Recombinant microorganism having enhanced 1,3-propanediol producing ability and method for producing 1,3-propanediol using the same
09932609 ยท 2018-04-03
Assignee
Inventors
Cpc classification
C12Y101/01304
CHEMISTRY; METALLURGY
C12N9/1022
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to a recombinant microorganism for producing 1,3-propanediol, wherein a pathway converting pyruvate into 2,3-butanediol is inhibited in a microorganism having a pyruvate and acetyl CoA biosynthetic pathway. In addition, the present invention relates to a method for producing 1,3-propanediol by using the recombinant microorganism.
Claims
1. A recombinant Klebsiella for producing 1,3-propanediol, wherein a pathway for converting pyruvate into -acetolactate catalyzed by -acetolactate synthase, a pathway for converting -acetolactate into acetoin catalyzed by -acetolactate decarboxylase, a pathway for converting acetoin into 2,3-butanediol catalyzed by acetoin reductase, a pathway for converting pyruvate into lactate catalyzed by lactate dehydrogenase, and a pathway for converting pyruvate into acetyl-CoA catalyzed by pyruvate-formate lyase are suppressed in recombinant Klebsiella, and the recombinant Klebsiella has no ability to produce succinic acid.
2. The recombinant Klebsiella for producing 1,3-propanediol according to claim 1, wherein a gene encoding lactate dehydrogenase, a gene encoding pyruvate-formate lyase, a gene encoding transcription activation factor, a gene encoding -acetolactate decarboxylase, a gene encoding -acetolactate synthase, and a gene encoding acetoin reductase are suppressed in the recombinant Klebsiella.
3. The recombinant Klebsiella for producing 1,3-propanediol according to claim 1, wherein a gene having the nucleotide sequence of SEQ ID NO: 1, a gene having the nucleotide sequence of SEQ ID NO: 9 and a gene having the nucleotide sequence of SEQ ID NO: 33 are suppressed in the recombinant Klebsiella.
4. The recombinant Klebsiella for producing 1,3-propanediol according to claim 1, wherein a pathway for converting pyruvate into formic acid catalyzed by pyruvate-formate lyase is further suppressed.
5. A method for producing 1,3-propanediol, comprising: growing the recombinant Klebsiella according to claim 1 in a culture medium; and harvesting 1,3-propanediol from the culture medium.
6. A recombinant Klebsiella for producing 1,3-propanediol, wherein a gene having the nucleotide sequence of SEQ ID NO: 1, a gene having the nucleotide sequence of SEQ ID NO: 9 and a gene having the nucleotide sequence of SEQ ID NO: 33 are suppressed in the recombinant Klebsiella.
7. The recombinant Klebsiella for producing 1,3-propanediol according to claim 6, wherein the recombinant Klebsiella has no ability to produce succinic acid.
8. A recombinant Klebsiella for producing 1,3-propanediol, wherein a gene encoding lactate dehydrogenase, a gene encoding pyruvate-formate lyase, a gene encoding transcription activation factor, a gene encoding -acetolactate decarboxylase, a gene encoding -acetolactate synthase, and a gene encoding acetoin reductase are suppressed in the recombinant Klebsiella.
9. The recombinant Klebsiella for producing 1,3-propanediol according to claim 8, wherein the recombinant Klebsiella has no ability to produce succinic acid.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
(7) The present invention relates to
(8) a recombinant microorganism for producing 1,3-propanediol,
(9) wherein a pathway for converting pyruvate into 2,3-butanediol is suppressed in a microorganism having pyruvate and acetyl-CoA biosynthetic pathways.
(10) In addition, the present invention relates to a method for producing 1,3-propanediol, including:
(11) culturing the recombinant microorganism according to the present invention; and
(12) harvesting 1,3-propanediol from the culture solution.
(13) Hereinafter, the present invention will be described in detail.
(14) Microorganism Having Pyruvate and Acetyl-CoA Biosynthetic Pathways
(15) The microorganism according to the present invention has pyruvate and acetyl-CoA biosynthetic pathways. Herein, the acetyl-CoA biosynthetic pathway refers to a pathway for synthesizing acetyl-CoA from a specific metabolite in a microorganism. The acetyl-CoA biosynthetic pathway may also refer to a pathway for synthesizing acetyl-CoA from pyruvate. The pyruvate biosynthetic pathway refers to a pathway for synthesizing pyruvate from a specific metabolite in a microorganism. The pyruvate biosynthetic pathway may also refer to a pathway for synthesizing pyruvate from phosphoenol pyruvic acid (PEP). Preferably, the microorganism according to the present invention has pyruvate and acetyl-CoA biosynthetic pathways from a carbon source such as glycerol.
(16) The microorganism having pyruvate and acetyl-CoA biosynthetic pathways according to the present invention is not particularly limited as long as the microorganism has the aforementioned biosynthetic pathways. In addition, the microorganism according to the present invention may be a microorganism having wild type pyruvate and acetyl-CoA biosynthetic pathways or a recombinant microorganism having pyruvate and acetyl-CoA biosynthetic pathways by genetic recombination. Preferably, the microorganism has an ability to produce 1,3-propanediol. The microorganism may be selected from the group consisting of genus Klebsiella, genus Enterobacter, and genus Lactobacillus. The microorganism is preferably a microorganism belonging to genus Klebsiella, more preferably Klebsiella pneumoniae.
(17) Recombinant Microorganism for Producing 1,3-Propanediol
(18) The recombinant microorganism for producing 1,3-propanediol according to the present invention has high 1,3-propanediol productivity and yield, and is characterized by a higher concentration of 1,3-propanediol in a fermented solution than a wild type microorganism upon fermentation. In addition, in the recombinant microorganism according to the present invention, production of oxidative byproducts such as lactate, formic acid, 2,3-butanediol and succinic acid is suppressed. Particularly, in the recombinant microorganism according to the present invention, production of 2,3-butanediol is suppressed wherein 2,3-butanediol has a boiling point similar to 1,3-propanediol as a target product, which renders purification difficult and thus lowers final purification yield. Preferably, the recombinant microorganism according to the present invention has no ability to produce formic acid, 2,3-butanediol, and succinic acid. The expression having no ability to produce formic acid, 2,3-butanediol, and succinic acid means that the microorganism does not produce substantial amounts of formic acid, 2,3-butanediol, and succinic acid, and means that there is no need for a separate process for removing formic acid, 2,3-butanediol, and succinic acid.
(19) Preferably, the recombinant microorganism for producing 1,3-propanediol according to the present invention is a recombinant microorganism wherein a pathway for converting pyruvate into 2,3-butanediol is suppressed in a microorganism having pyruvate and acetyl-CoA biosynthetic pathways. More preferably, a pathway for converting pyruvate into 2,3-butanediol and a pathway for converting pyruvate into lactate are suppressed, or a pathway for converting pyruvate into 2,3-butanediol and a pathway for converting pyruvate into formic acid are suppressed. Still more preferably, a pathway for converting pyruvate into 2,3-butanediol, a pathway for converting pyruvate into lactate, and a pathway for converting pyruvate into formic acid are suppressed.
(20) Preferably, the recombinant microorganism for producing 1,3-propanediol according to the present invention produces 1,3-propanediol with a yield of 0.40 g/g or more and a productivity of 1.5 g/L/hr or more on the basis of batch fermentation. Preferably, the recombinant microorganism has a ratio of 1,3-propanediol of 80 wt % or more in fermentation products when calculated in accordance with the following Equation 1. More preferably, the recombinant microorganism has a ratio of 1,3-propanediol of 85 wt % or more, still more preferably 88 wt % or more. Preferably, the recombinant microorganism has a ratio of lactate of less than 5 wt % in fermentation products, more preferably less than 2 wt %. Preferably, the recombinant microorganism has a ratio of formic acid of less than 1 wt % in fermentation products, more preferably less than 0.2 wt %, still more preferably less than 0.1 wt %. Preferably, the recombinant microorganism has a ratio of 2,3-butanediol of less than 1 wt % in fermentation products, more preferably less than 0.2 wt %, still more preferably less than 0.1 wt %. Preferably, the recombinant microorganism has a ratio of succinic acid of less than 1 wt % in fermentation products, more preferably less than 0.2 wt %, still more preferably less than 0.1 wt %.
Ratio of specific product in fermentation products={Concentration of specific product in fermentation products/(Total sum of concentrations of 1,3-propanediol, lactate, formic acid, 2,3-butanediol, ethanol, acetic acid, and succinic acid in fermentation products)}100<Equation 1>
(21) Suppression of Pathway for Converting Pyruvate into 2,3-Butanediol
(22) Microorganisms capable of producing 2,3-butanediol from pyruvate include a series of conversion enzymes such as -acetolactate synthase, -acetolactate decarboxylase, and acetoin reductase, as shown in
(23) <Pathway 1>
(24) Pyruvate.fwdarw.-acetolactate.fwdarw.acetoin.fwdarw.2,3-butanediol
(25) Transcription of genes encoding enzymes involved in 2,3-butanediol synthesis is regulated by transcription activation factors, and genes encoding 2,3-butanediol synthase and transcription activation factors is present in a gene family, which is called the 2,3-butanediol operon, as shown in
(26) Preferably, in the recombinant microorganism according to the present invention, the pathway for converting pyruvate into 2,3-butanediol is suppressed by suppressing one or more enzymes among -acetolactate decarboxylase, -acetolactate synthase, and acetoin reductase. More preferably, -acetolactate decarboxylase, -acetolactate synthase, and acetoin reductase are suppressed. Still more preferably, expression of budR, budA, budB and budC which are genes encoding the aforementioned enzymes and regulating expression thereof is suppressed.
(27) Suppression of Pathway for Converting Pyruvate into Acetyl-CoA and Formic Acid
(28) Pyruvate-formate lyase normally catalyzes conversion of pyruvate into acetyl-CoA and formic acid under anaerobic conditions (pathway 2).
(29) <Pathway 2>
(30) Pyruvate.fwdarw.acetyl-CoA+formic acid
(31) A pathway for converting pyruvate into acetyl-CoA may be suppressed by suppressing pyruvate-formate lyase. Suppression of pyruvate-formate lyase may be performed by expression suppression of pyruvate-formate lyase, suppression of enzyme activity of pyruvate-formate lyase, and the like. For example, those skilled in the art could easily suppress pyruvate-formate lyase by selecting suitable methods, such as deleting pflB which is a gene encoding pyruvate-formate lyase, causing mutations in the gene (mutations such as suppression of normal gene expression through modifying, substituting or deleting a partial nucleotide sequence or introducing a partial nucleotide sequence), regulating gene expression during transcription or translation, and the like.
