector, provided that the vector is capable of autonomous replication in the microorganism; examples of the vector include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above vectors. 
     In cases where a nucleic acid for genome integration is used in the present invention to introduce the nucleic acid or to enhance the expression of the polypeptide, the nucleic acid for genome integration is introduced by site-specific homologous recombination. The method for site-specific homologous recombination is not limited to a particular method, and examples of the method include a method in which λ Red recombinase and FLP recombinase are used (Proc Natl Acad Sci U.S.A. 2000 Jun. 6; 97 (12): 6640-6645.), and a method in which X Red recombinase and the soeB gene are used (Biosci Biotechnol Biochem. 2007 December; 71 (12):2905-11.). 
     The method of introducing the expression vector or the nucleic acid for genome integration is not limited to a particular method, provided that the method is for introduction of a nucleic acid into a microorganism; examples of the method include the calcium ion method (Journal of Molecular Biology, 53, 159 (1970)), and electroporation (N M Calvin, P C Hanawalt. J. Bacteriol, 170 (1988), pp. 2796-2801). 
     In the present invention, a genetically modified microorganism in which a nucleic acid encoding a 3-oxoadipyl-CoA reductase is introduced or expression of the corresponding polypeptide is enhanced is cultured in a culture medium, preferably a liquid culture medium, containing a carbon source as a material for fermentation which can be used by ordinary microorganisms. The culture medium used contains, in addition to the carbon source that can be used by the genetically modified microorganism, appropriate amounts of a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients such as amino acids and vitamins. Any of natural and synthetic culture media can be used as long as the medium contains the above-described nutrients. 
     The material for fermentation is a material that can be metabolized by the genetically modified microorganism. The term “metabolize” refers to conversion of a chemical substance, which a microorganism has taken up from the extracellular environment or intracellularly generated from a different chemical substance, to another chemical substance through an enzymatic reaction. Sugars can be suitably used as the carbon source. Specific examples of the sugars include monosaccharides, such as glucose, sucrose, fructose, galactose, mannose, xylose, and arabinose; disaccharides and polysaccharides formed by linking these monosaccharides; and saccharified starch solution, molasses, and saccharified solution from cellulose-containing biomass, each containing any of those saccharides. 
     Other than the above sugars, succinic acid, a substrate of the CoA transferase, can also be added to the culture medium for efficient production of 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid. 
     The above-listed carbon sources may be used individually or in combination. When a carbon source is added, the concentration of the carbon source in the culture medium is not particularly limited, and can be appropriately selected depending on the type of the carbon source; in the case of sugars, the concentration is preferably from 5 gL to 300 gL; in the case of succinic acid, the concentration is preferably from 0.1 gL to 100 gL. 
     As the nitrogen source used for culturing the genetically modified microorganism, for example, ammonia gas, aqueous ammonia, ammonium salts, urea, nitric acid salts, other supportively used organic nitrogen sources, such as oil cakes, soybean hydrolysate, casein degradation products, other amino acids; vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, and bacterial cells and hydrolysate of various fermentative bacteria can be used. The concentration of the nitrogen source in the culture medium is not particularly limited, and is preferably from 0.1 gL to 50 gL. 
     As the inorganic salts used for culturing the genetically modified microorganism, for example, phosphoric acid salts, magnesium salts, calcium salts, iron salts, and manganese salts can be appropriately added to the culture medium and used. 
     The culture conditions for the genetically modified microorganism to produce 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid are set by appropriately adjusting or selecting, for example, the culture medium with the above composition, culture temperature, stirring speed, pH, aeration rate, and inoculation amount, depending on, for example, the species of the genetically modified microorganism and external conditions. 
     The pH range of the culture is not specifically limited, provided that the genetically modified microorganism can be grown in the pH range. However, the pH range is preferably from pH 5 to 8, more preferably from pH 5.5 to 6.8. 
     Although the range of aeration rates in the culture is not specifically limited, as long as 3-hydroxyadipic acid, α-hydromuconic acid, andor adipic acid can be produced under the aeration conditions. It is desired that oxygen remain in the gaseous phase andor liquid phase in a culture container for good growth of the mutant microorganism at least at the start of incubation. 
     In cases where foam is formed in a liquid culture, an antifoaming agent such as a mineral oil, silicone oil, or surfactant may be appropriately added to the culture medium. 
     After a recoverable amount of 3-hydroxyadipic acid, ct-hydromuconic acid, andor adipic acid is produced during culturing of the microorganism, the produced products can be recovered. The produced products can be recovered, for example isolated, according to a commonly used method, in which the culturing is stopped once a product of interest is accumulated to an appropriate level, and the fermentation product is collected from the culture. Specifically, the products can be isolated from the culture by separation of bacterial cells through, for example, centrifugation or filtration prior to, for example, column chromatography, ion exchange chromatography, activated charcoal treatment, crystallization, membrane separation, or distillation. More specifically, examples include, but are not limited to, a method in which an acidic component is added to salts of the products, and the resulting precipitate is collected; a method in which water is removed from the culture by concentration using, for example, a reverse osmosis membrane or an evaporator to increase the concentrations of the products and the products andor salts of the products are then crystallized and precipitated by cooling or adiabatic crystallization to recover the crystals of the products andor salts of the products by, for example, centrifugation or filtration; and a method in which an alcohol is added to the culture to produce esters of the products and the resulting esters of the products are subsequently collected by distillation and then hydrolyzed to recover the products. These recovery methods can be appropriately selected and optimized depending on, for example, physical properties of the products. 
     EXAMPLES 
     The present invention will be specifically described below with reference to examples. 
     Reference Example 1 
     Production of plasmids each expressing an enzyme catalyzing a reaction to generate 3-oxoadipyl-CoA and coenzyme A (the reaction A), an enzyme catalyzing a reaction to generate 3-hydroxyadipic acid from 3-hydroxyadipyl-CoA (the reaction E) and a reaction to generate α-hydromuconic acid from 2,3-dehydroadipyl-CoA (the reaction F), and a polypeptide represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 
     The pBBR1MCS-2 vector, which is capable of autonomous replication in  E. coil  (ME Kovach, (1995). Gene 166: 175-176), was cleaved with Xhol to obtain pBBR1MCS-2Xhol. To integrate a constitutive expression promoter into the vector, primers (SEQ ID NOs: 187 and 188) were designed to amplify the upstream 200-b region (SEQ ID NO: 186) of gapzI (NCBI Gene ID: NC 000913.3) by PCR using the genomic DNA of  Escherichia coil  K-12 MG1655 as a template, and a PCR reaction was performed in accordance with routine procedures. The obtained fragment and pF3BR1MCS-2XhoI were ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into  E. coli  strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant  E. coli  strain was confirmed in accordance with routine procedures, and the plasmid was designated as pBBRIMCS-2::Pgap. Then, the pBBR1MCS-2::Pgap was cleaved with Seal to obtain pBBR1MCS-2::PgapScaI. For amplification of a gene encoding an enzyme catalyzing the reaction A, primers (SEQ ID NOs: 190 and 191) were designed to amplify the full length of the acyl transferase gene pcaF (NCBI Gene ID: 1041755, SEQ ID NO: 189) by PCR using the genomic DNA of  Pseudomonas putida  strain KT2440 as a template, and a PCR reaction was performed in accordance with routine procedures. The obtained fragment and the pBBR1MCS-2::PgapScaI were ligated together using the In-Fusion HD Cloning Kit, and the resulting plasmid was introduced into  E. coli  strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures, and the plasmid was designated as pBBR1MCS-2::AT. Then, the pBBR1MCS-2::AT was cleaved with Hpal to obtain pBBR1MCS-2::ATHpaI. For amplification of a gene encoding an enzyme catalyzing the reactions D and F, primers (SEQ ID NOs: 194 and 195) were designed to amplify a continuous sequence including the full lengths of genes together encoding a CoA transferase, peal and peal (NCBI Gene IDs: 1046613 and 1046612, SEQ ID NOs: 192 and 193) by PCR using the genomic DNA of  Pseudomonas putida  strain KT2440 as a template, and a PCR reaction was performed in accordance with routine procedures. The obtained fragment and the pBBR1MCS-2::ATHpal were ligated together using the In-Fusion HD Cloning Kit, and the resulting plasmid was introduced into  E. coli  strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures, and the plasmid was designated as pBBR1MCS-2::ATCT. 
     The pBBR1MCS-2::ATCT was cleaved with Seal to obtain pBBR1MCS-2::ATCTSca1. For amplification of a nucleic acid encoding a polypeptide represented by SEQ ID NO: 1, primers (SEQ ID NOs: 196 and 197) were designed to amplify the nucleic acid represented by SEQ ID NO: 87 through PCR using the genomic DNA of Serratia marcescens strain ATCC13880 as a template, and a PCR reaction was performed in accordance with routine procedures. For amplification of a nucleic acid encoding a polypeptide represented by SEQ ID NO: 2, primers (SEQ ID NOs: 198 and 199) were designed to amplify the nucleic acid represented by SEQ ID NO: 88 through PCR using the genomic DNA of Serratia nematodiphila strain DSM21420 as a template, and a PCR reaction was performed in accordance with routine procedures. For amplification of a nucleic acid encoding a polypeptide represented by SEQ ID NO: 3, primers (SEQ ID NOs: 200 and 201) were designed to amplify the nucleic acid represented by SEQ ID NO: 89 through PCR using the genomic DNA of Serratia plymuthica strain NBRC102599 as a template, and a PCR reaction was performed in accordance with routine procedures. For amplification of a nucleic acid encoding a polypeptide represented by SEQ ID NO: 4, primers (SEQ ID NOs: 202 and 203) were designed to amplify the nucleic acid represented by SEQ ID NO: 90 through PCR using the genomic DNA of  Serratia proteamaculans  strain 568 as a template, and a PCR reaction was performed in accordance with routine procedures. For amplification of a nucleic acid encoding a polypeptide represented by SEQ ID NO: 5, primers (SEQ ID NOs: 204 and 205) were designed to amplify the nucleic acid represented by SEQ ID NO: 91 through PCR using the genomic DNA of  Serratia ureilytica  strain Lr54 as a template, and a PCR reaction was performed in accordance with routine procedures. For amplification of a nucleic acid encoding a polypeptide represented by SEQ ID NO: 6, primers (SEQ ID NOs: 206 and 207) were designed to amplify the nucleic acid represented by SEQ ID NO: 92 through PCR using the genomic DNA of  Serratia  sp. strain BW106 as a template, and a PCR reaction was performed in accordance with routine procedures. For amplification of a nucleic acid encoding a polypeptide represented by SEQ ID NO: 7, primers (SEQ ID NOs: 208 and 209) were designed to amplify the nucleic acid represented by SEQ ID NO: 93 through PCR using the genomic DNA of  Serratia liquefaciens  strain FK01 as a template, and a PCR reaction was performed in accordance with routine procedures. Each of the obtained fragments and the pBBR1MCS-2::ATCTScaI were ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and each of the resulting plasmids was introduced into  E. coli  strain DH5a. The nucleotide sequence on the plasmid isolated from each of the obtained recombinant strains was confirmed in accordance with routine procedures. 
     The plasmid for expression of the polypeptide represented by SEQ ID NO: 1 was designated as “pBBR1MCS-2::ATCTOR1”; the plasmid for expression of the polypeptide represented by SEQ ID NO: 2 was designated as “pBBR1MCS-2::ATCTOR2”; the plasmid for expression of the polypeptide represented by SEQ ID NO: 3 was designated as “pBFIR1MCS-2::ATCTOR3”; the plasmid for expression of the polypeptide represented by SEQ ID NO: 4 was designated as “pBBR1N1CS-2::ATCTOR4”; the plasmid for expression of the polypeptide represented by SEQ ID NO: 5 was designated as “pBBR1MCS-2::ATCTOR5”; the plasmid for expression of the polypeptide represented by SEQ ID NO: 6 was designated as “pBBR1MCS-2::ATCTOR6”; and the plasmid for expression of the polypeptide represented by SEQ ID NO: 7 was designated as “pBBR1MCS-2::ATCTOR7”; and these plasmids are listed in Table 6. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                 ID 
               
