KR20130128241A - Recombinant microorganism expressing carbonic anhydrase from dunaliella sp and phosphoenolpyruvate carboxylase from p.tricornutum ccmp 637, and method for producing organic acid using the same - Google Patents

Abstract

The present invention relates to recombinant microorganisms which express carbonic anhydrase derived from Dunaliella sp. and phosphoenolpyruvate carboxylase derived from Phaeodactylum tricornutum CCMP 632 and a method for producing organic acids using the same. EscherichiacoliBL21(DE3)::DspGCPtP2 strains according to the present invention have superior productivity of organic acids such as succinic acids and lactic acids and are used in metabolic engineering using microorganisms.

Description

Technical Field [0001] The present invention relates to a recombinant microorganism expressing a phosphoenol skin acid carboxylase derived from Dna nulliella and a Pseudodylum triconitum CCMP637 and a method for producing an organic acid using the recombinant microorganism expressing carbonic anhydrase from Dunaliella sp. And phosphoenolpyruvate carboxylase from P. tricornutum CCMP 637, and method for producing organic acid using the same}

The present invention is directed to a method of producing < RTI ID = 0.0 & sp. ) and Pseudodecyltricornitum ( P. tricornutum CCMP 632) and a method for producing an organic acid using the recombinant microorganism.

The microalgae living in the ocean have a carbon concentration mechanism (CCM) for fixing carbon dioxide for photosynthesis, and physiologically and morphologically evolved to adapt to a wide range of carbon dioxide concentrations.

Until now, studies on the carbonic anhydrase (CA) in the CCM mechanism of marine microalgae have not been well known. In the early stage of research, a unique CA has been reported due to physiological adaptation to high salinity. CA, CA-CA, CA-CA, and CA-CA (Hewett-Emmett and Tashian, 1996) Trip et al., 2001), and it is believed that each group evolved through different routes because there is no similarity in the primary structure of the proteins. CA is located on the membrane and introduces the carbon dioxide present inside the microorganism into the inside of the organism. The carbonic acid produced at this time is converted into bicarbonate ion or carbonate ion by the surrounding environment.

On the other hand, another enzyme, phosphoenolpyruvate carboxylase (PEP carboxylase), is an enzyme that produces intermediates that play a key role in the glycolysis process of microorganisms. Phosphoeolpyruvate (PEP), a substrate for the enzyme, is converted to lactate, formate, acetate and ethanol under anaerobic conditions. Oxaloacetate is formed by pyruvate carboxylase (PYC) together with the above reaction. Phosphoenolactic acid carboxylase produces oxaloacetae, a four carbon material, using water and carbon dioxide as a substrate in the enzyme reaction. The oxalate is converted to a succinic acid salt or the like via a metabolic process.

Carbonic anhydrase, an enzyme that catalyzes the conversion of inorganic carbon, bicarbonate (HCO ₃ ) to CO ₂ , plays an important role in carbon dioxide immobilization. The phosphoenolpyruvate carboxylase promotes the metabolism leading to the conversion of bicarbonate and water molecules to oxaloacetate in the TCA circuit, resulting in the promotion of succinic acid production.

The present inventors are microalgae of the two analytic Ella (Dunaliella sp.) Carbonic anhydrase (carbonic anhydrase) coding ca and payioh deck tilrum tricot nyutum CCMP 632 (Phaeodactylum tricornutum CCMP 632) play the skin acid on phospholipase derived from that of the A recombinant plasmid containing ppc encoding a phosphoenolpyruvate carboxylase was prepared and transformed into Escherichia coli to produce a recombinant microorganism having excellent ability to produce an organic acid, thereby completing the present invention.

It is an object of the present invention to provide a method for the production of a < RTI ID = 0.0 > Ca < / RTI > Plasmid containing the gene and the phosphoenolpyruvate carboxylase of Pseudodylium triconatum CCMP 632, which encodes a phosphoenolpyruvate carboxylase The present invention provides a recombinant microorganism transformed with a plasmid containing the < RTI ID = 0.0 > ppc < / RTI >

It is still another object of the present invention to provide a method for producing an organic acid using the recombinant microorganism.

In order to solve the above-mentioned object, the present invention relates to a method for screening a gene encoding a Ca (II) -containing carbonic anhydrase Gene encoding a phosphoenolpyruvate carboxylase derived from a plasmid and a Pseudodylium triconitum CCMP 632 ppc A recombinant microorganism transformed with a plasmid containing the gene is provided.

