Recombinant microorganism and method for production of formic acid by using same

Abstract

The present invention relates to a recombinant microorganism for producing formic acid, which has a formate dehydrogenase 1 alpha subunit (FDH1α)-encoding endogenous gene deleted therefrom and an FDH1-encoding exogenous gene introduced thereinto, and a method for production of formic acid by using the microorganism.

Claims

1. A recombinant microorganism comprising a genetic modification that increases production of formate, wherein the genetic modification comprises: a deletion of an endogenous gene encoding formate dehydrogenase 1 alpha subunit (FDH1α) and an introduction of an exogenous gene encoding a homologous formate dehydrogenase 1 (FDH1), wherein the recombinant microorganism is modified from a microorganism of the genus Methylobacterium, and the microorganism of the genus Methylobacterium produces formate, wherein the microorganism of the genus Methylobacterium is selected from the group consisting of M. adhaesivum, M. chloromethanicum, M. suomiense, M. platani, M. soli, and M. extorquens.

2. The recombinant microorganism of claim 1, wherein the microorganism of the genus Methylobacterium is Methylobacterium extorquens.

3. The recombinant microorganism of claim 1, wherein the recombinant microorganism is cultured in an electrical or electrochemical system.

4. The recombinant microorganism of claim 3, wherein the electrochemical system is an electrochemical carbon dioxide reduction system.

5. The recombinant microorganism of claim 1, wherein the exogenous gene is introduced by a vector.

6. The recombinant microorganism of claim 5, wherein the vector comprises a PmxaF promoter.

7. The recombinant microorganism of claim 1, wherein an amount of formate produced by the recombinant microorganism is regulated by a methanol concentration.

8. The recombinant microorganism of claim 1, wherein the recombinant microorganism is cultured in an environment in which tungstate is present at a concentration of more than 30 μM and less than 120 μM.

9. The recombinant microorganism of claim 1, wherein the recombinant microorganism is cultured in an environment in which methyl viologen, ethyl viologen, or a combination thereof is present.

10. The recombinant microorganism of claim 1, wherein the recombinant microorganism is Accession No. KCTC 13388BP.

11. A method of producing formate, the method comprising culturing the recombinant microorganism of claim 1 in a medium.

12. The method of claim 11, further comprising saturating the medium with carbon dioxide; and electrically or electrochemically treating the medium.

13. The method of claim 11, wherein the medium further comprises methanol.

14. The method of claim 11, wherein the medium further comprises tungsten.

15. The method of claim 11, wherein the medium further comprises an electron mediator that transfers electrons to FDH1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates a method of preparing mutant strains of M. extorquens AM1;

(2) FIG. 2 is a graph showing formate production of various recombinant strains in an electrochemical reaction system (reaction conditions: 0.6 g wet-cell, 10 mM MV, pH 6.0; CO.sub.2 gas purging, 99.999%, rate: 1 mL/s);

(3) FIG. 3A shows a protein crude extract of a recombinant microorganism F1A-P1 loaded by SDS-PAGE (10%) and then stained with Coomassie blue (here, M indicates markers), and FIG. 3B shows Western blotting of FDH1α and FDH1β among proteins of the recombinant microorganism F1A-P1;

(4) FIG. 4A shows Western blotting of FDH1α and FDH1β among proteins of the recombinant microorganism F1A-P1 at various concentrations of MeOH, and FIG. 4B is a graph showing formate production of the recombinant microorganism F1A-P1 at various concentrations of MeOH (reaction conditions: 0.6 g wet-cell, 10 mM MV, pH 6.0; CO.sub.2 gas purging, 99.999%, rate: 1 mL/s);

(5) FIG. 5A shows Western blotting of FDH1α and FDH1β among proteins of the recombinant microorganism F1A-P1 at various concentrations of tungstate, and FIG. 5B is a graph showing formate production of the recombinant microorganism F1A-P1 at various concentrations of tungstate (reaction conditions: 0.6 g wet-cell, 10 mM MV, 0.5% (v/v) MeOH, pH 6.0; CO.sub.2 gas purging, 99.999%, rate: 1 mL/s);

(6) FIG. 6 is a graph showing formate production of the wild-type and the recombinant microorganism F1A-P1 under optimized conditions (reaction conditions: 0.6 g wet-cell, 10 mM MV, pH 6.0; CO.sub.2 gas purging, 99.999%, rate: 1 mL/s);

(7) FIG. 7 shows a comparison of formate production of the recombinant microorganism F1A-P1 under culture conditions containing various electron mediators (MV; methyl viologen, EV; ethyl viologen, FMN; flavin mononucleotide, and NR; neural red);

(8) FIG. 8 illustrates an electrochemical carbon dioxide reduction system;

(9) FIG. 9 shows formate concentrations for comparing formate productions of wild-type, F1A-P1, and wild-type+FDH1 under various conditions; and

(10) FIG. 10 shows formate productivity of various microorganisms of the genus Methylobacterium under electrochemical conditions.