               
                 Plasmid 
                 Originating organism 
                 Gene ID 
                 NO: 
               
               
                   
               
             
            
               
                 pBBR1MCS- 
                   Serratia marcescens  ATCC 
                 JMPQ01000047.1 
                 87 
               
               
                 2::ATCTOR1 
                 13880 
                   
                   
               
               
                 pBBR1MCS- 
                 
                   Serratia nematodiphila 
                 
                 JPUX00000000.1 
                 88 
               
               
                 2::ATCTOR2 
                 DSM21420 
                   
                   
               
               
                 pBBR1MCS- 
                 
                   Serratia plymuthica 
                 
                 BCTU01000013.1 
                 89 
               
               
                 2::ATCTOR3 
                 NBRC102599 
                   
                   
               
               
                 pBBR1MCS- 
                 
                   Serratia proteamaculans 
                 
                 CP000826.1 
                 90 
               
               
                 2::ATCTOR4 
                 568 
                   
                   
               
               
                 pBBR1MCS- 
                   Serratia ureilytica  Lr5/4 
                 JSFB01000001 
                 91 
               
               
                 2::ATCTOR5 
                   
                   
                   
               
               
                 pBBR1MCS- 
                   Serratia  sp. BW106 
                 MCGS01000002.1 
                 92 
               
               
                 2::ATCTOR6 
                   
                   
                   
               
               
                 pBBR1MCS- 
                   Serratia liquefaciens  FK01 
                 CP006252.1 
                 93 
               
               
                 2::ATCTOR7 
               
               
                   
               
            
           
         
       
     