(32) Suppression of Pathway for Converting Pyruvate into Lactate
(33) Lactate dehydrogenase catalyzes conversion of pyruvate into lactate. The pathway for converting pyruvate into lactate may be suppressed by suppressing the lactate dehydrogenase. Suppression of lactate dehydrogenase may be performed by expression suppression of lactate dehydrogenase, suppression of enzyme activity of lactate dehydrogenase, and the like. For example, those skilled in the art could easily suppress lactate dehydrogenase by selecting suitable methods, such as deleting ldhA which is a gene encoding lactate dehydrogenase, causing mutations in the gene (mutations such as suppression of normal gene expression through modifying, substituting or deleting a partial nucleotide sequence or introducing a partial nucleotide sequence), regulating gene expression during transcription or translation, and the like. The recombinant microorganism in which the aforementioned pathways are suppressed exhibits lactate ratio of less than 12 wt %, more preferably less than 8 wt %, still more preferably less than 5 wt % in the fermentation products.
(34) Method for Producing 1,3-Propanediol
(35) The present invention relates to a method for producing 1,3-propanediol, including: culturing the recombinant microorganism according to the present invention; and harvesting 1,3-propanediol from the culture solution.
(36) The recombinant microorganism according to the present invention may be cultured under aerobic conditions, preferably under microaerobic conditions. For example, the cultivation may be performed by supplying oxygen, namely, air, during cultivation. Specifically, the cultivation is performed by stirring, without being limited thereto.
Mode for Invention
(37) The advantages and features of the present invention and methods for accomplishing the same will become apparent from the following examples. It should be understood that the present invention is not limited to the following examples and may be embodied in different ways, and the following examples are given to provide complete disclosure of the present invention and to provide a thorough understanding of the present invention to those skilled in the art. The present invention should be defined only by the accompanying claims and equivalents thereof.
(38) <Materials and Methods> Concentration of 1,3-propanediol (g/L): Amounts of 1,3-propanediol produced per unit volume Yield of 1,3-propanediol (g/g): Produced amount of 1,3-propanediol (g)/carbon source (g) Productivity of 1,3-propanediol (g/L/h): Amounts of 1,3-propanediol produced per unit time and unit volume
(39) Strain of Klebsiella pneumoniae GSC123 ldhA (Kp ldhA)
(40) A strain of Klebsiella pneumoniae GSC123 ldhA (Kp ldhA) in which a lactate dehydrogenase gene (ldhA) was deleted was constructed as follows. Firstly, in order to clone a lactate dehydrogenase gene of Klebsiella pneumoniae, a homologous region 1 (SEQ ID NO: 2) of a target gene ldhA (SEQ ID NO: 1) was amplified using primers of SEQ ID NOs: 3 and 4 by polymerase chain reaction (PCR). Further, a homologous region 2 (SEQ ID NO: 5) was amplified using primers of SEQ ID NOs: 6 and 7 by PCR. Next, the homologous regions 1 and 2 were amplified using the same as templates for PCR, thereby obtaining a completed DNA fragment (SEQ ID NO: 8) in which the homologous regions 1 and 2 were ligated (Table 1).
(41) The completed DNA fragment may include antibiotic resistance genes and the like in order to enhance the probability of recombination of target genes. Further, the completed DNA fragment may include a sacB gene encoding levansucrase in order to remove antibiotic resistance genes recombined in the chromosomes.
(42) The prepared DNA fragment was transferred to wild type Klebsiella pneumoniae through electroporation (25 F, 200 , 18 kV/cm), in which the target gene was deleted by a homologous recombination mechanism indigenous to the microorganism.
(43) TABLE-US-00001 TABLE1 SEQ ID NO Sequence 1 ATGAAAATCGCGGTTTATAGTACGAAGCAGTACGATAAAAAGTAC CTGCAGCACGTTAATGATGCATACGGCTTTGAACTGGAATTCTTC GATTTCCTGCTGACAGCGAAGACTGCCAAAACCGCCAACGGTTGC GAAGCGGTATGTATCTTCGTCAATGACGACGGCAGCCGCCCGGTG CTGGAAGAGCTGAAGGCCCACGGGGTGAAATATATCGCCCTGCGC TGCGCCGGGTTTAACAACGTCGACCTTGAGGCGGCAAAGGAGCTT GGCCTGCGCGTCGTGCGCGTTCCAGCTTACTCTCCGGAAGCGGTC GCTGAGCATGCGATCGGTATGATGATGTCGCTCAACCGCCGCATC CACCGCGCTTACCAGCGTACCCGCGATGCCAATTTCTCCCTCGAA GGCCTCACCGGCTTCACCATGTACGGCAAAACCGCCGGGGTGATC GGCACCGGGAAAATTGGCGTAGCGATGTTGCGGATCCTCAAAGGC TTCGGCATGCGCCTGCTGGCGTTCGACCCGTACCCAAGCGCCGCC GCGCTGGAGCTGGGGGTGGAATATGTTGACCTCGCCACGCTGTAC AAGGAATCGGACGTGATCTCCCTGCACTGTCCGCTGACCGACGAA AACTACCACCTGCTCAATCGCGAAGCTTTCGATCAGATGAAAGAC GGGGTGATGGTGATCAACACCAGCCGCGGCGCCCTGATCGACTCT CAGGCGGCCATCGACGCCCTGAAGCACCAGAAAATTGGCGCGCTG GGGCTGGACGTTTATGAGAACGAACGCGATCTGTTCTTTGAAGAC AAATCCAACGACGTGATCCAGGACGATGTCTTCCGCCGCCTCTCC GCCTGCCATAACGTGCTGTTTACCGGCCACCAGGCGTTCCTCACC GCCGAGGCGCTGATCAGCATTTCGGAGACCACTCTGGGTAACCTG CAGCAGGTCGCCAACGGCGAAACCTGTCCGAACGCCATCGTC 2 CAAGCGTGCGCGGTGAACCGGGAGAGGGATCGCTGGCCGGCAGTT TGCTCAGGCAGGCGCTGTTGATCTCCAGCTGGCCAATATGCAGCC GCCAGCGGCTGGGACGCGAGAGACGGGCATCGGTCACCCGGGCGA TTTCACAGTCGCCCACCAGATAACGCAGATCGGGGATCAGCAGGG CCGACCGCGTCAGGCGCGGGCTCTCCTGCAAAGAGATACGCGTGC CCACGGGCAGCCAGATGCCCGCCAGCGTCGGCACCCAGTGGGTTA GCGTCAACAGCAGGGTTAGCGGCAATAACACCAGAACTAACACCA GCGCGATGGCGGCTTTATATTTACCCTTCATGGGCAGTTAATATC CTGATTCAACATAAGTAAAAGCCGAAAGGCGTCCATTGTGACACG TTCGACCAGTGAGTGAAAGTTTACGGCCTGTTAAAGCATAGTTGC CAGCCGGACTCGCGGCGCGACGTTCGGCCATTATCATTTAACTGT TGTTTAAGTCGCCCCTGCCACACTCCAGCCAGACGGGAATAGCTT GCGGGAGAGGCGGTGTCGTTAATTATCTCGCTCATAGAGAGCGCA CAGGACCACTATCCATGGGTATTGCTGATTGTTTTTCTGCTTACC TTCACTAAATCCTGCGCATTGGTCTCGCTGGCAATCCCCGGCACC TCCGGCCTGCTGCTGCTGGGGACATTCGCTTCCGCCAGCCTCGGA CATTTCCTGTTAATGTGGTCCAGCGCCAGCCTCGGCGCCATCGGC