The present invention also provides a method for producing an organic acid using the recombinant microorganism.

The recombinant microorganism of the present invention is excellent in the ability to produce organic acids such as succinic acid, lactic acid and the like, and can be usefully used for metabolism using microorganisms.

1 is a ppc Fig. 2 is a diagram showing the sugar-coding process of the gene encoding the phosphoenol skin cancer carboxylase.
Figure 2 is ca Fig. 2 shows the structure of a plasmid containing a gene.
Figure 3 is a ppc Fig. 2 shows the structure of a plasmid containing a gene.
Figure 4 is a ca The nucleotide sequence of the gene is analyzed.
Figure 5 ppc The nucleotide sequence of the gene is analyzed.
FIG. 6 is a cross- This shows the expression of carbonic anhydrase and phosphoenol skin cancer carboxylase in E. coli BL21 (DE3) :: DspGCPtP2 strain.

The present invention is directed to a method of producing < RTI ID = 0.0 & ca ) encoding a carbonic anhydrase derived from E. sp . Plasmid containing the gene, and Pseudodylum triconitum CCMP 632 ( P. tricornutum CCMP 632) encoding a phosphoenolpyruvate carboxylase. ppc A recombinant microorganism transformed with a plasmid containing the gene is provided.

The recombinant microorganism of the present invention expresses a carbonic anhydrase, and the enzyme plays a role of introducing carbon dioxide into an organism. The carbonic acid produced in this case is converted into bicarbonate ion or carbonate ion Can be switched.

In addition, the recombinant microorganism of the present invention expresses a phosphoenol skin acid carboxylase, and the enzyme uses PEP to produce oxaloacetate (4 carbon material) using water and carbon dioxide. The enzymes produce oxalate by fixing carbon dioxide, and the production of oxaloacetate leads to the production of succinic acid.

To prepare the recombinant microorganism of the present invention, Dunaliella < RTI ID = 0.0 > ca ) encoding a carbonic anhydrase derived from E. sp . Genes and Pseudodecyltrimonucleotides CCMP 632 ( P. tricornutum CCMP 632) encoding a phosphoenolpyruvate carboxylase. ppc Gene was amplified through specific primers through PCR. Thereafter, a ca of a carbonic anhydrase derived from dunalella A recombinant plasmid was constructed by using the restriction enzyme ( Sac I, Kpn I) to the pCDF-1b plasmid and named pCDF :: DspC.

It is also possible to use a phosphoenolpyruvate carboxylase encoding Pseudodylum triconitum CCMP 632 derived phosphoenolpyruvate carboxylase ppc Recombinant plasmids were constructed by pET-21a (+) plasmid with restriction enzymes ( Xho I, Eag I) and named pET :: PtP2.

The recombinant plasmids were transformed into E. coli BL21 strain to prepare recombinant microorganisms. The analysis of the nucleotide sequence of the recombinant microorganism is of the two analytic Ella derived ca The gene was confirmed to be a ν-ca gene encoding ν-carbonic anhydrase, and the nucleotide sequence is shown in SEQ ID NO: 1.

It is also possible to use a phosphoenolpyruvate carboxylase encoding Pseudodylum triconitum CCMP 632 derived phosphoenolpyruvate carboxylase The ppc gene was confirmed to be a gene encoding phosphoenolpyruvate carboxylase, and the nucleotide sequence is shown in SEQ ID NO: 2.

The microorganism in the present invention is preferably Escherichia coli, but is not limited thereto.

The recombinant microorganism of the present invention was transformed into Escherichia It was named coli BL21 (DE3) :: DspGCPtP2 and deposited with the KCTC of the Korea Research Institute of Bioscience and Biotechnology (KCTC) on April 27, 2012, and was given the accession number of KCTC 12198BP.

The Escherichia The E. coli BL21 (DE3) :: DspGCPtP2 strain is excellent in the ability to produce organic acids, and thus can be usefully used for metabolic engineering using microorganisms.

The present invention provides a method for producing an organic acid using the recombinant microorganism (Accession No. KCTC 12198BP).

The method for producing organic acid comprises:

1) Escherichia culturing Escherichia coli BL21 (DE3) :: DspGCPtP2 strain to the growth stasis;

2) treating isopropyl thio-beta-D galactoside (ITPG) and glucose under anaerobic conditions with the culture of step 1); And

3) treating the strain of step 2) with carbon dioxide to anaerobically ferment.