     Reference Example 2 
     Production of a plasmid for expression of an enzyme catalyzing a reaction to generate 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA (the reaction C) 
     The pMW119 expression vector (manufactured by Nippon Gene Co., Ltd.), which is capable of autonomous replication in  E. coli,  was cleaved with Sad to obtain pMW119SacI. To integrate a constitutive expression promoter into the vector, primers (SEQ ID NOs: 210 and 211) were designed to amplify the upstream 200-b region (SEQ ID NO: 186) of gapA (NCBI Gene ID: NC 000913.3) by PCR using the genomic DNA of Escherichia coil K-12 MG1655 as a template, and a PCR reaction was performed in accordance with routine procedures. The obtained fragment and the pMW119SacI were ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into  E. coli  strain DH5ct. The nucleotide sequence on the plasmid isolated from the obtained recombinant  E. coli  strain was confirmed in accordance with routine procedures, and the plasmid was designated as pMW1 19::Pgap. Then, the pMW1 19::Pgap was cleaved with Sphl to obtain pMW119::PgapSphl. For amplification of a gene encoding an enzyme catalyzing the reaction C, primers (SEQ ID NOs: 212 and 213) were designed to amplify the full length of the enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, SEQ ID NO: 176) by PCR using the genomic DNA of  Pseudomonas puticia  strain KT2440 as a template, and a PCR reaction was performed in accordance with routine procedures. The obtained fragment and the pMW119::PgapSphl were ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into E. coli strain DH5a. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures. The obtained plasmid was designated as “pMW119::EH”. 
     Reference Example 3 
     Production of plasmids each expressing an enzyme catalyzing a reaction to generate 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (the reaction A), an enzyme catalyzing a reaction to generate adipic acid from adipyl-CoA (the reaction G), and a polypeptide represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 
     For amplification of a gene encoding an enzyme catalyzing the reaction G, primers (SEQ ID NOs: 216 and 217) were designed to amplify a continuous sequence including the full lengths of genes together encoding a CoA transferase, dcaI and dcaf (NCBI Gene ID: CR543861.1, SEQ ID NOs: 214 and 215) by PCR using the genomic DNA of Acinetobacter baylyi strain ADPI as a template, and a PCR reaction was performed in accordance with routine procedures. The obtained fragment and each of the fragments obtained by cleaving the pBBR1MCS-2::ATCTORE pBBR1MCS-2::ATCTOR2, pBBR1MCS-2::ATCTOR3, pBBR1MCS-2::ATCTOR4, pBBR1MCS-2::ATCTOR5, pBBR1MCS-2::ATCTOR6, and pBBR1MCS-2::ATCTOR7 with Hpal, which were produced in Reference Example 1, were ligated together using the In-Fusion HD Cloning Kit, and each of the resulting plasmids was introduced into E. coli strain DH5u. The nucleotide sequences on the plasmids isolated from the obtained recombinant strains were confirmed in accordance with routine procedures, and the plasmids were designated as pBBR1MCS-2::ATCT2OR1, pBBIUMCS-2::ATCT2OR2, pBBR1MCS-2::ATCT2OR3, pBBR1MCS-2::ATCT2OR4, pBBR1MCS-2::ATCT2OR5, pBBR1MCS-2::ATCT2OR6, and pBBR1MCS-2::ATCT2OR7. 
     Reference Example 4 
     Production of a plasmid for expression of enzymes catalyzing a reaction to generate 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA (the reaction C) and a reaction to generate adipyl-CoA from 2,3-dehydroadipyl-CoA (the reaction D) 
     The pMW119::EH was cleaved with HindIll to obtain pMW119::EHHindIII. For amplification of a gene encoding an enzyme catalyzing the reaction D, primers (SEQ ID NOs: 219 and 220) were designed to amplify the full length of dcaA (NCBI-Protein ID: AAL09094.1, SEQ ID NO: 218) from Acinetobacter baylyi strain ADP1 by PCR, and a PCR reaction was performed in accordance with routine procedures. The obtained fragment and the pMW119::EHHindIII were ligated together using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.), and the resulting plasmid was introduced into  E. coli  strain DII5u. The nucleotide sequence on the plasmid isolated from the obtained recombinant strain was confirmed in accordance with routine procedures, and the plasmid was designated as pMW119::EHER. 
     Example 1 
     Generation of a Mutant Microorganism of the Genus  Serratia  with Impaired Pyruvate Kinase Function 
     Genes encoding the pyruvate kinase of a microorganism of the genus  Serratia,  pykF and pykA, were disrupted to generate a mutant microorganism of the genus  Serratia  with impaired pyruvate kinase function. 
     The procedure for disrupting pykF and pykA followed the method described in Proc Natl Acad Sci U S A., 2000 Jun. 6, 97(12): 6640-6645. 
     Generation of a Mutant Microorganism Of The Genus  Serratia  Deficient in pykF 
     A PCR reaction was performed using pKD4 as a template and oligo DNAs represented by SEQ ID NOs: 221 and 222 as primers to obtain a PCR fragment of 1.6 kb in length for disruption of pykF. A FRT recombinase expression plasmid, pKD46, was introduced into Serratia grime,sli strain NBRC13537, and an ampicillin-resistant strain was obtained. The obtained strain was inoculated into 5 mL of LB medium containing 500 ugmL ampicillin and was cultured at 30° C. with shaking for 1 day. Subsequently, 0.5 mL of the culture fluid was inoculated into 50 mL of LB medium containing 500 ugmI, ampicillin and 50 mM arabinose and was cultured in rotation at 30° C. for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cells were then washed with 10% (ww) glycerol three times. The washed pellet was suspended in 100 ,AL of 10% (ww) glycerol and mixed with 5 μL of the PCR fragment, and the mixture was then cooled in an electroporation cuvette on ice for 10 minutes. Electroporation was performed using a Gene Pulser electroporator (manufactured by Bio-Rad Laboratories, Inc.; 3 kV, 200 D, 25 μF), and 1 mL of SOC medium was added to the electroporation cuvette immediately after the electroporation, and the bacterial cells in the cuvette were incubated at 30° C. with shaking for 2 hours. The total volume of the culture was applied to LB agar medium containing 25 ugmL kanamycin and was incubated at 30° C. for 1 day. Direct colony PCR was performed on the resulting kanamycin-resistant strains to confirm the deletion of the gene of interest and the insertion of a kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 223 and 225 were used. 
     Subsequently, one of the kanamycin-resistant strains was inoculated into 5 mL of LB medium and was cultured at 37° C. and passaged twice to segregate away the pKD46 and to obtain an ampicillin-sensitive strain. The plasmid pCP20 was introduced into the ampicillin-sensitive strain, and ampicillin-resistant strains were again obtained. After culturing the obtained strains at 40° C., colony direct PCR was performed on the resulting strains to confirm the deletion of the kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ IT) NOs: 224 and 225 were used. Subsequently, one of the kanamycin-sensitive strains was inoculated into 5 mL of LB medium and was cultured at 37° C. and passaged twice to segregate away the pCP20. The obtained strain was designated as Serratia grimestiNBRC13537 zlpyk − F. 
     Generation of a Mutant Microorganism of the Genus  Serratia  Deficient in pykA 
     A PCR reaction was performed using pKD4 as a template and oligo DNAs represented by SEQ ID NOs: 226 and 227 as primers to obtain a PCR fragment of 1.6 kb in length for disruption of pykA. 
     By the same method as used for the generation of the pykA-deficient strain, pykA was disrupted in the  Serratia grimesii  NBRC13537 zipykE strain. After the plasmid pKD46 was introduced into the above strain, the PCR fragment used for disruption of pykA was introduced to the resulting strain. Direct colony PCR was performed on the resulting kanamycin-resistant strains to confirm the deletion of the gene of interest and the insertion of a kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 223 and 229 were used. 
     Subsequently, an ampicillin-sensitive strain was obtained by segregating away the pKD46. The plasmid pCP20 was introduced into the ampicillin-sensitive strain, and ampicillin-resistant strains were again obtained. Colony direct PCR was performed on the obtained strains to confirm the deletion of the kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 228 and 229 were used. The pCP20 was segregated away from one of the kanamycin-sensitive strains. The obtained strain was designated as SgΔPP. 
     Example 2 
     Generation of mutant microorganisms of the genus  Serratia  with impaired pyruvate kinase function and carrying a plasmid expressing enzymes that catalyze the reactions A, B, E, and F 
     Each of the plasmids produced in Reference Example 1 was introduced into the SgΔPP produced in Example 1 to generate mutant microorganisms of the genus Serratia. Additionally, a mutant microorganism of the genus  Serratia  was generated as a control by introducing the pBBR1MCS-2 empty vector into the SgΔPP. 
     The SgΔPP was inoculated into 5 mL of LB medium and cultured at 30° C. with shaking for I day. Subsequently, 0.5 mL of the culture fluid was inoculated into 5 mL of LB medium and was cultured at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cells were then washed with 10% (ww) glycerol three times. The washed pellet was suspended in 100 μL of 10% (ww) glycerol and mixed with 1 uL of the pBBR1MCS-2 (control), pBBR1MCS-2::ATCTOR1, pBBRIMCS-2::ATCTOR2, pBBR1MCS-2::ATCTOR3, pBBRIMCS-2::ATCTOR4, pBBR1MCS-2::ATCTOR5, pBBR1MCS-2::ATCTOR6, or pBBR1MCS-2::ATCTOR7, and the mixture was then cooled in an electroporation cuvette on ice for 10 minutes. Electroporation was performed using a Gene Pulser electroporator (manufactured by Bio-Rad Laboratories, Inc.; 3 kV, 200 Ω, 25 μF), and 1 mL of SOC medium was added to the electroporation cuvette immediately after the electroporation, and the bacterial cells in the cuvette were incubated at 30° C. with shaking for 1 hour. Fifty μL of the culture was applied to LB agar medium containing 25 μgmL kanamycin and was incubated at 30° C. for 1 day. The obtained strains were designated as SgΔPPpBBR (negative control), SgΔPP3HA1, SgΔPP31IA2, SgΔPP3HA3, SgΔPP3HA4, SgΔPP3HA5, SgΔPP3HA6, and SgΔPP3HA7. 
     Reference Example 5 
     Generation of Mutant Microorganisms of the Genus  Serratia  with Intact Pyruvate Kinase Function and Carrying a Plasmid Expressing Enzymes that Catalyze the Reactions A, B, E, and F 
     By the same method as in Example 2, the pBBR1MCS-2 (control), pBBR1MCS-2::ATCTOR1, pBBR1MCS-2::ATCTOR2, pBBR1MCS-2::ATCTOR3, pBBRl S-2::ATCTOR4,)1313R1MCS-2::AICTOR5, pBBR1MCS-2::ATCTOR6, or pBBRlMCS-2::ATCTOR7 was introduced into Serratia grimesii NBRC13537. The obtained strains were designated as SgpBBR (negative control), Sg3HA1, Sg31-1A2, Sg3HA3, Sg3HA4, Sg3HA5, Sg3HA6, and Sg3HA7. 
     Example 3 
     Production test of 3-hydroxyadipic acid and u-hydromuconic acid using mutant microorganisms of the genus  Serratia  with impaired pyruvate kinase function 
     The production test of 3-hydroxyadipic acid and α-hydromuconic acid was conducted using the mutant microorganisms of the genus  Serratia  produced in Example 2. 
     A loopful of each mutant produced in Example 2 was inoculated into 5 mL (in a glass test tube of 18-mm diameter with aluminum cap) of the culture medium 1 (10 gL Bacto Tryptone (manufactured by Difco Laboratories), 5 gL Bacto Yeast Extract (manufactured by Difco Laboratories), 5 gL sodium chloride, 25 μgmL kanamycin) adjusted to pH 7 and was cultured at 30° C. with shaking at 120 min −1  for 24 hours. Subsequently, 0.25 mL of the culture fluid was added to 5 mI, (in a glass test tube of 18-mm diameter with aluminum cap) of the culture medium II (50gL glucose. 1 gL ammonium sulfate, 50 mM potassium phosphate, 0.025 gL magnesium sulfate, 0.0625 mgL iron sulfate, 2.7 mgL manganese sulfate, 0.33 mgL calcium chloride, 1.25 gL sodium chloride, 2.5 gL Bacto Tryptone. 1.25 gL, Bacto Yeast Extract, 25 μgmL kanamycin) adjusted to p11 6.5 and was cultured at 30° C. with shaking at 120 min −I  for 24 hours. 
     Quantitative Analysis of Substrate and Product 
     The supernatant separated from bacterial cells by centrifugation of each culture fluid was processed by membrane treatment using Millex-GV (0.22 μm; PVDF; manufactured by Merck KGaA), and the resulting filtrate was analyzed by the following methods to quantify the concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium. The yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the above formula (2) from the measurement results are shown in Table 7. However, a concentration of not more than 0.1 mgL is considered to be below the detection limit in the quantitative LC-MSMS analysis and is hereinafter denoted in each table as N.D. 
     Quantitative Analysis of 3-Hydroxyadipic Acid and α-Hydromuconic Acid by LC-MSMS 
     
         
         HPLC: 1290 Infinity (manufactured by Agilent Technologies, Inc.) 
         Column: Synergi hydro-RP (manufactured by Phenomenex Inc.), length: 100 mm, internal diameter: 3 mm, particle size: 2.5 μm 
         Mobile phase: 0.1% aqueous formic acid solution methanol =7030 
         Flow rate: 0.3 mLmin 
         Column temperature: 40° C. 
         LC detector: 1260DAD VL+(210 nm) 
         MSMS: Triple-Quad LCMS (manufactured by Agilent Technologies, Inc.) 
         Ionization method: ESI in negative mode.
 
Quantitative analysis of organic acids by HPLC
 
         HPLC:LC-10A (manufactured by Shimadzu Corporation) 
         Column: Shim-pack SPR-H (manufactured by Shimadzu GLC Ltd.), length: 250 mm, internal diameter: 7.8 mm, particle size: 8 μm 
         Shim-pack SCR-101H (manufactured by Shimadzu GLC Ltd.) length: 250 mm, internal diameter: 7.8 mm, particle size: 10 um 
         Mobile phase: 5 mM p-toluenesulfonic acid 
         Reaction solution: 5 mMp-toluenesulfonic acid, 0.1 mM EDTA, 20 mM Bis-Tris 
         Flow rate: 0.8 mLmin 
         Column temperature: 45° C. 
         Detector: CDD-l0Avp (manufactured by Shimadzu Corporation) 
       
    
     Quantitative Analysis of Sugars and Alcohol by HPLC 
     
         
         HPLC: Shimazu Prominence (manufactured by Shimadzu Corporation) 
         Column: Shodex Sugar SII41011 (manufactured by Showa Denko K.K.), length: 300 mm, internal diameter: 8 mm, particle size: 6 μm 
         Mobile phase: 0.05M aqueous sulfuric acid solution 
         Flow rate: 0.6 mLmin 
         Column temperature: 65° C. 
         Detector: RID-10A (manufactured by Shimadzu Corporation). 
       