GGATTCTGGCTATCGTGGCGGCTGGGCATTCGCTACCGTCATCGC CTCACCCATCTACGCTGGCTGACCGCCGAGCGTCTGGCCCGCAGC CGCCTCTTCTTTCAGCGCTATGGCCCGTGGGCTATCTTTTTCAGC CGCTTTCTCTCTCCCCTGAGGGCTACGCTGCCCTTCGTTAGCGGC GCCAGCAGTCTGCCGCTGTGGTCGTTTCAGCTGGCTAACGTCAGC TCCGGTCTGCTGTGGCCGCTTCTGCTGCTCGCCCCCGGCGCTTTC AGCCTCAGTTTGTGGTGAAAAAACTTTGTCTTTCAAAGAGATTCC GCAAGTCCGCGATATGCTCTAGAATTAGGATTAGCACCCTCTCAT TAAACTATTTTTTAATAATTGTACGATTATTTTAAATATGCTACC GTGACGGTATAATCACTGGAGAAAAGTCTT 3 Kp_IdhA_FP1-TAGAGGATCCCAAGCGTGCGCGGTGAACCG 4 Kp_IdhA_RP1-GAGGAGCACAAAAGGGAAAGGCGAAGACTTTTC TCCAGTGATTATAC 5 CGCCTTTCCCTTTTGTGCTCCTCTCCCGGGGGGAGCACATTCAGA TAATCCCCACAGATCCCTGCTGCGATACCGTTACACTGGCTTGGT TTTATTAGTTATATGATTGTTTTGGAGTGAAAATGAACAAATTTG CGGCGCTTCTGGCGGCAGGTATGCTGCTGTCCGGCTGTGTCTATA ATAGTAAGGTGTCCACCGGTGCGGAACAGCTGCAGCATCATCGTT TCGTGCTGACCAGCGTCAACGGCCAGGCGGTCAACGCCAGCGACC GGCCGCTGGAGCTGAGCTTCGGTGAGAAGATGGCTATTACCGGCA AGATGTATGTATCCGGCAATATGTGCAACGGCTTTAGCGGGGAAG GTAAAGTGTCGGACGGCGAGCTGAAGGTCAAATCGCTGGCGATGA CCCGGATGCTGTGCCACGACGCCCAGCTCAATACCCTGGATGCGA CGATCGACAAGATGCTGCGCGAGGGTGCGCAGGTCGATCTGACGG AAAACCAGTTGACGCTGGCGACCGCCGACCAGACGCTGGTCTATA AGCTCGCCGACCTGATGCACTAGCCGGCGTTGAGGTGCCGCTGAC GCTGCCCCGCGACGGGGCCGCTGTTAGTAGCCGCAGCTGCCACCC GCCAGCGCCTGCTCGCTGCAGCGTTTGCCGTTCGGCAGCGCGCAC ATGCCAATCGCCGAACCATCGAGCTGACGAGCCACCGATAACGAG CCGCCTATCATGGCGCAGTTGGCCTGACCGGCGTCGCTCATCGCC GCCCGCATTCCCGGCGTGACGTGCGCCGCCGTGGCCTGCTGAACG GGTTCACTACTGCACGCGGACAGCAACAGCGCCGCACATCCTACT AACATCGCAGCTCGCATTCTCTCTCCCCTCGGAAACGTCTTAAAA AAGCAAACCCCAGAATAATAGGCAGCGTGGCGGGCGGCGTCGAGA GGGGAAGTACGTATTTATGCGCCTCATTAACATTTTCTAGCAAAT TTTCGCCTAAAGCTTGATCTGCCTCGGCCATGTCGCCCGGCGCAG GTGGTTCATCTCCCGGCAGGCAGCCATTTTCTCCGCGAACCACGC AAAATATTGATCTGGTCACGGGTACCCGGCGCATTGAGGACACAA ATGCAAAAATGGCGGGGTCAGCGGTTTGCTAAACTACCCCTTATA TAATTACAGGGCGCGTCGCGGTTTCACGC 6 Kp_IdhA_FP2-GTATAATCACTGGAGAAAAGTCTTCGCCTTTCC CTTTTGTGCTCCTC 7 Kp_IdhA_RP2-ATCGCGGCCGCGCGTGAAACCGCGACGCGCC 8 CAAGCGTGCGCGGTGAACCGGGAGAGGGATCGCTGGCCGGCAGTT TGCTCAGGCAGGCGCTGTTGATCTCCAGCTGGCCAATATGCAGCC GCCAGCGGCTGGGACGCGAGAGACGGGCATCGGTCACCCGGGCGA TTTCACAGTCGCCCACCAGATAACGCAGATCGGGGATCAGCAGGG CCGACCGCGTCAGGCGCGGGCTCTCCTGCAAAGAGATACGCGTGC CCACGGGCAGCCAGATGCCCGCCAGCGTCGGCACCCAGTGGGTTA GCGTCAACAGCAGGGTTAGCGGCAATAACACCAGAACTAACACCA GCGCGATGGCGGCTTTATATTTACCCTTCATGGGCAGTTAATATC CTGATTCAACATAAGTAAAAGCCGAAAGGCGTCCATTGTGACACG TTCGACCAGTGAGTGAAAGTTTACGGCCTGTTAAAGCATAGTTGC CAGCCGGACTCGCGGCGCGACGTTCGGCCATTATCATTTAACTGT TGTTTAAGTCGCCCCTGCCACACTCCAGCCAGACGGGAATAGCTT GCGGGAGAGGCGGTGTCGTTAATTATCTCGCTCATAGAGAGCGCA CAGGACCACTATCCATGGGTATTGCTGATTGTTTTTCTGCTTACC TTCACTAAATCCTGCGCATTGGTCTCGCTGGCAATCCCCGGCACC TCCGGCCTGCTGCTGCTGGGGACATTCGCTTCCGCCAGCCTCGGA CATTTCCTGTTAATGTGGTCCAGCGCCAGCCTCGGCGCCATCGGC GGATTCTGGCTATCGTGGCGGCTGGGCATTCGCTACCGTCATCGC CTCACCCATCTACGCTGGCTGACCGCCGAGCGTCTGGCCCGCAGC CGCCTCTTCTTTCAGCGCTATGGCCCGTGGGCTATCTTTTTCAGC CGCTTTCTCTCTCCCCTGAGGGCTACGCTGCCCTTCGTTAGCGGC GCCAGCAGTCTGCCGCTGTGGTCGTTTCAGCTGGCTAACGTCAGC TCCGGTCTGCTGTGGCCGCTTCTGCTGCTCGCCCCCGGCGCTTTC AGCCTCAGTTTGTGGTGAAAAAACTTTGTCTTTCAAAGAGATTCC GCAAGTCCGCGATATGCTCTAGAATTAGGATTAGCACCCTCTCAT TAAACTATTTTTTAATAATTGTACGATTATTTTAAATATGCTACC GTGACGGTATAATCACTGGAGAAAAGTCTTCGCCTTTCCCTTTTG TGCTCCTCTCCCGGGGGGAGCACATTCAGATAATCCCCACAGATC CCTGCTGCGATACCGTTACACTGGCTTGGTTTTATTAGTTATATG ATTGTTTTGGAGTGAAAATGAACAAATTTGCGGCGCTTCTGGCGG CAGGTATGCTGCTGTCCGGCTGTGTCTATAATAGTAAGGTGTCCA CCGGTGCGGAACAGCTGCAGCATCATCGTTTCGTGCTGACCAGCG TCAACGGCCAGGCGGTCAACGCCAGCGACCGGCCGCTGGAGCTGA GCTTCGGTGAGAAGATGGCTATTACCGGCAAGATGTATGTATCCG GCAATATGTGCAACGGCTTTAGCGGGGAAGGTAAAGTGTCGGACG GCGAGCTGAAGGTCAAATCGCTGGCGATGACCCGGATGCTGTGCC ACGACGCCCAGCTCAATACCCTGGATGCGACGATCGACAAGATGC TGCGCGAGGGTGCGCAGGTCGATCTGACGGAAAACCAGTTGACGC TGGCGACCGCCGACCAGACGCTGGTCTATAAGCTCGCCGACCTGA TGCACTAGCCGGCGTTGAGGTGCCGCTGACGCTGCCCCGCGACGG GGCCGCTGTTAGTAGCCGCAGCTGCCACCCGCCAGCGCCTGCTCG CTGCAGCGTTTGCCGTTCGGCAGCGCGCACATGCCAATCGCCGAA CCATCGAGCTGACGAGCCACCGATAACGAGCCGCCTATCATGGCG CAGTTGGCCTGACCGGCGTCGCTCATCGCCGCCCGCATTCCCGGC GTGACGTGCGCCGCCGTGGCCTGCTGAACGGGTTCACTACTGCAC GCGGACAGCAACAGCGCCGCACATCCTACTAACATCGCAGCTCGC ATTCTCTCTCCCCTCGGAAACGTCTTAAAAAAGCAAACCCCAGAA TAATAGGCAGCGTGGCGGGCGGCGTCGAGAGGGGAAGTACGTATT TATGCGCCTCATTAACATTTTCTAGCAAATTTTCGCCTAAAGCTT GATCTGCCTCGGCCATGTCGCCCGGCGCAGGTGGTTCATCTCCCG GCAGGCAGCCATTTTCTCCGCGAACCACGCAAAATATTGATCTGG TCACGGGTACCCGGCGCATTGAGGACACAAATGCAAAAATGGCGG GGTCAGCGGTTTGCTAAACTACCCCTTATATAATTACAGGGCGCG TCGCGGTTTCACGC
(44) Strain of Klebsiella pneumoniae GSC123 ldhA pflB (Kp ldhA pflB)
(45) A strain of Klebsiella pneumoniae GSC123 ldhA pflB (Kp ldhA pflB) in which a pyruvate-formate lyase gene (pflB) was further deleted was constructed as follows. Firstly, in order to clone a pyruvate-formate lyase gene of Klebsiella pneumoniae, a homologous region 1 (SEQ ID NO: 10) of a target gene pflB (SEQ ID NO: 9) was amplified using primers of SEQ ID NOs: 11 and 12 by polymerase chain reaction (PCR). Further, a homologous region 2 (SEQ ID NO: 13) was amplified using primers of SEQ ID NOs: 14 and 15 by PCR. Next, the homologous regions 1 and 2 were amplified using the same as templates for PCR, thereby obtaining a completed DNA fragment (SEQ ID NO: 16) in which the homologous regions 1 and 2 were ligated (Table 2).
(46) The completed DNA fragment may include antibiotic resistance genes and the like in order to enhance the probability of recombination of target genes. Further, the completed DNA fragment may include a sacB gene encoding levansucrase in order to remove antibiotic resistance genes recombined in the chromosomes.
(47) The prepared DNA fragment was transferred to lactate dehydrogenase (ldhA) deleted Klebsiella pneumoniae GSC123 ldhA (Kp ldhA) through electroporation (25 F, 200 , 18 kV/cm), in which the target gene was deleted by a homologous recombination mechanism indigenous to the microorganism.