The ITPG is a substance for inducing the expression of the transfected gene, and it is possible to treat doses commonly used in the art. The glucose is not limited thereto, but it can treat 5 to 60 g / l, preferably 10 g / l.

The organic acid produced by the recombinant microorganism of the present invention includes succinic acid, lactic acid, formic acid and the like, preferably succinic acid and lactic acid, and more preferably succinic acid.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easy understanding of the present invention, and the present invention is not limited by the examples.

Example  One: ca Gene and ppc  Production of recombinant plasmids containing the nucleotide sequence of the gene

1. Polymerase chain reaction PCR ) Gene sequence amplification

The gene amplification used in the present invention was carried out by polymerase chain reaction (PCR) using the primers shown in Table 1 below, ca Gene sequence and the ppc gene sequence were obtained. For the amplification of the gene sequence, denaturation temperature was 94 ^° C, annealing temperature was 68 ^° C, extension temperature was 72 ^° C, and total 35 reactions were performed. The polymerase used in the reaction was NEB (New England Biolabs Inc.).

gene Expression enzyme Gene source Use plasmid  primer Restriction enzyme ca karbonic anhydrase Dunaliella sp pCDF-1b (F)
5'-GTGGGTACC ATGTTACAGTCGTCATTT CGCCATGC-3 '
(R)
5'-TGGGACCTCTTACTTCTTAGGCCTTGTTGCAAGC-3 ' Sheet I, Kpn I ppc 포소노 로pyruvate carboxylase P. tricornutum CCMP 632 pET-21a (+) (F)
5'-CGGCCGATGATTGACGCCGCCAGCAAGC-3 '
(R)
5'-CCGCTCGAGCGGTTATCCACTGTTTCGCATTCCCT-3 ' Xho I, Eag I

2. Preparation of recombinant plasmids ( pCDF :: DspC  And pET :: PtP2 )

The gene nucleotide fragment and the protein expression vector obtained in Example 1-1 were cut using a restriction enzyme that selectively cleaves the terminal region, and the size was confirmed by electrophoresis. The nucleotides were digested with agarose gel at 50 ^° C, purified, and ligation was performed at 4 ^° C. The ligase used was the NEB product. Through the ligation reaction, two circular plasmids pCDF :: DspC (pCDF-1b and ca Plasmid containing the gene sequence) and pET :: PtP2 (pET-21a (+) and RTI ID = 0.0 > ppc < / RTI > gene sequence). Its structure is shown in Fig. 2 and Fig.

Example  2: Preparation of recombinant E. coli transformed with recombinant plasmid

Before the two recombinant plasmids pCDF :: DspC and pET :: PtP2 prepared in Example 1 were inserted into Escherichia coli BL21 (DE3), which is a heterologous microorganism, the stability of the vector and the efficiency of transformation into a host strain were improved E. coli was used as a competent cell.

Competition cells were prepared by the following procedure. Escherichia coli BL21 (DE3) cultured in liquid LB at 37 ° C was collected at exponential phase, sufficiently cooled at 0 ^° C, and then the supernatant was removed by centrifugation. The cell wall was treated with MgCl ₂ Solution was added and stored at 0 ^° C for 1 hour. The supernatant was removed by centrifugation again and CaCl ₂ The cells were untied using a solution and stored at 0 ^° C for 1 hour.

Divides the competent cell 100 ㎕ prepared as described above was reacted in the above Example 1 into the plasmid prepared in 0 ^o C for 30 min, 42 ^o heat treatment for 1 minute in the hot water tank of the C, and at 0 ^o C 5 The plasmids were transformed into competent cells by performing heat shock transformation in an ice bath for one minute.

The sample subjected to the thermal shock transformation method was incubated at 37 ° C for 1 hour to induce normal growth of the transformed cells. These cells were spread by a predetermined amount on solid LB medium (LB agar plate, tryptone 10 g / l, NaCl 5 g / l, yeast extract 5 g / l, agar 15 g / l) containing antibiotics ampicillin and streptomycin. The colonies were selected and cultured in 5 ml of LB medium containing ampicillin and streptomycin. The plasmid extracted using general plasmid extraction method was subjected to sequencing of ppc of Dunaliella sp. Ca and P. tricornutum CCMP 632. The Ca sequence of Dunaliella sp. Was assigned to SEQ ID NO: 1 and ppc of P. tricornutum CCMP 632 To SEQ ID NO: 2, and the results are shown in FIGS. 4 and 5. FIG. In addition, the recombinant strain Escherichia coli BL21 (DE3) :: DspGCPtP2) in the April 27, 2012, was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center, gene banks (KCTC), was given an accession number of KCTC 12198BP.