MODE OF DISCLOSURE

(11) Hereinafter, the present disclosure will be described in more detail.

(12) Unless defined otherwise, all technical terms used herein have the same meanings as those generally understood by one of ordinary skill in the art to which the present disclosure belongs. Further, although methods or samples are described herein, those similar or equivalent thereto are also incorporated in the scope of the present disclosure. The numerical values described herein are considered to include the meaning of “about”, unless otherwise specified. The contents of all the publications disclosed as references herein are incorporated in the present disclosure.

(13) Example 1: Preparation of Recombinant Microorganism

(14) To prepare a recombinant microorganism, Methylobacterium extorquens AM1 (ATCC 14781, GenBank accession No. CP001510.1) was cloned and modified, as illustrated in FIG. 1. Methylobacterium extorquens AM1 is known to contain three formate dehydrogenase-coding genes (fdh1, fdh2, fdh3). Among the three formate dehydrogenase-coding genes, the fdh1 gene for FDH1 (GenBank accession No. ACS42636.1(α-subunit), ACS42635.1(β-subunit)) was selected to prepare the recombinant microorganism of Methylobacterium extorquens AM1, because it was reported to play an important role during whole-cell oxidation of formate [17].

(15) For all the following cloning, one-step sequence and ligation-independent cloning (SLIC) was applied [25]. SLIC uses T4 DNA polymerase as exonuclease. This vector was linearized and amplified by restriction enzymes and a DNA amplifier. NEB 2.1 buffer (B7202S, BioLabs) and T4 polymerase were then added, and this mixture was incubated at room temperature for 2.5 min, then immediately incubated on ice for 10 min. Thereafter, 1 μl of the mixture was added to 100 μl of competent E. coli DH5α cells (RBC), and the DH5α cells were incubated on ice for 20 min. Then, 950 μl of LB medium was added and incubated at 37° C. for 16 hours.

(16) A preparation procedure of the recombinant microorganism is as follows.

(17) In detail, gene-knockout was performed according to a description of a paper [24]. First, according to the gene to be deleted, DNA located both upstream and downstream of FDH1α, and/or FDH1β gene (GenBank accession No. ACS42636.1(α-subunit), ACS42635.1(β-subunit)) of Methylobacterium extorquens AM1 was amplified. Primers used in the cloning are as in Table 1 below.

(18) TABLE-US-00001 TABLE 1 Primer sequence fdh1α knockout upstream F 5′- gccgccatatgcatccatggtaccCCGGCGGG TCGATGCGGTTGGAAA-3′ fdh1α knockout upstream R 5′- cacctgacgtctagatctg aattcTGGCCCGCG ACCTCACCGCGAACTACTT-3′ fdh1α knockout downstream F 5′- tggtcggctggatcctctagtgagctcTCTACGC CGAGGGCGTGAACGGACC-3′ fdh1α knockout downstream R 5′- gatccagcttatcgataccgcgggcccGAGGTG CCGATAGGCGTGGCGCGA-3′ fdh1β knockout upstream F 5′- gccgccatatgcatccatggtaccAATCTCTGT GTCCGCGCCT-3′ fdh1β knockout upstream R 5′- cacctgacgtctagatctgaattcGCTTCACCG CGTTCTTGAGGAA-3′ fdh1β knockout downstream F 5′- tggtcggctggatcctctagtgagctcGGCAGAG GTCTCGCCGTTGT-3′ fdh1β knockout downstream R gatccagcttatcgataccgcgggcccGACGCG ACCTGTGTTCCAACTAA-3′

(19) The amplified DNA was inserted to both sides of the loxP and kanamycin genes of pCM184 (Addgene plasmid 46012) and cloned. Methylobacterium extorquens AM1 was transformed with the cloned pCM184. When Methylobacterium extorquens AM1 is transformed with pCM184, allelic exchange occurs and Methylobacterium extorquens AM1 acquires loxP and kanamycin genes but loses a partial gene sequence of FDH1. The Methylobacterium extorquens AM1 was transformed with pCM157 (Addgene plasmid 45863), and the kanamycin gene between the IoxP sites was extracted by site-specific recombination using the cre recombinase expressed from pCM157, to produce a knockout microorganism. Thereafter, the recombinant plasmid was expressed in the knockout microorganism, as needed. In detail, the specific gene-knockout Methylobacterium extorquens AM1 was transformed with pCM110 containing a gene encoding FDH1 or a gene encoding FDH1a to recover expression of FDH1 or FDH1a.

(20) The following Table 2 shows bacterial strains and plasmids for knockout or for recombinant expression.