    
     Comparative Example 1 
     Production test of 3-hydroxyadipic acid and α-hydromuconic acid using mutant microorganisms of the genus  Serratia  with intact pyruvate kinase function 
     The mutant microorganisms of the genus  Serratia  produced in Reference Example 5 were cultured in the same manner as in Example 3. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the above formula (2) from the measurement results are shown in Table 7. 
     By comparing the results of Comparative Example 1 and Example 3, it was found that the yields of 3-hydroxyadipic acid and ct-hydromuconic acid were increased by impairing the function of pyruvate kinase in the microorganism of the genus  Serratia.    
     
       
         
           
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 Yield of 
                 Yield of 
               
               
                   
                 Strain 
                 3HA (%) 
                 HMA (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Example 3 
                 SgΔPP/ 
                 0.0362 
                 0.0113 
               
               
                   
                 pBBR 
                   
                   
               
               
                   
                 SgΔPP/3HA1 
                 3.47 
                 0.0782 
               
               
                   
                 SgΔPP/3HA2 
                 5.78 
                 0.0960 
               
               
                   
                 SgΔPP/3HA3 
                 5.24 
                 0.0846 
               
               
                   
                 SgΔPP/3HA4 
                 5.10 
                 0.0909 
               
               
                   
                 SgΔPP/3HA5 
                 6.21 
                 0.107 
               
               
                   
                 SgΔPP/3HA6 
                 6.28 
                 0.103 
               
               
                   
                 SgΔPP/3HA7 
                 4.96 
                 0.0638 
               
               
                 Comparative 
                 Sg/ 
                 N.D. 
                 N.D. 
               
               
                 Example 1 
                 pBBR 
                   
                   
               
               
                   
                 Sg/3HA1 
                 0.784 
                 0.0293 
               
               
                   
                 Sg/3HA2 
                 1.15 
                 0.0470 
               
               
                   
                 Sg/3HA3 
                 0.942 
                 0.0461 
               
               
                   
                 Sg/3HA4 
                 0.875 
                 0.0418 
               
               
                   
                 Sg/3HA5 
                 1.01 
                 0.0529 
               
               
                   
                 Sg/3HA6 
                 1.03 
                 0.0366 
               
               
                   
                 Sg/3HA7 
                 0.943 
                 0.0237 
               
               
                   
               
            
           
         
       
     
     Example 4 
     Generation of an  E. coli  mutant with impaired pyruvate kinase function 
     Genes encoding the pyruvate kinase of  E. coli,  pykF and pykA, were disrupted to generate an  E. coli  mutant with impaired pyruvate kinase function. The procedure for disrupting pykF and pykA followed the method described in Proc Natl Acad Sci USA., 2000 Jun. 6, 97(12): 6640-6645. 
     Generation of an  E. Coli  Mutant Deficient in pykF 
     A PCR reaction was performed using pKD4 as a template and oligo DNAs represented by SEQ ID NOs: 230 and 231 as primers to obtain a PCR fragment of 1.6 kb in length for disruption of pykF. 
     A FRT recombinase expression plasmid, pKD46, was introduced into  Escherichia coli  strain MG1655, and an ampicillin-resistant strain was obtained. The obtained strain was inoculated into 5 mL of LB medium containing 100 μgmL ampicillin and cultured at 30° C. with shaking for 1 day. Subsequently, 0.5 mL of the culture fluid was inoculated into 50 mL of LB medium containing 100 ugmL ampicillin and 50 mM arabinose, and was cultured in rotation at 30° C. for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cells were then washed with 10% (ww) glycerol three times. The washed pellet was suspended in 100 μL of 10% (ww) glycerol and mixed with 5 μL of the PCR fragment, and the mixture was then cooled in an electroporation cuvette on ice for 10 minutes. Electroporation was performed using a Gene Pulser electroporator (manufactured by Bio-Rad Laboratories, Inc.; 3 kV, 200 Q, 25 uF), and 1 mL of SOC medium was added to the electroporation cuvette immediately after the electroporation, and the bacterial cells in the cuvette were incubated at 30° C. with shaking for 2 hours. The total volume of the culture was applied to LB agar medium containing 25 μgmL kanamycin and was incubated at 30° C. for 1 day. Direct colony PCR was performed on the resulting kanamycin-resistant strains to confinn the deletion of the gene of interest and the insertion of a kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 223 and 233 were used. 
     Subsequently, one of the kanamycin-resistant strains was inoculated into 5 mL of LB medium and was cultured at 37° C. and passaged twice to segregate away the pKD46 and to obtain an ampicillin-sensitive strain. The plasmid pCP20 was introduced into the ampicillin-sensitive strain, and ampicillin-resistant strains were again obtained. After culturing the obtained strains at 40° C., direct colony PCR was performed on the resulting strains to confirm the deletion of the kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 232 and 233 were used. Subsequently, one of the kanamycin-sensitive strains was inoculated into 5 mL of LB medium and was cultured at 37° C. and passaged twice to segregate away the pCP20. The obtained strain was designated as Escherichia coli MG1655 zlpykF. 
     Generation of an  E. coli  Mutant Deficient in pykA 
     A PCR reaction was performed using pKD4 as a template and oligo DNAs represented by SEQ ID NOs: 234 and 235 as primers to obtain a PCR fragment of 1.6 kb in length for disruption of pykA. 
     By the same method as used for the generation of the pykF-deficient strain, pykA was disrupted in the  Escherichia coli  MG1655 zlpykF strain. After the plasmid pKD46 was introduced into the above strain, the PCR fragment used for disruption of pykA was introduced to the resulting strain. Direct colony PCR was performed on the resulting kanamycin-resistant strains to confirm the deletion of the gene of interest and the insertion of a kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 223 and 224 were used. 
     Subsequently, an ampicillin-sensitive strain was obtained by segregating away the pKD46. The plasmid pCP20 was introduced into the ampicillin-sensitive strain, and ampicillin-resistant strains were again obtained. Direct colony PCR was performed on the obtained strains to confirm the deletion of the kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 236 and 237 were used. The pCP20 was segregated away from one of the kanamycin-sensitive strains. The obtained strain was designated as EcΔPP. 
     Example 5 
     Generation of  E. coli  Mutants with Impaired Pyruvate Kinase Function and Carrying a Plasmid Expressing Enzymes that Catalyze the Reactions A, B, E, and F 
     Each of the plasmids produced in Reference Example 1 was introduced into the EcΔPP produced in Example 4 to generate  E. coli  mutants. 
     The EcΔPP was inoculated into 5 mL of LB medium and cultured at 30° C. with shaking for 1 day. Subsequently, 0.5 mL of the culture fluid was inoculated into 5 mL of LB medium and was cultured at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cells were then washed with 10% (ww) glycerol three times. The washed pellet was suspended in 100 μL of 10% (wvv) glycerol and mixed with 1 μL of the pBBR1MCS-2 (negative control), pBBR1MCS-2::ATCTOR1, pBBR1MCS-2::ATCTOR2, pBBR1MCS-2::ATCTOR3. pBBR1MCS-2::ATCTOR4, pBBR1MCS-2::ATCTOR5, pBBR1MCS-2::ATCTOR6, or pBBR1MCS-2::ATCTOR7, and the mixture was then cooled in an electroporation cuvette on ice for 10 minutes. Electroporation was performed using a Gene Pulser electroporator (manufactured by Bio-Rad Laboratories, Inc.; 3 kV, 200 Q, 25 μF). and 1 mL of SOC medium was added to the electroporation cuvette immediately after the electroporation, and the bacterial cells in the cuvette were incubated at 30° C. with shaking for 1 hour. Fifty μL of the culture was applied to LB agar medium containing 25 pgmL kanamycin and was incubated at 30° C. for 1 day. The obtained strains were designated as EcΔPPpBBR (negative control), EcΔPP3HA1, EcΔPP3HA2, EcΔPP31-1A3, EcΔPP3HA4, EcΔPP3HA5, EcΔPP3HA6, and EcΔPP3IIA7. 
     Reference Example 6 
     Generation of  E. Coli  Mutants with Intact Pyruvate Kinase Function and Carrying a Plasmid Expressing Enzymes that Catalyze the Reactions A, B, E, and F 
     By the same method as in Example 5, the pBBR1MCS-2 (control), pBBR1MCS-2::ATCTOR1, pBBR1MCS-2::ATCTOR2, pBBR1MCS-2::ATCTOR3, pBBR1MCS-2::ATCTOR4, pBBR1MCS-2::ATCTORS, pBBR1MCS-2::ATCTOR6, or pBBR1MCS-2::ATCTOR7 was introduced into  Escherichia coli  MG1655. The obtained strains were designated as EepBBR (negative control), Ec3IIA1, Ec3HA2, Ec3HA2, Ec3HA4, Ec3HA5, Ec3HA6, and Ec3HA7. 
     Example 6 
     Production Test of 3-Hydroxyadipic Acid and α-Hydromuconic Acid Using  E. Coli  Mutants with Impaired Pyruvate Kinase Function 
     The mutants produced in Example 5 were cultured in the same manner as in Example 3. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the above formula (2) from the measured values are shown in Table 8. 
     Comparative Example 2 
     Production Ttest of 3-Hydroxyadipic Acid and α-Hydromuconic Acid Using  E. Coli  Mutants with Intact Pyruvate Kinase Function 
     The mutants produced in Reference Example 6 were cultured in the same manner as in Example 6. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yields of 3-hydroxyadipic acid and ci-hydromuconic acid calculated using the above formula (2) from the measured values are shown in Table 8. 
     By comparing the results of Comparative Example 2 and Example 6. it was found that the yields of 3-hydroxyadipic acid and 0.-hydromuconic acid were increased by impairing the function of pyruvate kinase in  E. coli.    
     