As shown in Fig. 4 and Fig. 5, it was confirmed that the ca gene of SEQ ID NO: 1 was a ν-ca gene encoding ν-carbonic anhydrase, and ppc The gene was confirmed to be a gene encoding phosphoenolpyruvate carboxylase.

Experimental Example  1. Identification of recombinant E. coli protein

The recombinant microorganism Escherichia < RTI ID = 0.0 > To confirm protein expression of E. coli BL21 (DE3) :: DspGCPtP2 strain, the strain was added to 50 ml of liquid LB medium containing antibiotics and incubated at 37 ^° C in a shaking incubator at a rate of 200 rpm until the exponential phase. At the exponential phase, isopropyl thio-β-D galactoside (IPTG) was added to express the inserted gene sequence and the protein was incubated at 26 ^° C. for 6 hours to form an easily stereoscopic structure for observation. After centrifugation to obtain proteins from the cultured Escherichia coli, the cells were suspended in PBS buffer. Thereafter, ultrasonic treatment was performed for 2 minutes with AMP at a concentration of 30%, and the cycle of stopping the ultrasonic treatment for 1 minute was repeated 10 times. Thereafter, the soluble supernatant was separated by centrifugation, the remaining debris was suspended in PBS buffer, sonicated for 2 minutes, and sonicated for 1 minute, and the cycle was repeated 5 times. The supernatant and the residue were then electrophoresed on SDS-PAGE. The gel used in SDS-PAGE consisted of 6% resolving and 5% stacking. The SDS-PAGE electrophoresis results are shown in FIG.

As shown in Figure 5, Escherichia It was confirmed that the protein expressed in the E. coli BL21 (DE3) :: DspGCPtP2 strain coincided with the protein size of the carbonic anhydrase and the phosphoenolpyruvate carboxylase, and CA (carbonic anhydrase) was expressed in a water - insoluble form and phosphoenolpyruvate carboxylase was expressed in a water - soluble form.

Experimental Example  2. Escherichia coli BL21 ( DE3 ) :: DspGCPtP2  (Access number: KCTC  12198 BP ) Production of Organic Acids Using

The recombinant microorganism Escherichia < RTI ID = 0.0 > To confirm the production of the organic acid of E. coli BL21 (DE3) :: DspGCPtP2 strain, a phase transition fermentation method was performed. In 200 ml of liquid LB medium in a 500 ml size fermenter, Escherichia < RTI ID = 0.0 > E. coli BL21 (DE3) :: DspGCPtP2 strain was cultured and antibiotic ampicillin and streptomycin were added. Incubation at 37 ^o C, 150 rpm, and MgCO ₃ were added for pH titration. Escherichia coli was aerobically cultured. When the growth of bacteria reached a congested state, a solution of glucose at 10 g / l for IPTG and organic acid production for gene expression was added. Two solutions were added and the incubator was fully anaerobically fermented with carbon dioxide gas. After the carbon source of the culture broth was consumed, 1 ml of the sample was collected and centrifuged and filtered. Thereafter, high performance liquid chromatography was carried out. For high performance liquid chromatographic analysis, 5 mM sulfuric acid solution was used as the mobile phase. Samples were analyzed with a RI detector and the column used was Aminex HPX-87H (Biorad, USA) and placed in a 50 ^o C oven. The results of the analysis are shown in Table 2 below.

Organic acid (g / l) E. coli BL21 E. coli BL21 :: CPtP2 Succinic acid 0.97 3.84 Lactic acid 4.27 5.51

As shown in Table 2, it was confirmed that the production of succinic acid and lactic acid, which are organic acids, in the Escherichia coli BL21 (DE3) :: DspGCPtP2 strain of the present invention was significantly increased as compared with that of ordinary E. coli . More specifically, it was confirmed that succinic acid was increased by about 3.96 times and lactic acid was increased by 1.07 times, and the amount of total organic acid was increased by about 2 times.

Therefore, the recombinant microorganism of the present invention ( Escherichia coli BL21 (DE3) :: DspGCPtP2 strain), it was confirmed that a useful organic acid such as succinic acid, lactic acid and the like can be mass produced.