(48) TABLE-US-00002 TABLE2 SEQ ID NO Sequence 9 ATGTCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTTTT GCGAAAGGTGACTGGCAGAATGAAGTCAACGTCCGTGACTTTATT CAGAAAAACTACACCCCATATGAAGGCGACGAATCCTTCCTGGCT GGCGCGACTGAAGCGACCACCAAGCTGTGGGACACCGTAATGGAA GGTGTAAAACAGGAAAACCGCACTCACGCGCCTGTTGATTTTGAC ACTGCCCTGGCTTCCACCATCACCTCTCACGACGCGGGCTATATC GAGAAAGGTCTGGAAAAAATCGTTGGTCTGCAGACCGAAGCGCCG CTGAAACGTGCGATCATCCCGTTCGGTGGTATCAAAATGGTTGAA GGTTCCTGCAAAGCGTATAATCGCGAGCTGGACCCGATGCTGAAA AAAATCTTCACAGAGTACCGTAAAACTCACAACCAGGGCGTTTTC GACGTCTATACCCCGGACATTCTGCGCTGCCGTAAATCCGGCGTG CTGACGGGTCTGCCGGATGCTTACGGTCGTGGTCGTATCATCGGT GACTACCGTCGCGTTGCGCTGTACGGTATCGACTTCCTGATGAAA GACAAATTCGCCCAGTTCAACTCTCTGCAAGCGAAACTGGAAAGC GGCGAAGACCTGGAAGCGACCATCCGTCTGCGTGAAGAAATCGCT GAACAACACCGCGCACTGGGCCAGATCAAAGAGATGGCCGCTAAA TATGGCTATGACATCTCCGGTCCGGCGACCACCGCTCAGGAAGCG ATTCAGTGGACCTACTTCGGTTACCTGGCTGCCGTGAAATCTCAG AACGGCGCGGCAATGTCCTTCGGTCGTACCTCCAGCTTCCTGGAT ATCTACATCGAGCGTGACCTGCAGGCGGGTAAAATCACCGAGCAA GACGCGCAGGAAATGGTTGACCACCTGGTCATGAAACTGCGTATG GTTCGCTTCCTGCGTACCCCGGAATATGATGAACTGTTCTCCGGC GACCCGATTTGGGCAACGGAATCCATCGGCGGTATGGGCGTTGAC GGCCGTACTCTGGTGACCAAAAACAGCTTCCGCTTCCTGAACACC CTGTACACCATGGGGCCGTCTCCGGAGCCGAACATTACTATCCTG TGGTCTGAAAAACTGCCGCTGAGCTTCAAGAAATTCGCCGCTAAA GTGTCCATCGATACCTCTTCTCTGCAGTATGAGAACGATGACCTG ATGCGTCCGGACTTCAACAACGACGACTACGCTATCGCATGCTGC GTAAGCCCGATGGTTGTTGGTAAGCAAATGCAGTTCTTCGGCGCT CGCGCTAACCTCGCGAAAACCATGCTGTACGCTATCAACGGCGGC GTGGATGAAAAACTGAAAATGCAGGTTGGTCCGAAATCTGAACCG ATCAAAGGCGACGTCCTGAACTTCGACGAAGTAATGGATCGCATG GATCACTTCATGGACTGGCTGGCTAAACAGTACGTCACCGCGCTG AACATCATCCACTACATGCACGACAAGTACAGCTACGAAGCCTCT CTGATGGCGCTGCACGACCGTGACGTTATCCGCACCATGGCGTGT GGTATCGCTGGTCTGTCCGTTGCTGCTGACTCCCTGTCTGCTATC AAATATGCGAAAGTTAAACCGATTCGTGACGAAGACGGTCTGGCT ATCGACTTCGAAATCGAAGGCGAATACCCGCAGTTTGGTAACAAC GACCCTCGCGTCGATGACATGGCCGTTGACCTGGTTGAACGTTTC ATGAAGAAAATTCAGAAACTGCACACCTACCGCAACGCTATCCCG ACTCAGTCTGTTCTGACCATCACCTCTAACGTGGTGTACGGTAAG CCGGTAATACCCCAGACGGTCGTCGCGCTGGCGCGCCGTTCGGTC CAGGTGCTAACCCGATGCACGGCCGTGACCAGAAAGGCGCAGTAG CCTCTCTGACCTCCGTCGCTAAACTGCCGTTTGCTTACGCGAAAG ATGGTATCTCTTATACCTTCTCTATCGTGCCGAACGCGCTGGGTA AAGACGACGAAGTTCGTAAGACCAACCTGGCGGGTCTGATGGATG GTTACTTCCATCACGAAGCGTCCATCGAAGGTGGTCAGCACCTGA ACGTGAACGTCATGAACCGCGAAATGCTGCTCGACGCGATGGAAA ACCCGGAAAAATATCCGCAGCTGACCATCCGTGTATCTGGCTACG CCGTACGTTTTAACTCCCTGACCAAAGAACAGCAGCAGGATGTTA TTACCCGTACCTTCACTCAGACCATG 10 GTTTGTGCTGCTGATGTGGTTATCAGGCGAATATATGACTGCCAA CGGCGGCTGGGGGCTAAACGTTCTGCAGACCGCCGACCACAAAAT GCACCATACTTTTGTGGAGGCCGTGAGCCTGGGTATCCTCGCTAA CCTGATGGTTTGTCTCGCCGTATGGATGAGCTATTCCGGTCGTAG CCTGATGGATAAAGCGATGATCATGGTCCTGCCGGTAGCGATGTT CGTTGCCAGCGGCTTTGAGCACAGCATCGCCAACATGTTTATGAT CCCGATGGGTATCGTAATCCGCAACTTTGCAAGCCCGGAATTCTG GACCGCCATCGGTTCGACTCCGGAAAGTTTCTCTCACTTGACCGT TATGAACTTCATCACTGATAACCTGATTCCGGTAACTATCGGGAA CATTATCGGCGGGGGTCTGCTGGTCGGGTTGACATACTGGGTCAT TTACCTGCGTGGCAACGACCATCACTAAGGGTTGTTTCAGGCAGT AAATAAAAAATCCACTTAAGAAGGTAGGTGTTAC 11 Kp_pflB_FP1-GGATCCGTTTGTGCTGCTGATGTGGTTATCAG GC 12 Kp_pflB_RP1-CGCCTTTTCAGTCAGACAGGGAAGTAACACCTA CCTTCTTAAGTGG 13 TTCCCTGTCTGACTGAAAAGGCGTACAATAAAGGCCCCACATCAG TGGGGCCTTTTTAACAAGCATTCCCCGCCCCAGCCTGCTTTGCCA GTTATCTATACTTTGGGTACCTGTCAAAACAGACTCGACGCAGCC GCTGAGCTGCGCACCAACACGGCCCCGGATGGGCCACATCTGGAG AAAACACCGCAATGTCAGTTATTGGTCGCATTCACTCCTTTGAAT CCTGTGGCACCGTTGATGGCCCAGGCATCCGCTTTATTACCTTTT TCCAGGGCTGCCTGATGCGCTGCCTGTACTGCCATAACCGTGACA CCTGGGATACCCACGGCGGCAAAGAAATCACCGTTGAAGAATTAA TGAAAGAGGTGGTGACCTATCGTCACTTTATGAATGCTTCCGGCG GCGGCGTCACCGCCTCGGGCGGTGAGGCGATCCTGCAGGCGGAGT TTGTTCGCGACTGGTTCCGCGCGTGTAAGAAAGAAGGCATCCACA CCTGCCTGGATACCAACGGCTTCGTACGTCGCTACGATCCGGTTA TCGACGAGCTGCTGGAGGTAACAGACCTGGTGATGCTGGATCTCA AGCAGATGAAC 14 Kp_pflB_FP2-CCACTTAAGAAGGTAGGTGTTACTTCCCTGTCT GACTGAAAAGGCG 15 Kp_pflB_RP2-GCGGCCGCGTTCATCTGCTTGAGATCCAGCATC ACC 16 GTTTGTGCTGCTGATGTGGTTATCAGGCGAATATATGACTGCCAA CGGCGGCTGGGGGCTAAACGTTCTGCAGACCGCCGACCACAAAAT GCACCATACTTTTGTGGAGGCCGTGAGCCTGGGTATCCTCGCTAA CCTGATGGTTTGTCTCGCCGTATGGATGAGCTATTCCGGTCGTAG CCTGATGGATAAAGCGATGATCATGGTCCTGCCGGTAGCGATGTT CGTTGCCAGCGGCTTTGAGCACAGCATCGCCAACATGTTTATGAT CCCGATGGGTATCGTAATCCGCAACTTTGCAAGCCCGGAATTCTG GACCGCCATCGGTTCGACTCCGGAAAGTTTCTCTCACTTGACCGT TATGAACTTCATCACTGATAACCTGATTCCGGTAACTATCGGGAA CATTATCGGCGGGGGTCTGCTGGTCGGGTTGACATACTGGGTCAT TTACCTGCGTGGCAACGACCATCACTAAGGGTTGTTTCAGGCAGT AAATAAAAAATCCACTTAAGAAGGTAGGTGTTACTTCCCTGTCTG ACTGAAAAGGCGTACAATAAAGGCCCCACATCAGTGGGGCCTTTT TAACAAGCATTCCCCGCCCCAGCCTGCTTTGCCAGTTATCTATAC TTTGGGTACCTGTCAAAACAGACTCGACGCAGCCGCTGAGCTGCG CACCAACACGGCCCCGGATGGGCCACATCTGGAGAAAACACCGCA ATGTCAGTTATTGGTCGCATTCACTCCTTTGAATCCTGTGGCACC GTTGATGGCCCAGGCATCCGCTTTATTACCTTTTTCCAGGGCTGC CTGATGCGCTGCCTGTACTGCCATAACCGTGACACCTGGGATACC CACGGCGGCAAAGAAATCACCGTTGAAGAATTAATGAAAGAGGTG GTGACCTATCGTCACTTTATGAATGCTTCCGGCGGCGGCGTCACC GCCTCGGGCGGTGAGGCGATCCTGCAGGCGGAGTTTGTTCGCGAC TGGTTCCGCGCGTGTAAGAAAGAAGGCATCCACACCTGCCTGGAT ACCAACGGCTTCGTACGTCGCTACGATCCGGTTATCGACGAGCTG CTGGAGGTAACAGACCTGGTGATGCTGGATCTCAAGCAGATGAAC
(49) Strain of Klebsiella pneumoniae GSC123 dhA pflB budA (Kp ldhA pflB budA)
(50) A strain of Klebsiella pneumoniae GSC123 ldhA pflB budA (Kp ldhA pflB budA) in which a gene for converting -acetolactate into acetoin (-acetolactate decarboxylase gene, budA) on a pathway of synthesizing 2,3-butanediol was further deleted was constructed as follows. Firstly, in order to clone an -acetolactate decarboxylase gene of Klebsiella pneumoniae, a homologous region 1 (SEQ ID NO: 18) of a target gene budA (SEQ ID NO: 17) was amplified using primers of SEQ ID NOs: 19 and 20 by polymerase chain reaction (PCR). Further, a homologous region 2 (SEQ ID NO: 21) was amplified using primers of SEQ ID NOs: 22 and 23 by PCR. Next, the homologous regions 1 and 2 were amplified using the same as templates for PCR, thereby obtaining a completed DNA fragment (SEQ ID NO: 24) in which the homologous regions 1 and 2 were ligated (Table 3).
(51) The completed DNA fragment may include antibiotic resistance genes and the like in order to enhance the probability of recombination of target genes. Further, the completed DNA fragment may include a sacB gene encoding levansucrase in order to remove antibiotic resistance genes recombined in the chromosomes.
(52) The prepared DNA fragment was transferred to lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pflB) deleted Klebsiella pneumoniae GSC123 ldhA pflB (Kp ldhA pflB) through electroporation (25 F, 200 , 18 kV/cm), in which the target gene was deleted by a homologous recombination mechanism indigenous to the microorganism.