       
         
           
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
                   
                 Yield of 
                 Yield of 
               
               
                   
                 Strain 
                 3HA (%) 
                 HMA (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Example 6 
                 EcΔPP/ 
                 0.0427 
                 0.0132 
               
               
                   
                 pBBR 
                   
                   
               
               
                   
                 EcΔPP/3HA1 
                 2.54 
                 0.0292 
               
               
                   
                 EcΔPP/3HA2 
                 3.97 
                 0.0333 
               
               
                   
                 EcΔPP/3HA3 
                 3.64 
                 0.0273 
               
               
                   
                 EcΔPP/3HA4 
                 2.86 
                 0.0257 
               
               
                   
                 EcΔPP/3HA5 
                 3.67 
                 0.0269 
               
               
                   
                 EcΔPP/3HA6 
                 3.57 
                 0.0348 
               
               
                   
                 EcΔPP/3HA7 
                 3.13 
                 0.0274 
               
               
                 Comparative 
                 Ec/ 
                 N.D. 
                 N.D. 
               
               
                 Example 2 
                 pBBR 
                   
                   
               
               
                   
                 Ec/3HA1 
                 1.48 
                 0.0172 
               
               
                   
                 Ec/3HA2 
                 2.59 
                 0.0160 
               
               
                   
                 Ec/3HA3 
                 2.64 
                 0.0167 
               
               
                   
                 Ec/3HA4 
                 1.82 
                 0.0186 
               
               
                   
                 Ec/3HA5 
                 2.47 
                 0.0166 
               
               
                   
                 Ec/3HA6 
                 2.66 
                 0.0228 
               
               
                   
                 Ec/3HA7 
                 1.78 
                 0.0172 
               
               
                   
               
            
           
         
       
     
     Example 7 
     Generation of mutant microorganisms of the genus  Serratia  with impaired pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B, C, E, and F 
     The plasmid pMW119::EH produced in Reference Example 2 was introduced into each mutant microorganism of the genus  Serratia  produced in Example 2 to generate mutant microorganisms of the genus  Serratia  . Additionally, a mutant microorganism of the genus  Serratia  was generated as a control by introducing the pMW119 empty vector into the SgΔPPpBBR produced in Example 2. 
     The SgΔPPpBBR, SgΔPP3HA1 SgΔPP3HA2, SgΔPP3HA3, SgΔPP3HA4, SgΔPP3HA5, SgΔPP3HA6, or SgΔPP3HA7 was inoculated into 5 mi. of LB medium containing 25 μgmL kanamycin and cultured at 30° C. with shaking for 1 day. Subsequently, 0.5 mL of the culture fluid was inoculated into 5 mf. of LB medium containing 25 μgmL kanamycin and was cultured at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cells were then washed with 10% (ww) glycerol three times. The washed pellet was suspended in 100 μL of 10% (ww) glycerol and mixed with 1 μL of the pBBR1MCS-2 (control) or pMW119::EH, and the mixture was then cooled in an electroporation cuvette on ice for 10 minutes. Electroporation was performed using a Gene Pulser electroporator (manufactured by Bio-Rad Laboratories, Inc.; 3 kV, 200 Ω, 25 μF), and 1 mL of SOC medium was added to the electroporation cuvette immediately after the electroporation, and the bacterial cells in the cuvette were incubated at 30° C. with shaking for 1 hour. Fitly of the culture was applied to LB agar medium containing 500 ugmL ampicillin and 25 μgmL kanamycin and was incubated at 30° C. for 1 day. The obtained strains were designated as SgΔPPpBBRpMW (negative control), SgΔPPIIMA1, SgΔPPHMA2, SgΔPPHMA3, SgΔPPHMA4, SgΔPPHMAS, SgΔPPHMA6, and S APPHMA7. 
     Reference Example 7 
     Generation of mutant microorganisms of the genus  Serratia  with intact pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B, C, E, and F 
     By the same method as in Example 7, the pMW119 (control) or pMW119::EH was introduced into SgpBBR, Sg3HA1, Sg3HA2, Sg3HA3, Sg3HA4, Sg3HA5, Sg311A6, and Sg3HA7. The obtained strains were designated as SgpBBRpMW (negative control), SgHMA1, SgHMA2, SgHMA3, SgHMA4, SgHMA5, SgHMA6, and SgHMA7. 
     Example 8 
     Production test of α-hydromuconic acid using mutant microorganisms of the genus Semliki with impaired pyruvate kinase function 
     The mutants produced in Example 7 were cultured in the same manner as in Example 3, except that ampicillin was added to the culture medium to a final concentration of 500 μgmL. The concentrations of α-hydromuconic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of α-hydromuconic acid calculated using the above formula (2) from the measured values is shown in Table 9. 
     Comparative Example 3 
     Production test of α-hydromuconic acid using mutant microorganisms of the genus  Serratia  with intact pyruvate kinase function 
     The mutants produced in Reference Example 7 were cultured in the same manner as in Example 8. The concentrations of α-hydromuconic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of α-hydromuconie acid calculated using the above formula (2) from the measured values is shown in Table 9. 
     By comparing the results of Comparative Example 3 and Example 8, it was found that the yield of a-hydromuconic acid was increased by impairing the function of pyruvate kinase in the microorganism of the genus  Serratia.    
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                   
                   
                 Yield of 
               
               
                   
                   
                 Strain 
                 HMA (%) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 Example 8 
                 SgΔPP/ 
                 0.0119 
               
               
                   
                   
                 pBBRpMW 
                   
               
               
                   
                   
                 SgΔPP/HMA1 
                 0.156 
               
               
                   
                   
                 SgΔPP/HMA2 
                 0.179 
               
               
                   
                   
                 SgΔPP/HMA3 
                 0.153 
               
               
                   
                   
                 SgΔPP/HMA4 
                 0.118 
               
               
                   
                   
                 SgΔPP/HMA5 
                 0.217 
               
               
                   
                   
                 SgΔPP/HMA6 
                 0.241 
               
               
                   
                   
                 SgΔPP/HMA7 
                 0.140 
               
               
                   
                 Comparative 
                 Sg/ 
                 N.D. 
               
               
                   
                 Example 3 
                 pBBRpMW 
                   
               
               
                   
                   
                 Sg/HMA1 
                 0.0495 
               
               
                   
                   
                 Sg/HMA2 
                 0.0584 
               
               
                   
                   
                 Sg/HMA3 
                 0.0434 
               
               
                   
                   
                 Sg/HMA4 
                 0.0524 
               
               
                   
                   
                 Sg/HMA5 
                 0.0587 
               
               
                   
                   
                 Sg/HMA6 
                 0.0618 
               
               
                   
                   
                 Sg/HMA7 
                 0.0519 
               
               
                   
                   
               
            
           
         
       
     
     Example 9 
     Generation of  E. coli  mutants with impaired pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B, C, E, and F 
     The plasmid pMW119::EH produced in Reference Example 2 was introduced into each of the  E. coli  mutants produced in Example 5 to generate  E. coli  mutants. Additionally, an  E. coli  mutant was generated as a control by introducing the pMW119 empty vector into the EcΔPPpBBR produced in Example 5. 
     The EcΔPPpBBR. EcΔPP311 − A1, EcΔPPRHA2, EcΔPP3HA3, EcΔPP3HA4. EcΔPP3HA5, EcΔPP311A6, or EcΔPP3HA7 was inoculated into 5 mL of LB medium containing 25 μgmL kanamycin and cultured at 30° C. with shaking for 1 day. Subsequently, 0.5 mL of the culture fluid was inoculated into 5 mL of LB medium containing 25 μgmL kanamycin and was cultured at 30° C. with shaking for 2 hours. The culture fluid was cooled on ice for 20 minutes, and the bacterial cells were then washed with 10% (ww) glycerol three times. The washed pellet was suspended in 100 μL of 10% (ww) glycerol and mixed with 1 uL of the pBBR1MCS-2 (control) or pMW1 19::EH, and the mixture was then cooled in an electroporation cuvette on ice for 10 minutes. Electroporation was performed using a Gene Pulser electroporator (manufactured by Bio-Rad Laboratories, Inc.; 3 kV, 200 Ω, 25 μF.), and 1 mL of SOC medium was added to the electroporation cuvette immediately after the electroporation, and the bacterial cells in the cuvette were incubated at 30° C. with shaking for 1 hour. Fifty uL of the culture was applied to LB agar medium containing 100 μgmL ampicillin and 25 μgmL kanamycin and was incubated at 30° C. for 1 day. The obtained strains were designated as EcΔPPpBBRpMW (negative control), EcΔPPHMA1, EcΔPPHMA2, EcΔPPHMA3, EcΔPPHMA4 EcΔPPHMAS, EcΔPPHMA6, and EcΔPPHMA7. 
     Reference Example 8 
     Generation of  E. coli  mutants with intact pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B, C, E, and F 
     By the same method as in Example 9, the pMW119 (control) or pMW119::EH was introduced into the EcpBBR, Ec3HA1, Ec3HA2, Ec31IA3, Ec3HA4, Ec3HA5, Ec3HA6, and Ec3HA7. The obtained strains were designated as EcpBBRpMW (negative control), EcHMA1, EcHMA2, EcIIMA3, EcHMA4, EcHMAS, EcIIMA6. and EcHMA7. 
     Example 10 
     Production test of α-hydromuconic acid using  E. coli  mutants with impaired pyruvate kinase (Unction 
     The mutants produced in Reference Example 9 were cultured in the same manner as in Example 6, except that ampicillin was added to the culture medium to a concentration of 100 μgmL. The concentrations of α-hydromuconic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of α-hydromuconic acid calculated using the above formula (2) from the measured values is shown in Table 10. 
     Comparative Example 4 
     Production test of α-hydromuconic acid using  E. coli  mutants with intact pyruvate kinase function 
     The mutants produced in Reference Example 8 were cultured in the same manner as in Example 10. The concentrations of α-hydromuconic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of α-hydromuconic acid calculated using the above formula (2) from the measured values is shown in Table 10. 
     By comparing the results of Comparative Example 4 and Example 10, it was found that the yield of α-hydromuconic acid was increased by impairing the function of pyruvate kinase in  E. coli.    
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 10 
               
               
                   
                   
               
               
                   
                   
                   
                 Yield of 
               
               
                   
                   
                 Strain 
                 HMA (%) 
               
               
                   
                   
               
             
            
               
                   
                 Example 10 
                 EcΔPP/ 
                 0.0167 
               
               
                   
                   
                 pBBRpMW 
                   
               
               
                   
                   
                 EcΔPP/HMA1 
                 0.0511 
               
               
                   
                   
                 EcΔPP/HMA2 
                 0.0818 
               
               
                   
                   
                 EcΔPP/HMA3 
                 0.0717 
               
               
                   
                   
                 EcΔPP/HMA4 
                 0.0688 
               
               
                   
                   
                 EcΔPP/HMA5 
                 0.0765 
               
               
                   
                   
                 EcΔPP/HMA6 
                 0.0761 
               
               
                   
                   
                 EcΔPP/HMA7 
                 0.0599 
               
               
                   
                 Comparative 
                 Ec/ 
                 N.D. 
               