Korea Biotechnology Research Institute KCTC12198BP 20120427

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Recombinant microorganism expressing carbonic anhydrase from          Dunaliella sp and phosphoenolpyruvate carboxylase from          P.trcornutum CCMP 632, and method for producing organic acid          using the same <130> P1-74 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> it is DNA sequence coding carbonic anhydrase of Dunaliella sp <400> 1 atgttacagt cgtcatttcg ccatgcgggg tggagtttcc ctttccgcca cccccctgca 60 tccggagccc ttgagaagac cctggctggt gtaggctcct tgttccgtgt gctgggctct 120 gctattgatg gctttggagc cacgctgcaa gggcctggag cgttgagaga acaagttcag 180 cccaatctgg cctgggcccc caccaagctg gatgagcgct gcccgccctc gcgcggccag 240 gtggtgaacc tgccatccat ggccgcgatg ccgtctttga agcatgttgt gttgccgctc 300 aagggcgaca acgtctttat cgctcccaat gccaatgtga tgggtgacgt gaagattggc 360 gccaactcct ccatctggta tggagccgta ttgagaggtg acgtgaacag cattgaagta 420 ggcagcaaca ccaacattca ggacaacgcc atcatccacg tggccaagca cagcatcagt 480 ggcgatgcca agcccacaat cattggcaac aacgtgacaa ttgggcatgg agccactgtg 540 catgccgcca ccattgaaga caacgtgctc attgggatgg gggcgacagt gcttgatgga 600 tgcgtggtgg aggctggagc gattgtagca gcaggatcaa tggtaactcc agggaagaga 660 gtgcctgcag ggcaggtctg ggcaggcaac cccgcgcgct acctgaggga tgtggagcca 720 gaggagcacg gctttgtgga gtcaagtgcc tctaattatg ccgagctggc agacttgcat 780 aaattcgaga actcaaagac ctttgaggag ttgagcgcgg agcgagctat tgaggtcgac 840 cgctacgttg ccagtgactc caccaatagc gtgcaccaaa tgtggatctt tgataagcag 900 accttgcttg caacaaggcc taagaagtaa 930 <210> 2 <211> 2634 <212> DNA <213> Artificial Sequence <220> <223> it is DNA sequence coding phosphoenolpyruvate carboxylase of          P.tricornutum CCMP 632 <400> 2 atgattgacg ccgccagcaa gctcaccgcg acggaagccc tgggcgtgac gcgcgtcttt 60 tccatcatgc tcaatctcgt caacgccgcc gaagtccagc accgcaaccg acagattcgg 120 gcacacgagt ccaccaagga cccctccggt ggccctctcc ccaaaacgga agattccatt 180 cgcggaacca tggagacgct gttggaatcg aaacaggcga caccggaaga aatatttgcc 240 cagctgcaga agcaaaaagt ggaaatcgtc ctgacggctc atccgactca agtccagcgc 300 aaatcgcttc tgcgcaagta ccgtcgcgtt tcggagatgc tcgcttattt ggagcgaccc 360 gatttggatg gttttgaaaa gtcgtccgcc caaacgagct tgcaaacaat cttgagcagc 420 atttggggag ctgacgaaat tcgaagacaa aaaccgacac cacaacaaga ggccgcaggg 480 ggtaacgcaa tattggagtc ggttttgtgg gacgcggtgc cagcctatct gcgcaaattg 540 gatcaacagt gccgacttac cctggggcag tcgctgcccg tggacgtatg ccccatcaag 600 tttgcttcct ggatcggtgg ggatcgcgat ggtaacccca acgtgacgcc cgaagttacc 660 cgcgaggttg ttctgcaaca acgattgcgg gctgctcgtt tgcttctcaa ggacatgtac 720 gatttgatct ccgaattggc aatttctagc cgcttttcgc ccgccatgga tgccttggca 780 gattccgtca aggactcgca gcataagcgt gaaaagtacc gtcgtgtgat tggacacttg 840 atcaaacgtc tcgtcaaaac ggcccgtgaa tgtgaattag aattgtcgaa actcaacacc 900 tcagctagta tggtcagtca gactctcgtt gaggaagcag tggatggttg gcaagacgtc 960 gatgctcttg acgatgcgac tgatttgatc aagcctttgc gcataatgta cgattcgttg 1020 gttgaaacgg gcttcggttt ggtggccgac ggtttattgg tcgatatcat tcgtcgattg 1080 tatgtgtttg gtatgtccct cgtgcccttg gatattcgcg aggagagtac caagcacacg 1140 gaagcgttag atgccattac gcgttggttg ggaattggct cctatagtga atggaccgaa 1200 gaggctcgtc tcagctggtt gacttctgag ctttccaaca aacgtccctt gtaccgaatt 1260 cgcgaattgc ccaagctggg tttcaatgac agtgtcttga agacgctcaa cgtattcggc 1320 accatagcta ccctacgacc atcttgtttg ggagcctacg tcattagtca ggcgcagacc 1380 gcaagtgatg tcttggccgt catgcttttg caaaagcagt acggtatgac ggacaagaac 1440 agaaacatga tgcgtgtggt tccgttgttt gagaccttga atgacttgac caacgcgccc 1500 gacaaactcg aacagctctt cagtattccg ctttacgtcg gcgccgtcaa agggaaacag 1560 gaagtaatgg tcgggtatag tgacagtgcc aaggatgccg gacgtctggc tgcctgctgg 1620 gcgcagtaca actcgcaaga acgaatggtg aaggtagcgg cgaagcacaa cattgaattg 1680 actttcttcc acggcaaagg gggtaccgta ggacgtggcg gtaacccatc cgtctatcgt 1740 gccattatga gccatccgcc caataccatt aatggccgtt tccgggtgac ggaacagggt 1800 gaaatgataa cgcaaaactt tggagctccg tccattgctg aacgaacttt ggacatttac 1860 acggctggcg tatgtcgcga agctttttct gagcgcgtgg aaccgtcgca agcatggcgc 1920 gaccagatgc aacggatctc cgatgtgagt tgtgccgagt accgccactt agtccgtgag 1980 gaaccgcggt ttgttcccta ctttcgccag gcgacaccgg agttggaact cggaagtttg 2040 aacataggca gtcgtccggc caaacgtaac ccgaaaggcg gtattgaaag tctccgcgcg 2100 attccgtgga cctttgcttg gacgcagacg cgcacacact tatcggcgtg gctgggagtt 2160 ggcgctggtc tcacaacgac agatcaaagc gaattgaaga cgcttcgagc aatgtacatt 2220 gaatggcctt ggtttcgtga aactattgat ctaattgcca tgattgtatc caagacagac 2280 ttttccatat ccaaaaatta tgacgatcaa ctggtggaaa agaaagaagg tttgttgaag 2340 ctgggagacg aggtcaggga gaaaatggtg caaactcgtc aagctgttct tgatgtgacc 2400 gagtctacgg atgttgctgg ggctcacgtc gcccttatgc gagggtcgtc gaccattcgt 2460 catccatacg tcgatccggt caacgttatt caagccgaat tgctcaagcg attgcgagtc 2520 atggacaaga aaaagtctct gtcggcggat gaaatggaag aacaagaaat tttaaaggat 2580 gccctgatta tcagtatcaa tggcatcgct cagggaatgc gaaacagtgg ataa 2634