(53) TABLE-US-00003 TABLE3 SEQ ID NO Sequence 17 ATGAATCATTCTGCTGAATGCACCTGCGAAGAGAGTCTATGCGAA ACCCTGCGGGCGTTTTCCGCGCAGCATCCCGAGAGCGTGCTCTAT CAGACATCGCTCATGAGCGCCCTGCTGAGCGGGGTTTACGAAGGC AGCACCACCATCGCCGACCTGCTGAAACACGGCGATTTCGGCCTC GGCACCTTTAATGAGCTGGACGGGGAGCTGATCGCCTTCAGCAGT CAGGTCTATCAGCTGCGCGCCGACGGCAGCGCGCGCAAAGCCCAG CCGGAGCAGAAAACGCCGTTCGCGGTGATGACCTGGTTCCAGCCG CAGTACCGGAAAACCTTTGACCATCCGGTGAGCCGCCAGCAGCTG CACGAGGTGATCGACCAGCAAATCCCCTCTGACAACCTGTTCTGC GCCCTGCGCATCGACGGCCATTTCCGCCATGCCCATACCCGCACC GTGCCGCGCCAGACGCCGCCGTACCGGGCGATGACCGACGTACTC GACGATCAGCCGGTGTTCCGCTTTAACCAGCGCGAAGGGGTGCTG GTCGGCTTCCGGACCCCGCAGCATATGCAGGGGATCAACGTCGCC GGGTATCACGAGCATTTTATTACCGATGACCGCAAAGGCGGCGGT CACCTGCTGGATTACCAGCTCGACCACGGGGTGCTGACCTTCGGC GAAATTCACAAGCTGATGATCGACCTGCCCGCCGACAGCGCGTTC CTGCAGGCTAATCTGCATCCCGATAATCTCGATGCCGCCATCCGT TCCGTAGAAAGT 18 GCAGATTAAAGGCTTTACTGCTCTCGCACGGCAGGCGGACGAAGG CGATATCCAGCTCGGCCTCGCTCAGGGCGGTCATCAGATTGGCCA TATTGTCTTCCATCTGGTGCAGGGTCACCCCGGGGTGGTCGAGCT GAAAACGGTGCAGCAGCGTGAAGATTTGCGGATGGAAAGCATCAG AACTGGTAATGCCTAGCGACAGGCTGCCGTTCATCCCGCGCGCAA TGCCCTTGGCCTTCTCCAGCGCCGCATCGCTCATGGCGAGGATCT GGCGGGCATCCTCATAGAAAGACTCTCCCGCTTCCGTCAGCTCCA CCCCGCGGGTTAAACGCCGGAACAGCGGGGTCCCCACCTCGCGCT CAAGCCGCTGAATTTGCTGACTTAACGGAGGCTGTGAAATACCCA GCTCCTTGGCGGCCTGGGTGAAGTGCCGCGTCCTGGCGACGGCGA CAAAATAGCGAAGATAACGAAGTTCCATATCGAAAACGTCTCAAA CCAGCATGGTTTCTATATTGGAACTGTGAGCTGAATCGGGTCAAC ATTTATTTAACCTTTCTTATATTTGTTGAACGAGGAAGTGGTATA TGAATCATTCTGCTGAATGCACCTGCGAACCCGATAATCTCGATG CCGCCATCCGTTCCGTAGAAAGT 19 Kp_budA_FP1-TCTAGAGGATCCGCAGATTAAAGGCTTTACTGC TCTC 20 Kp_budA_RP1-CGGATGGCGGCATCGAGATTATCGGGTTCGCAG GTGCATTCAGCAGAATGATTC 21 ATGAATCATTCTGCTGAATGCACCTGCGAACCCGATAATCTCGAT GCCGCCATCCGTTCCGTAGAAAGTTAAGGGGGTCACATGGACAAA CAGTATCCGGTACGCCAGTGGGCGCACGGCGCCGATCTCGTCGTC AGTCAGCTGGAAGCACAGGGGGTACGCCAGGTGTTCGGCATCCCC GGCGCCAAAATCGACAAGGTCTTCGATTCACTGCTGGATTCCTCC ATTCGCATTATTCCGGTACGCCACGAAGCCAACGCCGCATTTATG GCCGCCGCCGTCGGACGTATTACCGGCAAAGCGGGCGTGGCGCTG GTCACCTCCGGTCCGGGTTGTTCTAACCTGATCACCGGCATGGCC ACCGCGAACAGCGAAGGCGACCCGGTGGTGGCCCTGGGCGGCGCG GTAAAACGCGCCGATAAAGCCAAACAGGTCCACCAGAGTATGGAT ACGGTGGCGATGTTCAGCCCGGTCACCAAATACGCCGTCGAGGTG ACGGCGCCGGATGCGCTGGCGGAAGTGGTCTCCAACGCCTTCCGC GCCGCCGAGCAGGGCCGGCCGGGCAGCGCGTTCGTTAGCCTGCCG CAGGATGTGGTCGATG 22 Kp_budA_FP2-GAATCATTCTGCTGAATGCACCTGCGAACCCGA TAATCTCGATGCCGCCATCCG 23 Kp_budA_RP2-GATCGCGGCCGCCATCGACCACATCCTGCGGCA GG 24 GCAGATTAAAGGCTTTACTGCTCTCGCACGGCAGGCGGACGAAGG CGATATCCAGCTCGGCCTCGCTCAGGGCGGTCATCAGATTGGCCA TATTGTCTTCCATCTGGTGCAGGGTCACCCCGGGGTGGTCGAGCT GAAAACGGTGCAGCAGCGTGAAGATTTGCGGATGGAAAGCATCAG AACTGGTAATGCCTAGCGACAGGCTGCCGTTCATCCCGCGCGCAA TGCCCTTGGCCTTCTCCAGCGCCGCATCGCTCATGGCGAGGATCT GGCGGGCATCCTCATAGAAAGACTCTCCCGCTTCCGTCAGCTCCA CCCCGCGGGTTAAACGCCGGAACAGCGGGGTCCCCACCTCGCGCT CAAGCCGCTGAATTTGCTGACTTAACGGAGGCTGTGAAATACCCA GCTCCTTGGCGGCCTGGGTGAAGTGCCGCGTCCTGGCGACGGCGA CAAAATAGCGAAGATAACGAAGTTCCATATCGAAAACGTCTCAAA CCAGCATGGTTTCTATATTGGAACTGTGAGCTGAATCGGGTCAAC ATTTATTTAACCTTTCTTATATTTGTTGAACGAGGAAGTGGTATA TGAATCATTCTGCTGAATGCACCTGCGAACCCGATAATCTCGATG CCGCCATCCGTTCCGTAGAAAGTTAAGGGGGTCACATGGACAAAC AGTATCCGGTACGCCAGTGGGCGCACGGCGCCGATCTCGTCGTCA GTCAGCTGGAAGCACAGGGGGTACGCCAGGTGTTCGGCATCCCCG GCGCCAAAATCGACAAGGTCTTCGATTCACTGCTGGATTCCTCCA TTCGCATTATTCCGGTACGCCACGAAGCCAACGCCGCATTTATGG CCGCCGCCGTCGGACGTATTACCGGCAAAGCGGGCGTGGCGCTGG TCACCTCCGGTCCGGGTTGTTCTAACCTGATCACCGGCATGGCCA CCGCGAACAGCGAAGGCGACCCGGTGGTGGCCCTGGGCGGCGCGG TAAAACGCGCCGATAAAGCCAAACAGGTCCACCAGAGTATGGATA CGGTGGCGATGTTCAGCCCGGTCACCAAATACGCCGTCGAGGTGA CGGCGCCGGATGCGCTGGCGGAAGTGGTCTCCAACGCCTTCCGCG CCGCCGAGCAGGGCCGGCCGGGCAGCGCGTTCGTTAGCCTGCCGC AGGATGTGGTCGATG
(54) Strain of Klebsiella pneumoniae GSC123 ldhA pflB budC (Kp ldhA pflB budC)
(55) A strain of Klebsiella pneumoniae GSC123 ldhA pflB budC (Kp ldhA pflB budC) in which acetoin reductase gene, budC for converting acetoin into 2,3-butanediol on a pathway of synthesizing 2,3-butanediol, was further deleted was constructed as follows. Firstly, in order to clone an acetoin reductase gene of Klebsiella pneumoniae, a homologous region 1 (SEQ ID NO: 26) of a target gene budC (SEQ ID NO: 25) was amplified using primers of SEQ ID NOs: 27 and 28 by polymerase chain reaction (PCR). Further, a homologous region 2 (SEQ ID NO: 29) was amplified using primers of SEQ ID NOs: 30 and 31 by PCR. Next, the homologous regions 1 and 2 were amplified using the same as templates for PCR, thereby obtaining a completed DNA fragment (SEQ ID NO: 32) in which the homologous regions 1 and 2 were ligated (Table 4).
(56) The completed DNA fragment may include antibiotic resistance genes and the like in order to enhance the probability of recombination of target genes. Further, the completed DNA fragment may include a sacB gene encoding levansucrase in order to remove antibiotic resistance genes recombined in the chromosomes.
(57) The prepared DNA fragment was transferred to lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pflB) deleted Klebsiella pneumoniae GSC123 ldhA pflB (Kp ldhA pflB) through electroporation (25 F, 200 , 18 kV/cm), in which the target gene was deleted by a homologous recombination mechanism indigenous to the microorganism.