               
                   
                 Example 4 
                 pBBRpMW 
                   
               
               
                   
                   
                 Ec/HMA1 
                 0.0362 
               
               
                   
                   
                 Ec/HMA2 
                 0.0636 
               
               
                   
                   
                 Ec/HMA3 
                 0.0569 
               
               
                   
                   
                 Ec/HMA4 
                 0.0624 
               
               
                   
                   
                 Ec/HMA5 
                 0.0621 
               
               
                   
                   
                 Ec/HMA6 
                 0.0640 
               
               
                   
                   
                 Ec/HMA7 
                 0.0491 
               
               
                   
                   
               
            
           
         
       
     
     Example 11 
     Generation of mutant microorganisms of the genus  Serratia  with impaired pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B. C, D, and G 
     By the same method as in Example 2, the pBBR1MCS-2::ATCT2OR1, pBBR1MCS-2::ATCT2OR2, pBBR1MCS-2::ATCT2OR3, pBBR1.MCS-2::ATCT2OR4, pBBR1MCS-2::ATCT2OR5, pBBR1MCS-2::ATCT2OR6, or pBBR1MCS-2::ATCT2OR7 produced in Reference Example 3 was introduced into the SgΔPP. By the same method as in Example 7, the plasmid pMW119::EIIER produced in Reference Example 4 was introduced into each of the obtained mutants to generate mutant microorganisms of the genus  Serratia  . The obtained strains were designated as SgΔPPADA1, SgΔPPADA2, SgΔPPADA3, SgΔPPADA4, SgΔPPADAS, SgΔPPADA6, and SgΔPPADA7. 
     Reference Example 9 
     Generation of mutant microorganisms of the genus  Serratia  with intact pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B, C, D, and G 
     By the same method as in Example 11, the pBBR1MCS-2::ATCT2OR1, pBBR1MCS-2::ATCT2OR2, pBBR1MCS-2::ATCT2OR3, pBBR1MCS-2::ATCT2OR4, pBBR1MCS-2::ATCT2OR5, pBBR1MCS-2::ATCT2OR6, or pBBR1MCS-2::ATCT2OR7 produced in Reference Example 3 was introduced into Serratia grimesii NBRC13537. By the same method as in Example 7, the plasmid pMW119::EHER produced in Reference Example 4 was introduced into each of the obtained mutants to generate mutant microorganisms of the genus  Serratia  . The obtained strains were designated as SgADA1, SgADA2, SgADA3, SgADA4, SgADAS, SgADA6, and SgADA7. 
     Example 12 
     Production test of adipic acid using mutant microorganisms of the genus  Serratia  with impaired pyruvate kinase function 
     The mutants produced in Example 11 and the SgΔPPpBBRpMW (negative control) were cultured in the same manner as in Example 8. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The quantification of adipic acid was performed using LC-MSMS under the same conditions for the quantification of 3-hydroxyadipic acid and α-hydromuconic acid. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 11. 
     Comparative Example 5 
     Production test of adipic acid using mutant microorganisms of the genus Serratia with intact pyruvate kinase function 
     The mutants produced in Reference Example 9 and the SgpBBRpMW were cultured in the same manner as in Example 8. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 11. 
     By comparing the results of Comparative Example 5 and Example 12, it was found that the yield of adipic acid was increased by impairing the function of pyruvate kinase in the microorganism of the genus  Serratia  . 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 11 
               
               
                   
                   
               
               
                   
                   
                   
                 Yield of 
               
               
                   
                   
                 Strain 
                 ADA (%) 
               
               
                   
                   
               
             
            
               
                   
                 Example 12 
                 SgΔPP/ 
                 N.D. 
               
               
                   
                   
                 pBBRpMW 
                   
               
               
                   
                   
                 SgΔPP/ADA1 
                 0.0783 
               
               
                   
                   
                 SgΔPP/ADA2 
                 0.110 
               
               
                   
                   
                 SgΔPP/ADA3 
                 0.0861 
               
               
                   
                   
                 SgΔPP/ADA4 
                 0.116 
               
               
                   
                   
                 SgΔPP/ADA5 
                 0.108 
               
               
                   
                   
                 SgΔPP/ADA6 
                 0.136 
               
               
                   
                   
                 SgΔPP/ADA7 
                 0.0958 
               
               
                   
                 Comparative 
                 Sg/ 
                 N.D. 
               
               
                   
                 Example 5 
                 pBBRpMW 
                   
               
               
                   
                   
                 Sg/ADA1 
                 0.0244 
               
               
                   
                   
                 Sg/ADA2 
                 0.0325 
               
               
                   
                   
                 Sg/ADA3 
                 0.0254 
               
               
                   
                   
                 Sg/ADA4 
                 0.0246 
               
               
                   
                   
                 Sg/ADA5 
                 0.0314 
               
               
                   
                   
                 Sg/ADA6 
                 0.0264 
               
               
                   
                   
                 Sg/ADA7 
                 0.0289 
               
               
                   
                   
               
            
           
         
       
     
     Example 13 
     Generation of E. coli mutants with impaired pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B, C, D, and G 
     By the same method as in Example 5, the PBBR1MCS-2::ATCT2OR1, pBBR1MCS-2::ATCT2OR2, pBBR1MCS-2::ATCT2OR3, pBBR1MCS-2::ATCT2OR4, pBBR1MCS-2::ATCT2OR5, pBBR1MCS-2::ATCT2OR6, or pBBR1MCS-2::ATCT2OR7 produced in Reference Example 3 was introduced into the EeAPP. By the same method as in Example 9, the plasmid pMW119::EHER produced in Reference Example 4 was introduced into each of the obtained mutants to generate  E. coli  mutants. The obtained strains were designated as EcΔPPADA1, EcΔPPADA2, EcΔPPADA3, EcΔPPADA4, EcΔPPADAS, EcΔPPADA6, and EcΔPPADA7. 
     Reference Example 10 
     Generation of E. coli mutants with intact pyruvate kinase function and carrying plasmids expressing enzymes that catalyze the reactions A, B, C, D, and G 
     By the same method as in Example 13, the pBBR1MCS-2::ATCT2OR1, pBBR1MCS-2::ATCT2OR2, pBBR1MCS-2::ATCT2OR3, pBBR1MCS-2::ATCT2OR4, pBBR1MCS-2::ATCT2OR5, pBBR1MCS-2::ATCT2OR6, or pBBR1MCS-2::ATCT2OR7 produced in Reference Example 3 was introduced into  Escherichia coli  MG1655. By the same method as in Example 9, the plasmid pMW119::EHER produced in Reference Example 4 was introduced into each of the obtained mutants to generate  E. coli  mutants. The obtained strains were designated as EcADA1, EcADA2, EcADA3, EcADA4, EcADAS, EcADA6, and EcADA7. 
     Example 14 
     Production test of adipic acid using  E. coli  mutants with impaired pyruvate kinase function 
     The mutants produced in Example 13 and the EcΔPPpBBRpMW were cultured in the same manner as in Example 10. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The quantification of adipic acid was performed using LC-MSMS under the same conditions for the quantification of 3-hydroxyadipic acid and α-hydromuconic acid. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 12. 
     Comparative Example 6 
     Production test of adipic acid using  E. coli  mutants with intact pyruvate kinase function 
     The mutants produced in Reference Example 10 and the EcpBBRpMW were cultured in the same manner as in Example 10. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 12. 
     By comparing the results of Comparative Example 6 and Example 14, it was found that the yield of adipic acid was increased by impairing the function of pyruvate kinase in  E. coli.    
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                   
                   
                 Yield of 
               
               
                   
                   
                 Strain 
                 ADA (%) 
               
               
                   
                   
               
             
            
               
                   
                 Example 14 
                 EcΔPP/ 
                 N.D. 
               
               
                   
                   
                 pBBRpMW 
                   
               
               
                   
                   
                 EcΔPP/ADA1 
                 0.0213 
               
               
                   
                   
                 EcΔPP/ADA2 
                 0.0338 
               
               
                   
                   
                 EcΔPP/ADA3 
                 0.0293 
               
               
                   
                   
                 EcΔPP/ADA4 
                 0.0315 
               
               
                   
                   
                 EcΔPP/ADA5 
                 0.0382 
               
               
                   
                   
                 EcΔPP/ADA6 
                 0.0407 
               
               
                   
                   
                 EcΔPP/ADA7 
                 0.0235 
               
               
                   
                 Comparative 
                 Ec/ 
                 N.D. 
               