Claims (8)

Dunaliella ca ) encoding a carbonic anhydrase derived from E. sp . Recombinant transformed with recombinant plasmid comprising plasmid containing gene and ppc gene encoding phosphoenolpyruvate carboxylase from P. tricornutum CCMP 632 Microorganism (Accession Number: KCTC 12198BP). The method of claim 1, wherein ca encoding the carbonic anhydrase Recombinant microorganism, characterized in that the gene has a nucleotide sequence of SEQ ID NO: 1. The recombinant microorganism according to claim 1, wherein the ppc gene encoding the phosphoenolpiburic acid carboxylase has a nucleotide sequence of SEQ ID NO: 2. The recombinant microorganism of claim 1, wherein the recombinant microorganism has a production capacity of succinic acid and lactic acid. The recombinant microorganism of claim 1, wherein the microorganism is Escherichia coli. 1) aerobic culture of the recombinant microorganism (Accession No .: KCTC 12198BP) of claim 1 until the growth plateau;
2) treating the culture of step 1) with isopropyl thio-β-D galactoside and glucose under anaerobic conditions; And
3) anaerobic fermentation by treating carbon dioxide to the strain of step 2); characterized in that it comprises a. The method of claim 6, wherein the organic acid is at least one selected from the group consisting of succinic acid, lactic acid, and formic acid. The method of claim 6, wherein the microorganism is Escherichia coli.

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