(58) TABLE-US-00004 TABLE4 SEQ ID NO Sequence 25 ATGAAAAAAGTCGCACTTGTTACCGGCGCCGGCCAGGGGATTGGT AAAGCTATCGCCCTTCGTCTGGTGAAGGATGGATTTGCCGTGGCC ATTGCCGATTATAACGACGCCACCGCCAAAGCGGTCGCCTCCGAA ATCAACCAGGCCGGCGGCCGCGCCATGGCGGTGAAAGTGGATGTT TCTGACCGCGACCAGGTATTTGCCGCCGTCGAACAGGCGCGCAAA ACGCTGGGCGGCTTCGACGTCATCGTCAACAACGCCGGCGTGGCG CCATCCACGCCGATCGAGTCCATTACCCCGGAGATTGTCGACAAA GTCTACAACATCAACGTCAAAGGGGTGATCTGGGGCATCCAGGCA GCGGTCGAGGCCTTTAAGAAAGAGGGTCACGGCGGGAAAATCATC AACGCCTGTTCCCAGGCCGGCCACGTCGGCAACCCGGAGCTGGCG GTATATAGCTCGAGTAAATTCGCGGTACGCGGCTTAACCCAGACC GCCGCTCGCGACCTCGCGCCGCTGGGCATCACGGTCAACGGCTAC TGCCCGGGGATTGTCAAAACGCCGATGTGGGCCGAAATTGACCGC CAGGTGTCCGAAGCCGCCGGTAAACCGCTGGGCTACGGTACCGCC GAGTTCGCCAAACGCATCACCCTCGGCCGCCTGTCCGAGCCGGAA GATGTCGCCGCCTGCGTCTCCTATCTTGCCAGCCCGGATTCTGAT TATATGACCGGTCAGTCATTGCTGATCGACGGCGGCATGGTGTTT AAC 26 GCTGCGTATCGTTCGCGCCATGCAGGACATCGTCAACAGCGACGT CACGTTGACCGTGGACATGGGCAGCTTCCATATCTGGATTGCCCG CTACCTGTACAGCTTCCGCGCCCGCCAGGTGATGATCTCCAACGG CCAGCAGACCATGGGCGTCGCCCTGCCCTGGGCCATCGGCGCCTG GCTGGTCAATCCTGAGCGCAAAGTGGTCTCCGTCTCCGGCGACGG CGGCTTCCTGCAGTCGAGCATGGAGCTGGAGACCGCCGTCCGCCT GAAAGCCAACGTGCTGCACCTGATCTGGGTCGATAACGGCTACAA CATGGTGGCCATTCAGGAAGAGAAAAAATACCAGCGCCTGTCCGG CGTCGAGTTTGGGCCGATGGATTTTAAAGCCTATGCCGAATCCTT CGGCGCGAAAGGGTTTGCCGTGGAAAGCGCCGAGGCGCTGGAGCC GACCCTGCGCGCGGCGATGGACGTCGACGGCCCGGCGGTAGTGGC CATCCCGGTGGATTATCGCGATAACCCGCTGCTGATGGGCCAGCT GCATCTGAGTCAGATTCTGTAAGTCATCACAATAAGGAAAGAAAA ATGAAAAAAGTCGCACTTGTTACCGGCGCCATGACCGGTCAGTCA TTGCTGATCG 27 KpbudC_FP1-TCTAGAGGATCCGCTGCGTATCGTTCGCGC CATGC 28 Kp_budC_RP1-CGATCAGCAATGACTGACCGGTCATGGCGCCGG TAACAAGTGCGACTT 29 AAGTCGCACTTGTTACCGGCGCCATGACCGGTCAGTCATTGCTGA TCGACGGCGGCATGGTGTTTAACTAATAAAAAAAAGCTCTGACAT GGCTTGCCCCTGCTTTCGCGCAGGGGCTTTTTTTGGTTTGGGTGT AAGTGTAAGCATCCCGGAGAAACGAAGCATCGATATTTGAGGGCT TCTGGCGTTCTCACTTACGCTTCGACACGACGTGGGCAATCTGAC TGGGATGAAGGTCTGATTTGAGCGAGGAGCGGAAGTTCGGGAACG GGATAGCTCTGACCTGCCACCAGGATTAGATACAACCGTCAGTTA GTAAGGTCGGTTTGTTTACCTTCACATTTTCCATTTCGCCACCGT GCTGCAAACTCTGATGGCGTCTGATAATTCAGTGCTGAATGTGGA CGACACTCGTTATAATCCTGCCGCCAGTCATTAATGATTTTCCTT GCGTGAACGATATCGCTGAACCAGTGCTCATTCAGGCATTCATCG CGAAATCGTCCGTTAAAGCTCTCAATAAATCCGTTCTGCGTTGGC TTGCCCGGCTGGATTAAGCGCAACTCAACACCATGCTCAAAGGCC CATTGATCCAGTGCACGGCAAGTGAACTCCGGCCCCTGG 30 Kp_budC_FP2-AAGTCGCACTTGTTACCGGCGCCATGACCGGTC AGTCATTGCTGATCG 31 Kp_budC_RP2-GCGGCCGCCCAGGGGCCGGAGTTCACTTGCC 32 GCTGCGTATCGTTCGCGCCATGCAGGACATCGTCAACAGCGACGT CACGTTGACCGTGGACATGGGCAGCTTCCATATCTGGATTGCCCG CTACCTGTACAGCTTCCGCGCCCGCCAGGTGATGATCTCCAACGG CCAGCAGACCATGGGCGTCGCCCTGCCCTGGGCCATCGGCGCCTG GCTGGTCAATCCTGAGCGCAAAGTGGTCTCCGTCTCCGGCGACGG CGGCTTCCTGCAGTCGAGCATGGAGCTGGAGACCGCCGTCCGCCT GAAAGCCAACGTGCTGCACCTGATCTGGGTCGATAACGGCTACAA CATGGTGGCCATTCAGGAAGAGAAAAAATACCAGCGCCTGTCCGG CGTCGAGTTTGGGCCGATGGATTTTAAAGCCTATGCCGAATCCTT CGGCGCGAAAGGGTTTGCCGTGGAAAGCGCCGAGGCGCTGGAGCC GACCCTGCGCGCGGCGATGGACGTCGACGGCCCGGCGGTAGTGGC CATCCCGGTGGATTATCGCGATAACCCGCTGCTGATGGGCCAGCT GCATCTGAGTCAGATTCTGTAAGTCATCACAATAAGGAAAGAAAA ATGAAAAAAGTCGCACTTGTTACCGGCGCCATGACCGGTCAGTCA TTGCTGATCGACGGCGGCATGGTGTTTAACTAATAAAAAAAAGCT CTGACATGGCTTGCCCCTGCTTTCGCGCAGGGGCTTTTTTTGGTT TGGGTGTAAGTGTAAGCATCCCGGAGAAACGAAGCATCGATATTT GAGGGCTTCTGGCGTTCTCACTTACGCTTCGACACGACGTGGGCA ATCTGACTGGGATGAAGGTCTGATTTGAGCGAGGAGCGGAAGTTC GGGAACGGGATAGCTCTGACCTGCCACCAGGATTAGATACAACCG TCAGTTAGTAAGGTCGGTTTGTTTACCTTCACATTTTCCATTTCG CCACCGTGCTGCAAACTCTGATGGCGTCTGATAATTCAGTGCTGA ATGTGGACGACACTCGTTATAATCCTGCCGCCAGTCATTAATGAT TTTCCTTGCGTGAACGATATCGCTGAACCAGTGCTCATTCAGGCA TTCATCGCGAAATCGTCCGTTAAAGCTCTCAATAAATCCGTTCTG CGTTGGCTTGCCCGGCTGGATTAAGCGCAACTCAACACCATGCTC AAAGGCCCATTGATCCAGTGCACGGCAAGTGAACTCCGGCCCCTG G
(59) Strain of Klebsiella pneumoniae GSC123 ldhA pflB budRABC (Kp ldhA pflB budRABC)
(60) A strain of Klebsiella pneumoniae GSC123 ldhA pflB budRABC (Kp ldhA pflB budRABC) in which genes (budRABC) constituting a 2,3-butanediol operon, namely, a gene for transcription activation factors (budR), a gene for -acetolactate decarboxylase (budA), a gene for -acetolactate synthase (budB), and a gene for acetoin reductase (budC) were further deleted was constructed as follows. Firstly, in order to clone a gene for a 2,3-butanediol operon of Klebsiella pneumoniae, a homologous region 1 (SEQ ID NO: 34) of a target gene budRABC (SEQ ID NO: 33) was amplified using primers of SEQ ID NOs: 35 and 36 by polymerase chain reaction (PCR). Further, a homologous region 2 (SEQ ID NO: 37) was amplified using primers of SEQ ID NOs: 38 and 39 by PCR. Next, the homologous regions 1 and 2 were amplified using the same as templates for PCR, thereby obtaining a completed DNA fragment (SEQ ID NO: 40) in which the homologous regions 1 and 2 were ligated (Table 5).
(61) The completed DNA fragment may include antibiotic resistance genes and the like in order to enhance the probability of recombination of target genes. Further, the completed DNA fragment may include a sacB gene encoding levansucrase in order to remove antibiotic resistance genes recombined in the chromosomes.
(62) The prepared DNA fragment was transferred to lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pflB) deleted Klebsiella pneumoniae GSC123 ldhA pflB (Kp ldhA pflB) through electroporation (25 F, 200 , 18 kV/cm) in which the target gene was deleted by a homologous recombination mechanism indigenous to the microorganism.
(63) TABLE-US-00005 TABLE5 SEQ ID NO Sequence 33 GAACATCGCCAGAAAGCGTTTCACCGTACGCGAGCGCTCGAAGCG CCGCCAGGCGATGGCGATATCGGTCTTCAGCGGCGCCCCGCTAAG CGGGTGATAGCTGACGTTCGGCTGCTGGATGCAGGTCATCGACTG CGGAACCAGCGCGAAGCCGAAGCCAGCATTGACCATGCTCAGCGA CGACGAAATTTGCGACGACTGCCAGGCGCGCTCCATATCGATCCC GGCGCGCAGACAGCTGTTGTACACCAGCTCATACAGCCCGGGGGC CACCTCCCGCGGGAAGAGGATCGGCGCCACGTCGCGCAGCTGCTC CAGGGCCAGGGTCGGCTGCGTCGCCAGCGGGTTATCGCGCGGCAG CGCGATAACCATCGGCTCCTCATCGATAATCCGCAGATTAAAGGC TTTACTGCTCTCGCACGGCAGGCGGACGAAGGCGATATCCAGCTC GGCCTCGCTCAGGGCGGTCATCAGATTGGCCATATTGTCTTCCAT CTGGTGCAGGGTCACCCCGGGGTGGTCGAGCTGAAAACGGTGCAG CAGCGTGAAGATTTGCGGATGGAAAGCATCAGAACTGGTAATGCC TAGCGACAGGCTGCCGTTCATCCCGCGCGCAATGCCCTTGGCCTT CTCCAGCGCCGCATCGCTCATGGCGAGGATCTGGCGGGCATCCTC ATAGAAAGACTCTCCCGCTTCCGTCAGCTCCACCCCGCGGGTTAA ACGCCGGAACAGCGGGGTCCCCACCTCGCGCTCAAGCCGCTGAAT TTGCTGACTTAACGGAGGCTGTGAAATACCCAGCTCCTTGGCGGC CTGGGTGAAGTGCCGCGTCCTGGCGACGGCGACAAAATAGCGAAG ATAACGAAGTTCCATATCGAAAACGTCTCAAACCAGCATGGTTTC TATATTGGAACTGTGAGCTGAATCGGGTCAACATTTATTTAACCT TTCTTATATTTGTTGAACGAGGAAGTGGTATATGAATCATTCTGC TGAATGCACCTGCGAAGAGAGTCTATGCGAAACCCTGCGGGCGTT TTCCGCGCAGCATCCCGAGAGCGTGCTCTATCAGACATCGCTCAT