               
                   
                 Example 6 
                 pBBRpMW 
                   
               
               
                   
                   
                 Ec/ADA1 
                 0.0148 
               
               
                   
                   
                 Ec/ADA2 
                 0.0153 
               
               
                   
                   
                 Ec/ADA3 
                 0.0107 
               
               
                   
                   
                 Ec/ADA4 
                 0.0192 
               
               
                   
                   
                 Ec/ADA5 
                 0.0139 
               
               
                   
                   
                 Ec/ADA6 
                 0.0147 
               
               
                   
                   
                 Ec/ADA7 
                 0.0167 
               
               
                   
                   
               
            
           
         
       
     
     Example 15 
     Production test 2 of 3-hydroxyadipic acid and α-hydromuconic acid using mutant microorganisms of the genus  Serratia  with impaired pyruvate kinase function 
     The production test of 3-hydroxyadipic acid and a.-hydromuconic acid was conducted using the mutant microorganisms of the genus  Serratia  produced in Example 2 under anaerobic conditions. 
     The mutant microorganisms of the genus  Serratia  produced in Example 2 were cultured in the same manner as in Example 3, except that the mutant microorganisms were cultured statically using the culture medium II. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the above formula (2) from the measured values are shown in Table 13. 
     Comparative Example 7 
     Production test 2 of 3-hydroxyadipic acid and α-hydromuconic acid using mutant microorganisms of the genus  Serratia  with intact pyruvate kinase function 
     The mutants produced in Reference Example 5 were cultured in the same manner as in Example 15. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. 
     The yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the above formula (2) from the measured values are shown in Table 13. 
     By comparing the results of Comparative Example 7 and Example 15, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were increased even under anaerobic conditions by impairing the function of pyruvate kinase in the microorganism of the genus  Serratia  . 
     
       
         
           
               
               
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                   
                   
                 Yield of 
                 Yield of 
               
               
                   
                 Strain 
                 3HA (%) 
                 HMA (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Example 15 
                 SgΔPP/ 
                 0.0485 
                 0.0224 
               
               
                   
                 pBBR 
                   
                   
               
               
                   
                 SgΔPP/3HA1 
                 4.84 
                 0.159 
               
               
                   
                 SgΔPP/3HA2 
                 6.07 
                 0.171 
               
               
                   
                 SgΔPP/3HA3 
                 5.99 
                 0.143 
               
               
                   
                 SgΔPP/3HA4 
                 5.30 
                 0.195 
               
               
                   
                 SgΔPP/3HA5 
                 5.84 
                 0.180 
               
               
                   
                 SgΔPP/3HA6 
                 6.02 
                 0.202 
               
               
                   
                 SgΔPP/3HA7 
                 5.98 
                 0.160 
               
               
                 Comparative 
                 Sg/ 
                 N.D. 
                 N.D. 
               
               
                 Example 7 
                 pBBR 
                   
                   
               
               
                   
                 Sg/3HA1 
                 1.68 
                 0.0482 
               
               
                   
                 Sg/3HA2 
                 2.46 
                 0.0577 
               
               
                   
                 Sg/3HA3 
                 1.94 
                 0.0471 
               
               
                   
                 Sg/3HA4 
                 1.99 
                 0.0527 
               
               
                   
                 Sg/3HA5 
                 2.29 
                 0.0523 
               
               
                   
                 Sg/3HA6 
                 2.95 
                 0.0627 
               
               
                   
                 Sg/3HA7 
                 1.66 
                 0.0595 
               
               
                   
               
            
           
         
       
     
     Example 16 
     Production test 2 of 3-hydroxyadipic acid and u-hydromuconic acid using  E. coli  mutants with impaired pyruvate kinase function 
     The production test of 3-hydroxyadipic acid and u-hydromuconic acid was conducted under anaerobic conditions using the K coli mutants produced in Example 5. 
     The  E. coli  mutants produced in Example 5 were cultured in the same manner as in Example 6, except that the mutants were cultured statically using, the culture medium II. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the above formula (2) from the measured values are shown in Table 14. 
     Comparative Example 8 
     Production test 2 of 3-hydroxyadipic acid and α-hydromuconic acid using  E. coli  mutants with intact pyruvate kinase function 
     The mutants produced in Reference Example 6 were cultured in the same manner as in Example 16. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the above formula (2) from the measured values are shown in Table 14. 
     By comparing the results of Comparative Example 8 and Example 16, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were increased even under anaerobic conditions by impairing the function of pyruvate kinase in  E. coli.    
     
       
         
           
               
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                   
                   
                 Yield of 
                 Yield of 
               
               
                   
                 Strain 
                 3HA (%) 
                 HMA (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Example 16 
                 EcΔPP/ 
                 0.0669 
                 0.0113 
               
               
                   
                 pBBR 
                   
                   
               
               
                   
                 EcΔPP/3HA1 
                 13.2 
                 0.0213 
               
               
                   
                 EcΔPP/3HA2 
                 14.9 
                 0.0277 
               
               
                   
                 EcΔPP/3HA3 
                 13.9 
                 0.0268 
               
               
                   
                 EcΔPP/3HA4 
                 14.1 
                 0.0224 
               
               
                   
                 EcΔPP/3HA5 
                 14.3 
                 0.0259 
               
               
                   
                 EcΔPP/3HA6 
                 14.7 
                 0.0226 
               
               
                   
                 EcΔPP/3HA7 
                 13.2 
                 0.0213 
               
               
                 Comparative 
                 Ec/ 
                 N.D. 
                 N.D. 
               
               
                 Example 8 
                 pBBR 
                   
                   
               
               
                   
                 Ec/3HA1 
                 1.32 
                 0.0171 
               
               
                   
                 Ec/3HA2 
                 2.00 
                 0.0154 
               
               
                   
                 Ec/3HA3 
                 1.82 
                 0.0130 
               
               
                   
                 Ec/3HA4 
                 1.47 
                 0.0136 
               
               
                   
                 Ec/3HA5 
                 2.17 
                 0.0138 
               
               
                   
                 Ec/3HA6 
                 1.77 
                 0.0166 
               
               
                   
                 Ec/3HA7 
                 1.14 
                 0.0179 
               
               
                   
               
            
           
         
       
     
     Example 17 
     Production test 2 of adipic acid using mutant microorganisms of the genus  Serratia  with impaired pyruvate kinase function 
     The production test of adipic acid was conducted using the mutant microorganisms of the genus  Serratia  produced in Example 11 under anaerobic conditions. 
     The mutant microorganisms of the genus  Serratia  produced in Example 11 were cultured in the same manner as in Example 12. except that the mutant microorganisms were cultured statically using the culture medium II. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 15. 
     Comparative Example 9 
     Production test 2 of adipic acid using mutant microorganisms of the genus  Serratia  with intact pyruvate kinase function 
     The mutants produced in Reference Example 9 were cultured in the same manner as in Example 17. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 15. 
     By comparing the results of Comparative Example 9 and Example 17, it was found that the yield of adipic acid was increased even under anaerobic conditions by impairing the function of pyruvate kinase in the microorganism of the genus  Serratia  . 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 15 
               
               
                   
                   
               
               
                   
                   
                   
                 Yield of 
               
               
                   
                   
                 Strain 
                 ADA (%) 
               
               
                   
                   
               
             
            
               
                   
                 Example 17 
                 SgΔPP/ 
                 N.D. 
               
               
                   
                   
                 pBBRpMW 
                   
               
               
                   
                   
                 SgΔPP/ADA1 
                 0.0359 
               
               
                   
                   
                 SgΔPP/ADA2 
                 0.0480 
               
               
                   
                   
                 SgΔPP/ADA3 
                 0.0379 
               
               
                   
                   
                 SgΔPP/ADA4 
                 0.0395 
               
               
                   
                   
                 SgΔPP/ADA5 
                 0.0431 
               
               
                   
                   
                 SgΔPP/ADA6 
                 0.0490 
               
               
                   
                   
                 SgΔPP/ADA7 
                 0.0375 
               
               
                   
                 Comparative 
                 Sg/ 
                 N.D. 
               
               
                   
                 Example 9 
                 pBBRpMW 
                   
               
               
                   
                   
                 Sg/ADA1 
                 0.0152 
               
               
                   
                   
                 Sg/ADA2 
                 0.0181 
               
               
                   
                   
                 Sg/ADA3 
                 0.0188 
               
               
                   
                   
                 Sg/ADA4 
                 0.0179 
               
               
                   
                   
                 Sg/ADA5 
                 0.0135 
               
               
                   
                   
                 Sg/ADA6 
                 0.0130 
               
               
                   
                   
                 Sg/ADA7 
                 0.0093 
               
               
                   
                   
               
            
           
         
       
     
     Example 18 
     Production test 2 of adipic acid using E. coli mutants with impaired pyruvate kinase function 
     The production test of adipic acid was conducted using the  E. coli  mutants produced in Example 13 under anaerobic conditions. 
     The  E. coli  mutants produced in Example 13 were cultured in the same manner as in Example 14, except that the mutants were cultured statically using the culture medium II. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 16. 
     Comparative Example 10 
     Production test 2 of adipic acid using  E. coli  mutants with intact pyruvate kinase function 
     The mutants produced in Reference Example 10 were cultured in the same manner as in Example 18. The concentrations of adipic acid and other products accumulated in the culture supernatant and the concentration of sugars remaining unused in the culture medium were quantified. The yield of adipic acid calculated using the above formula (2) from the measured values is shown in Table 16. 
     By comparing the results of Comparative Example 10 and Example 18, it was found that the yield of adipic acid was increased even under anaerobic conditions by impairing the function of pyruvate kinase in  E. coli.    
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 16 
               
               
                   
                   
               
               
                   
                   
                   
                 Yield of 
               
               
                   
                   
                 Strain 
                 ADA (%) 
               
               
                   
                   
               
             
            
               
                   
                 Example 18 
                 EcΔPP/ 
                 N.D. 
               