GAGCGCCCTGCTGAGCGGGGTTTACGAAGGCAGCACCACCATCGC CGACCTGCTGAAACACGGCGATTTCGGCCTCGGCACCTTTAATGA GCTGGACGGGGAGCTGATCGCCTTCAGCAGTCAGGTCTATCAGCT GCGCGCCGACGGCAGCGCGCGCAAAGCCCAGCCGGAGCAGAAAAC GCCGTTCGCGGTGATGACCTGGTTCCAGCCGCAGTACCGGAAAAC CTTTGACCATCCGGTGAGCCGCCAGCAGCTGCACGAGGTGATCGA CCAGCAAATCCCCTCTGACAACCTGTTCTGCGCCCTGCGCATCGA CGGCCATTTCCGCCATGCCCATACCCGCACCGTGCCGCGCCAGAC GCCGCCGTACCGGGCGATGACCGACGTACTCGACGATCAGCCGGT GTTCCGCTTTAACCAGCGCGAAGGGGTGCTGGTCGGCTTCCGGAC CCCGCAGCATATGCAGGGGATCAACGTCGCCGGGTATCACGAGCA TTTTATTACCGATGACCGCAAAGGCGGCGGTCACCTGCTGGATTA CCAGCTCGACCACGGGGTGCTGACCTTCGGCGAAATTCACAAGCT GATGATCGACCTGCCCGCCGACAGCGCGTTCCTGCAGGCTAATCT GCATCCCGATAATCTCGATGCCGCCATCCGTTCCGTAGAAAGTTA AGGGGGTCACATGGACAAACAGTATCCGGTACGCCAGTGGGCGCA CGGCGCCGATCTCGTCGTCAGTCAGCTGGAAGCACAGGGGGTACG CCAGGTGTTCGGCATCCCCGGCGCCAAAATCGACAAGGTCTTCGA TTCACTGCTGGATTCCTCCATTCGCATTATTCCGGTACGCCACGA AGCCAACGCCGCATTTATGGCCGCCGCCGTCGGACGTATTACCGG CAAAGCGGGCGTGGCGCTGGTCACCTCCGGTCCGGGTTGTTCTAA CCTGATCACCGGCATGGCCACCGCGAACAGCGAAGGCGACCCGGT GGTGGCCCTGGGCGGCGCGGTAAAACGCGCCGATAAAGCCAAACA GGTCCACCAGAGTATGGATACGGTGGCGATGTTCAGCCCGGTCAC CAAATACGCCGTCGAGGTGACGGCGCCGGATGCGCTGGCGGAAGT GGTCTCCAACGCCTTCCGCGCCGCCGAGCAGGGCCGGCCGGGCAG CGCGTTCGTTAGCCTGCCGCAGGATGTGGTCGATGGCCCGGTCAG CGGCAAAGTACTGCCGGCCAGCGGGGCCCCGCAGATGGGCGCCGC GCCGGATGATGCCATCGACCAGGTGGCGAAGCTTATCGCCCAGGC GAAGAACCCGATCTTCCTGCTCGGCCTGATGGCCAGCCAGCCGGA AAACAGCAAGGCGCTGCGCCGTTTGCTGGAGACCAGCCATATTCC AGTCACCAGCACCTATCAGGCCGCCGGAGCGGTGAATCAGGATAA CTTCTCTCGCTTCGCCGGCCGGGTTGGGCTGTTTAACAACCAGGC CGGGGACCGTCTGCTGCAGCTTGCCGACCTGGTGATCTGCATCGG CTACAGCCCGGTGGAATACGAACCGGCGATGTGGAACAGCGGCAA CGCGACGCTGGTGCACATCGACGTGCTGCCCGCCTATGAAGAGCG CAACTACACCCCGGATGTCGAGCTGGTAGGCGATATCGCCGGCAC TCTCAACAAGCTGGCGCAAAATATCGATCATCGGCTGGTGCTCTC CCCGCAGGCAGCGGAGATCCTCCGCGACCGCCAGCACCAGCGCGA GCTGCTGGACCGCCGCGGCGCGCAGCTCAACCAGTTTGCCCTGCA TCCGCTGCGTATCGTTCGCGCCATGCAGGACATCGTCAACAGCGA CGTCACGTTGACCGTGGACATGGGCAGCTTCCATATCTGGATTGC CCGCTACCTGTACAGCTTCCGCGCCCGCCAGGTGATGATCTCCAA CGGCCAGCAGACCATGGGCGTCGCCCTGCCCTGGGCCATCGGCGC CTGGCTGGTCAATCCTGAGCGCAAAGTGGTCTCCGTCTCCGGCGA CGGCGGCTTCCTGCAGTCGAGCATGGAGCTGGAGACCGCCGTCCG CCTGAAAGCCAACGTGCTGCACCTGATCTGGGTCGATAACGGCTA CAACATGGTGGCCATTCAGGAAGAGAAAAAATACCAGCGCCTGTC CGGCGTCGAGTTTGGGCCGATGGATTTTAAAGCCTATGCCGAATC CTTCGGCGCGAAAGGGTTTGCCGTGGAAAGCGCCGAGGCGCTGGA GCCGACCCTGCGCGCGGCGATGGACGTCGACGGCCCGGCGGTAGT GGCCATCCCGGTGGATTATCGCGATAACCCGCTGCTGATGGGCCA GCTGCATCTGAGTCAGATTCTGTAAGTCATCACAATAAGGAAAGA AAAATGAAAAAAGTCGCACTTGTTACCGGCGCCGGCCAGGGGATT GGTAAAGCTATCGCCCTTCGTCTGGTGAAGGATGGATTTGCCGTG GCCATTGCCGATTATAACGACGCCACCGCCAAAGCGGTCGCCTCC GAAATCAACCAGGCCGGCGGCCGCGCCATGGCGGTGAAAGTGGAT GTTTCTGACCGCGACCAGGTATTTGCCGCCGTCGAACAGGCGCGC AAAACGCTGGGCGGCTTCGACGTCATCGTCAACAACGCCGGCGTG GCGCCATCCACGCCGATCGAGTCCATTACCCCGGAGATTGTCGAC AAAGTCTACAACATCAACGTCAAAGGGGTGATCTGGGGCATCCAG GCAGCGGTCGAGGCCTTTAAGAAAGAGGGTCACGGCGGGAAAATC ATCAACGCCTGTTCCCAGGCCGGCCACGTCGGCAACCCGGAGCTG GCGGTATATAGCTCGAGTAAATTCGCGGTACGCGGCTTAACCCAG ACCGCCGCTCGCGACCTCGCGCCGCTGGGCATCACGGTCAACGGC TACTGCCCGGGGATTGTCAAAACGCCGATGTGGGCCGAAATTGAC CGCCAGGTGTCCGAAGCCGCCGGTAAACCGCTGGGCTACGGTACC GCCGAGTTCGCCAAACGCATCACCCTCGGCCGCCTGTCCGAGCCG GAAGATGTCGCCGCCTGCGTCTCCTATCTTGCCAGCCCGGATTCT GATTATATGACCGGTCAGTCATTGCTGATCGACGGCGGCATGGTG TTTAAC 34 CCAGCTGGTGCTCAATGGCTTCGGCGACAGCAGCCACGCCCGGGC TGAAGTCGCCGCGCTGGGCAAGATCCCCGGCTATCACGACGCCGA CCTGCGCGACGTCGGGCAGATCGAGGCGATGATGCGCTATGCCGA AAGCACCTTCGGCGGCGTCGATATCGTGATCAATAACGCCGGCAT CCAGCACGTGGCCCCGGTGGAGCAGTTCCCGGTGGACAAATGGAA CGATATTCTCGCCATCAATCTCTCCAGCGTCTTCCACACCACCCG CCTGGCGCTGCCGGGTATGCGCCAGCGCAACTGGGGGCGCATCAT CAACATTGCCTCAGTGCATGGCCTGGTGGCGTCGAAAGAGAAATC GGCCTACGTCGCCGCCAAGCACGCGGTGGTCGGGCTGACCAAAAC CGTGGCCCTGGAAACCGCGCGCAGCGGTATCACCTGCAACGCCAT CTGCCCTGGCTGGGTGCTAACCCCGCTGGTGCAGCAGCAGATCGA CAAACGCATCGCCGAGGGGGTCGACCCGGAGCAGGCCAGCGCCCA GCTGCTGGCGGAAAAACAGCCCTCCGGGGAGTTTGTCACCCCGCA GCAGCTGGGCGAAATGGCGCTGTTTCTGTGCAGCGATGCCGCCGC CCAGGTGCGCGGCGCCGCATGGAACATGGATGGCGGCTGGGTGGC GCAGTAAGCCGCTGGCGCCGCGAAGA 35 Kp_budRABC_FP1-TAGAGGATCCCCAGCTGGTGCTCAATGGCT TCG 36 Kp_budRABC_RP1-CAAGCCATGTCAGAGCTTTTTTTTATCTTC GCGGCGCCAGCGGC 37 TAAAAAAAAGCTCTGACATGGCTTGCCCCTGCTTTCGCGCAGGGG CTTTTTTTGGTTTGGGTGTAAGTGTAAGCATCCCGGAGAAACGAA GCATCGATATTTGAGGGCTTCTGGCGTTCTCACTTACGCTTCGAC ACGACGTGGGCAATCTGACTGGGATGAAGGTCTGATTTGAGCGAG GAGCGGAAGTTCGGGAACGGGATAGCTCTGACCTGCCACCAGGAT TAGATACAACCGTCAGTTAGTAAGGTCGGTTTGTTTACCTTCACA TTTTCCATTTCGCCACCGTGCTGCAAACTCTGATGGCGTCTGATA ATTCAGTGCTGAATGTGGACGACACTCGTTATAATCCTGCCGCCA GTCATTAATGATTTTCCTTGCGTGAACGATATCGCTGAACCAGTG CTCATTCAGGCATTCATCGCGAAATCGTCCGTTAAAGCTCTCAAT AAATCCGTTCTGCGTTGGCTTGCCCGGCTGGATTAAGCGCAACTC AACACCATGCTCAAAGGCCCATTGATCCAGTGCACGGCAAGTGAA CTCCGGCCCCTGGTCAGTTCTTATCGTCGCCGGATAGCCTCGAAA CAGTGCAATGCTGTCCAGAATACGCGAGACCTGAACGCCTGAAAT CCCAAAGGCAACAGTGACCGTCAGGCATTCCTTTGTGAAATCATC GACGCAGGTAAGACACTTGATCCTGC 38 Kp_budRABC_FP2-GCCGCTGGCGCCGCGAAGATAAAAAAAAGC TCTGACATGGCTTG 39 Kp_budRABC_RP2-GATCGCGGCCGCGCAGGATCAAGTGTCTTA CCTGCG 40 CCAGCTGGTGCTCAATGGCTTCGGCGACAGCAGCCACGCCCGGGC TGAAGTCGCCGCGCTGGGCAAGATCCCCGGCTATCACGACGCCGA CCTGCGCGACGTCGGGCAGATCGAGGCGATGATGCGCTATGCCGA AAGCACCTTCGGCGGCGTCGATATCGTGATCAATAACGCCGGCAT CCAGCACGTGGCCCCGGTGGAGCAGTTCCCGGTGGACAAATGGAA CGATATTCTCGCCATCAATCTCTCCAGCGTCTTCCACACCACCCG CCTGGCGCTGCCGGGTATGCGCCAGCGCAACTGGGGGCGCATCAT CAACATTGCCTCAGTGCATGGCCTGGTGGCGTCGAAAGAGAAATC GGCCTACGTCGCCGCCAAGCACGCGGTGGTCGGGCTGACCAAAAC CGTGGCCCTGGAAACCGCGCGCAGCGGTATCACCTGCAACGCCAT CTGCCCTGGCTGGGTGCTAACCCCGCTGGTGCAGCAGCAGATCGA CAAACGCATCGCCGAGGGGGTCGACCCGGAGCAGGCCAGCGCCCA GCTGCTGGCGGAAAAACAGCCCTCCGGGGAGTTTGTCACCCCGCA GCAGCTGGGCGAAATGGCGCTGTTTCTGTGCAGCGATGCCGCCGC CCAGGTGCGCGGCGCCGCATGGAACATGGATGGCGGCTGGGTGGC GCAGTAAGCCGCTGGCGCCGCGAAGATAAAAAAAAGCTCTGACAT GGCTTGCCCCTGCTTTCGCGCAGGGGCTTTTTTTGGTTTGGGTGT AAGTGTAAGCATCCCGGAGAAACGAAGCATCGATATTTGAGGGCT TCTGGCGTTCTCACTTACGCTTCGACACGACGTGGGCAATCTGAC TGGGATGAAGGTCTGATTTGAGCGAGGAGCGGAAGTTCGGGAACG GGATAGCTCTGACCTGCCACCAGGATTAGATACAACCGTCAGTTA GTAAGGTCGGTTTGTTTACCTTCACATTTTCCATTTCGCCACCGT GCTGCAAACTCTGATGGCGTCTGATAATTCAGTGCTGAATGTGGA CGACACTCGTTATAATCCTGCCGCCAGTCATTAATGATTTTCCTT GCGTGAACGATATCGCTGAACCAGTGCTCATTCAGGCATTCATCG CGAAATCGTCCGTTAAAGCTCTCAATAAATCCGTTCTGCGTTGGC TTGCCCGGCTGGATTAAGCGCAACTCAACACCATGCTCAAAGGCC CATTGATCCAGTGCACGGCAAGTGAACTCCGGCCCCTGGTCAGTT CTTATCGTCGCCGGATAGCCTCGAAACAGTGCAATGCTGTCCAGA ATACGCGAGACCTGAACGCCTGAAATCCCAAAGGCAACAGTGACC GTCAGGCATTCCTTTGTGAAATCATCGACGCAGGTAAGACACTTG ATCCTGC
(64) The genotypes of recombinant strains of Klebsiella pneumoniae constructed for the present invention are as summarized in Table 6.
(65) TABLE-US-00006 TABLE 6 Recombinant strains Description KpldhA Klebsiella pneumoniae GSC123 in which a gene for lactate dehydrogenase (ldhA) is deleted KpldhA pflB Klebsiella pneumoniae GSC123 in which a gene for lactate dehydrogenase (ldhA) and a gene for pyruvate-formate lyase (pflB) are deleted KpldhA pflBbudA Klebsiella pneumoniae GSC123 in which a gene for lactate dehydrogenase (ldhA), a gene for pyruvate-formate lyase (pflB) and a gene for - acetolactate decarboxylase (budA) are deleted KpldhA pflB budC Klebsiella pneumoniae GSC123 in which a gene for lactate dehydrogenase (ldhA), a gene for pyruvate-formate lyase (pflB) and a gene for acetoin reductase (budC) are deleted KpldhA pflB Klebsiella pneumoniae GSC123 in which a gene budRABC for lactate dehydrogenase (ldhA), a gene for pyruvate-formate lyase (pflB) and a gene for 2,3-butanediol operon (budRABC) are deleted
(66) The recombinant strains constructed in Experimental Example 1 were cultured, thereby producing 1,3-propanediol. As a control for comparison, a wild type Klebsiella pneumoniae GSC123 (Kp wt) was used.
(67) 250 ml of a complex medium was inoculated with each recombinant strain, followed by culturing at 37 C. for 16 hours. 3 L of complex medium was inoculated with the resulting culture solution, and subjected to fermentation. The fermentation conditions were as follows: microaerobic conditions (aeration rate of 1 vvm, stirring speed of 200 rpm), 46 g/L of glycerol (500 mM glycerol), pH 7.0, and cultivation temperature of 37 C. While fermenting, ammonia (NH.sub.3) was used in order to adjust pH. Samples were taken while fermenting using the recombinant Klebsiella. The growth rate was determined by measuring OD600 (optical density) of the sampled specimens. The sampled specimens were subjected to centrifugation at 13,000 rpm for 10 minutes, followed by assaying the concentration of metabolites and 1,3-propanediol in the supernatant by high performance liquid chromatography (HPLC).
(68) As a result, the recombinant strain (Kp ldhA) in which ldhA was deleted produced a remarkably reduced amount of lactate which was a major byproduct of the wild type Klebsiella pneumoniae (Kp wt). However, the strain produced an increased amount of other byproducts such as formic acid, 2,3-butanediol, ethanol, acetic acid, and succinic acid, which in turn decreased production concentration and production yield of final 1,3-propanediol. In addition, the recombinant strain (KpldhA pflB) in which both ldhA and NW were deleted at the same time showed greatly reduced byproducts except 2,3-butanediol while improved production concentration and production yield of 1,3-propanediol as compared to the wild type Klebsiella pneumoniae GSC123 (Kp wt) or the ldhA deleted Klebsiella pneumoniae Kp ldhA. However, concentration of 2,3-butanediol was also greatly increased.
(69) As compared to recombinant strains, namely, Kp ldhA pflB budA or Kp ldhA pflB budC in which a part of enzyme family related to 2,3-butanediol synthesis was deleted, the recombinant strain Kp ldhA pflB budRABC in which the entire operon of 2,3-butanediol was deleted exhibited the highest 1,3-propanediol concentration and the lowest byproduct production. In view of production yield and productivity, the recombinant strain Kp ldhA pflB budRABC showed the best results. Meanwhile, the recombinant strain Kp ldhA pflB budA in which budA was deleted in order to reduce accumulation of 2,3-butanediol and the recombinant strain Kp ldhA pflB budRABC in which budRABC were deleted in order to reduce accumulation of 2,3-butanediol were found to be effective. However, the recombinant strain Kp ldhA pflB budC in which budC was deleted was found to have no effect. The recombinant strain Kp ldhA pflB budA in which budA was deleted showed poor fermentation performance, exhibiting residual glycerol after 24 hours of fermentation which was close to the end point of fermentation. The recombinant strain Kp ldhA pflB budRABC in which budRABC was deleted showed fermentation patterns that were similar to those of parent strain Kp ldhA pflB, thereby not producing 2,3-butanediol (Tables 7 and 8,
(70) TABLE-US-00007 TABLE 7 Fermentation products (g/L) 1,3- Formic 2,3- Acetic Succinic Strains propanediol Lactate acid butanediol Ethanol acid acid Kp wt 17.3 11.5 6.1 0.0 4.7 3.8 1.8 Kp ldhA 16.3 0.4 8.4 2.6 6.2 4.6 2.4 Kp ldhA pflB 20.0 0.3 0.0 7.0 1.4 0.4 2.2 Kp ldhA pflB 17.7 0.0 0.7 0.0 1.5 0.2 0.1 budA Kp ldhA pflB 19.4 0.7 0.0 7.9 1.2 0.1 0.9 budC KpldhA pflB 22.1 0.3 0.0 0.0 1.7 0.1 0.0 budRABC
(71) TABLE-US-00008 TABLE 8 Production result of 1,3-propanediol Yield Final concentration Productivity Strains (g/g) (g/L) (g/L/hr) Kp wt 0.36 17.3 1.7 Kp ldhA 0.33 16.3 1.6 Kp ldhA pflB 0.41 20.0 1.4 Kp ldhA pflB budA 0.36 17.7 0.7 Kp ldhA pflB budC 0.38 19.4 0.8 KpldhApflB budRABC 0.47 22.1 1.6
INDUSTRIAL APPLICABILITY
(72) The present invention relates to a recombinant microorganism for producing 1,3-propanediol, wherein a pathway for converting pyruvate into 2,3-butanediol is suppressed in a microorganism having pyruvate and acetyl-CoA biosynthetic pathways. In addition, the present invention relates to a method for producing 1,3-propanediol using the recombinant microorganism.
(73) [Brief Description of the Sequences Provided in the Sequence]
(74) SEQ ID NO: 1 is a nucleotide sequence of ldhA gene. SEQ ID NO: 2 is a homologous region 1 of ldhA gene, and SEQ ID NOs: 3 and 4 are primers for amplification of it. SEQ ID NO: 5 is a homologous region 2 of ldhA gene, and SEQ ID NOs: 6 and 7 are primers for PCR amplification of it. SEQ ID NO: 8 is a DNA fragment in which the homologous regions 1 and 2 of ldhA gene are ligated.
(75) SEQ ID NO: 9 is a nucleotide sequence of pflB gene. SEQ ID NO: 10 is a homologous region 1 of pflB gene, and SEQ ID NOs: 11 and 12 are primers for amplification of it. SEQ ID NO: 13 is a homologous region 2 of pflB gene, and SEQ ID NOs: 14 and 15 are primers for PCR amplification of it. SEQ ID NO: 16 is a DNA fragment in which the homologous regions 1 and 2 of pflB gene are ligated.
(76) SEQ ID NO: 17 is a nucleotide sequence of budA gene. SEQ ID NO: 18 is a homologous region 1 of budA gene, and SEQ ID NOs: 19 and 20 are primers for amplification of it. SEQ ID NO: 21 is a homologous region 2 of budA gene, and SEQ ID NOs: 22 and 23 are primers for PCR amplification of it. SEQ ID NO: 24 is a DNA fragment in which the homologous regions 1 and 2 of budA gene are ligated.
(77) SEQ ID NO: 25 is a nucleotide sequence of budC gene. SEQ ID NO: 26 is a homologous region 1 of budC gene, and SEQ ID NOs: 27 and 28 are primers for amplification of it. SEQ ID NO: 29 is a homologous region 2 of budC gene, and SEQ ID NOs: 30 and 31 are primers for PCR amplification of it. SEQ ID NO: 32 is a DNA fragment in which the homologous regions 1 and 2 of budC gene are ligated.
(78) SEQ ID NO: 33 is a nucleotide sequence of budRABC gene. SEQ ID NO: 34 is a homologous region 1 of budRABC gene, and SEQ ID NOs: 35 and 36 are primers for amplification of it. SEQ ID NO: 37 is a homologous region 2 of budRABC gene, and SEQ ID NOs: 38 and 39 are primers for PCR amplification of it. SEQ ID NO: 40 is a DNA fragment in which the homologous regions 1 and 2 of budRABC gene are ligated.