               
                   
                   
                 pBBRpMW 
                   
               
               
                   
                   
                 EcΔPP/ADA1 
                 0.0255 
               
               
                   
                   
                 EcΔPP/ADA2 
                 0.0254 
               
               
                   
                   
                 EcΔPP/ADA3 
                 0.0269 
               
               
                   
                   
                 EcΔPP/ADA4 
                 0.0248 
               
               
                   
                   
                 EcΔPP/ADA5 
                 0.0212 
               
               
                   
                   
                 EcΔPP/ADA6 
                 0.0278 
               
               
                   
                   
                 EcΔPP/ADA7 
                 0.0212 
               
               
                   
                 Comparative 
                 Ec/ 
                 N.D. 
               
               
                   
                 Example 10 
                 pBBRpMW 
                   
               
               
                   
                   
                 Ec/ADA1 
                 0.0166 
               
               
                   
                   
                 Ec/ADA2 
                 0.0180 
               
               
                   
                   
                 Ec/ADA3 
                 0.0140 
               
               
                   
                   
                 Ec/ADA4 
                 0.0161 
               
               
                   
                   
                 Ec/ADA5 
                 0.0148 
               
               
                   
                   
                 Ec/ADA6 
                 0.0219 
               
               
                   
                   
                 Ec/ADA7 
                 0.0123 
               
               
                   
                   
               
            
           
         
       
     
     Example 19 
     Generation of a mutant microorganism of the genus  Serratia  with defects in genes encoding pyruvate kinase and a phosphotransferase system enzyme 
     A mutant microorganism of the genus  Serratia  with impaired function of both pyruvate kinase and a phosphotransferase system enzyme was generated by disrupting a gene encoding a phosphotransferase, ptsG, in the SgΔPP strain produced in Example 1. 
     A PCR reaction was performed using pKD4 as a template and oligo DNAs represented by SEQ ID NOs: 239 and 240 as primers to obtain a PCR fragment of 1.6 kb in length for disruption of ptsG. The introduction of pKD46 into the above strain was followed by the introduction of the PCR fragment for disruption of ptsG into the resulting strain. Direct colony PCR was performed on the resulting kanamycin-resistant strains to confirm the deletion of the gene of interest and the insertion of a kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 223 and 242 were used. 
     Subsequently, an ampicillin-sensitive strain was obtained by segregating away the pKD46. The plasmid pCP20 was introduced into the ampicillin-sensitive strain, and ampicillin-resistant strains were again obtained. Direct colony PCR was performed on the obtained strains to confirm the deletion of the kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 241 and 242 were used. The pCP20 was segregated away from one of the kanamycin-sensitive strains. The obtained strain is hereinafter referred to as SgΔPPG. 
     Example 20 
     Generation of a mutant microorganism of the genus  Serratia  with defects in genes encoding pyruvate kinase and a phosphotransferase system enzyme and carrying a plasmid expressing enzymes that catalyze the reactions A, B, E, and F By the same method as in Example 2, a plasmid produced in Reference Example 1, pBBR1MCS-2::ATCTOR1, was introduced into the SgΔPPG strain produced in Example 19, and the obtained mutant microorganism of the genus  Serratia  was designated as SgΔPPG3HA1. 
     Example 21 
     Production test of 3-hydroxyadipic acid and α-hydromuconic acid using a mutant microorganism of the genus  Serratia  with impaired function of both pyruvate kinase and a phosphotransferase system enzyme and carrying a plasmid expressing enzymes that catalyze the reactions A, B, E, and F 
     By the same method as in Example 15, the production test of 3-hydroxyadipic acid and α-hydromuconic acid was conducted using the mutant microorganism of the genus  Serratia  produced in Example 20. 
     Comparative Example 11 
     Production test of 3-hydroxyadipic acid and α-hydromuconic acid using a mutant microorganism of the genus  Serratia  with intact pyruvate kinase function and intact phosphotransferase system enzyme function and carrying a plasmid expressing enzymes that catalyze the reactions A, B. E, and F 
     By the same method as in Comparative Example 7, the production test of 3-hydroxyadipic acid and α-hydromuconic acid was conducted using the Sg314A1 strain produced in Reference Example 5. 
     By comparing the results of Example 21 and Example 15, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were further increased in the mutant microorganism of the genus  Serratia  with defects in the genes encoding pyruvate kinase and the phosphotransferase system enzyme and with enhanced activity of an enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. Additionally, by comparing the results of Example 21 and Comparative Example 11, it was found that the yields of acetic acid and ethanol, both of which were generated by conversion of acetyl-CoA, were also increased in the mutant with defects in the genes encoding pyruvate kinase and the phosphotransferase system enzyme. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
                 TABLE 17 
               
               
                   
                   
               
               
                   
                   
                   
                   
                 Yield of 
                 Yield of 
                   
               
               
                   
                   
                 Yield of 
                 Yield of 
                 succinic 
                 acetic 
                 Yield of 
               
               
                   
                 Strain 
                 3HA (%) 
                 HMA (%) 
                 acid (%) 
                 acid (%) 
                 ethanol (%) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Example 21 
                 SgΔPPG/3HA1 
                 6.06 
                 0.180 
                 60.6 
                 36.8 
                 52.2 
               
               
                 Comparative 
                 Sg/3HA1 
                 1.68 
                 0.0482 
                 7.26 
                 35.1 
                 38.8 
               
               
                 Example 11 
               
               
                   
               
            
           
         
       
     
     Example 22 
     Generation of an  E. coli  mutant with defects in genes encoding pyruvate kinase and a phosphotransferase system enzyme 
     An  E. coli  mutant with impaired function of both pyruvate kinase and a phosphotransferase system enzyme was generated by disrupting a gene encoding a phosphotransferase,ptsG, in the EcΔPP produced in Example 4. 
     A PCR reaction was performed using pKD4 as a template and oligo DNAs represented by SEQ ID NOs: 243 and 244 as primers to obtain a PCR fragment of 1.6 kb in length for disruption of ptsG. The introduction of pKD46 into the above strain was followed by the introduction of the PCR fragment for disruption of ptsG into the resulting strain. Direct colony PCR was performed on the resulting kanamycin-resistant strains to confirm the deletion of the gene of interest and the insertion of a kanamycin resistance gene from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 223 and 246 were used. 
     Subsequently, an ampicillin-sensitive strain was obtained by segregating away the pKD46. The plasmid pCP20 was introduced into the ampicillin-sensitive strain, and ampicillin-resistant strains were again obtained. Direct colony PCR was performed on the obtained strains to confirm the deletion of the kanamycin resistance gene :from the length of the amplified band. Oligo DNA primers represented by SEQ ID NOs: 245 and 246 were used. The pCP20 was segregated away from one of the kanamycin-sensitive strains. The obtained strain is hereinafter referred to as EcΔPPG. 
     Example 23 
     Generation of an  E. coli  mutant with defects in genes encoding pyruvate kinase and a phosphotransferase system enzyme and carrying a plasmid expressing enzymes that catalyze the reactions A, B, E, and F 
     By the same method as in Example 5, the pBBIUMCS-2::ATCTOR1 produced in Reference Example 1 was introduced into the EcΔPPG strain produced in Example 22, and the obtained  E. coli  mutant was designated as EcΔPPG3HA1. 
     Example 24 
     Production test of 3-hydroxyadipic acid and u-hydromuconic acid using an  E. coli  mutant with impaired function of both pyruvate kinase and a phosphotransferase system enzyme and carrying a plasmid expressing enzymes that catalyze the reactions A, B, E, and F 
     By the same method as in Example 16, the production test of 3-hydroxyadipic acid and α-hydromuconic acid was conducted using the  E. coli  mutant produced in Example 23. 
     Comparative Example 12 
     Production test of 3-hydroxyadipic acid and α-hydromuconic acid using an  E. coli  mutant with intact pyruvate kinase function and intact phosphotransferase system enzyme function and carrying a plasmid expressing enzymes that catalyze the reactions A, B, E, and F 
     By the same method as in Comparative Example 8, the production test of 3-hydroxyadipic acid and α-hydromuconic acid was conducted using the Ec/3HA1 produced in Reference Example 6. 
     By comparing the results of Example 24 and Example 16, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were further increased in the E. coli mutant with defects in the genes encoding pyruvate kinase and the phosphotransferase system enzyme and with enhanced activity of an enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. Additionally, by comparing the results of Example 24 and Comparative Example 12, it was found that the yields of acetic acid and ethanol, both of which were generated by conversion of acetyl-CoA, were also increased in the mutant with defects in the genes encoding pyruvate kinase and the phosphotransferase system enzyme. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
                 TABLE 18 
               
               
                   
                   
               
               
                   
                   
                   
                   
                 Yield of 
                 Yield of 
                   
               
               
                   
                   
                 Yield of 
                 Yield of 
                 succinic 
                 acetic 
                 Yield of 
               
               
                   
                 Strain 
                 3HA (%) 
                 HMA (%) 
                 acid (%) 
                 acid (%) 
                 ethanol (%) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Example 24 
                 EcΔPPG/3HA1 
                 15.4 
                 0.0439 
                 60.2 
                 51.3 
                 58.0 
               
               
                 Comparative 
                 Ec/3HA1 
                 1.32 
                 0.0171 
                 12.6 
                 36.7 
                 40.7 
               
               
                 Example 12