(21) TABLE-US-00002 TABLE 2 Deleted Recombinant Selectable Strain gene Knockout plasmid plasmid antibiotic Wild- — — — Rif type F1A Δfdh1α pCM184(Dfdh1α) — Rif, Kan F1A-P1 Δfdh1α pCM184(Δfdh1α) pCM110(fdh1) Rif, Tet pCM157(cre) F1AB- Δfdh1αβ pCM184(Δfdh1α) pCM110(fdh1α) Rif, Kan, P1B pCM157(cre) Tet pCM184(Δfdh1β)

(22) Among the prepared microorganisms, F1A-P1 was deposited under the terms of the Budapest Treaty at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, 181, Ipsin-gil, Jeongeup-si, Jeolllabuk-do 56212, Republic of Korea, on Nov. 10, 2017, with Accession No. KCTC 13388BP. A basic culture medium for the microorganisms included 16 g/L of succinate as a carbon source and a minimal salt medium (1.62 g/L NH.sub.4Cl, 0.2 g/L MgSO.sub.4, 2.21 g/L K.sub.2HPO.sub.4, and 1.25 g/L NaH.sub.2PO.sub.4.Math.2H.sub.2O). As a selectable antibiotic to select the recombinant microorganisms, 50 pg/mL rifamycin (Rif), 50 pg/mL kanamycin (Kan), or 10 μg/mL tetracycline (Tet) was used. Each of the microorganisms was cultured in a 1 L Erlenmeyer shake flask with 200 mL volume at 26° C. and 200 rpm.

(23) Example 2: Identification of Essential Enzymes for Production of formate from Carbon Dioxide

(24) Amounts of formate produced by the recombinant microorganisms prepared in Example 1 were measured under various conditions and compared.

(25) As a formate production condition, an electrochemical carbon dioxide reduction system was used according to a previous paper [16]. The system includes a copper plate (2 cm×1.5 cm), a reference electrode (Ag/AgCl), and a platinum wire as an anode. In the system, the platinum wire generates both electrons and cations (e.g., protons) in a 1 mM sulfate aqueous solution (initial volume: 10 mL), and the generated cations pass through a proton-exchange membrane (Nafion®, 0.005-inch thickness, 30 cm×30 cm, Sigma-Aldrich, USA) to the cathode during carbon dioxide reduction reactions. The cathode section includes 0.6 g of wet-cell, 200 mM potassium phosphate buffer (pH 6.0), and 10 mM methyl viologen (MV) (initial volume: 10 mL), and worked to reduce carbon dioxide to formate by using electrons and cations supplied in the aqueous solution. For reduction reaction, the cathode section solution containing the microorganism was saturated with high purity carbon dioxide gas (99.999%, purging rate: 1 mL/s) and stirred at 300 rpm and room temperature. When the Ag/AgCl electrode (MF-2079, BASi) was used as a reference electrode, the electric potential (−0.75 V) of redox was constantly controlled by a potentiometer (MultiEnStat3, PalmSens, Netherlands), and the microorganism was cultured for an indicated time. Thereafter, the concentration of formate produced by whole-cell catalysis reaction was analyzed with HPLC. HPLC analysis was performed at 30° C. using a refractive index detector (RID) with an Aminex HPX 87-H Ion Exclusion Column (300 mm×7.8 mm, Bio-Rad) (mobile phase: 5 mM sulfuric acid, flow rate: 0.6 mL/min).

(26) Results are shown in FIG. 2. As shown in FIG. 2, the wild-type and the recombinant microorganism F1A-P1 were able to produce formate from carbon dioxide in the electrochemical reduction system, but neither F1A which is a FDH1α knockout mutant nor F1AB-P1B which is an FDH1β knockout mutant produced any detectible level of formate. These results support that FDH1 is the key enzyme responsible for the conversion of carbon dioxide to formate, and both FDH1α and β are simultaneously required for FDH1 to function properly.

(27) Further, formate productivity of F1A-P1 was 0.98 mM/hr/g-wet cell, and formate productivity of the wild-type was 0.68 mM/hr/g-wet cell, indicating that formate productivity of F1A-P1 is higher than that of the wild-type. The F1A-P1 strain is homologously expressed by a plasmid pCM10(fdh1), which contains PmxaF as a strong inducible promoter. This promoter is able to significantly increase the expression of FDH1 because it has higher inducibility than other promoters [20]. Based on this result, it is supposed that FDH1 expression in cells may directly affect formate production.

(28) Further, fdh1 was transformed into the wild-type and overexpressed therein, as in Example 1, and then compared with F1A-P1. Surprisingly, when fdh1 was simply overexpressed (MeAM1(WT)+pCM110), it did not produce formate like F1A-P1 (FIG. 9).

(29) Further, when the same electrochemical carbon dioxide reduction system was applied to various microorganisms of the genus Methylobacterium, many microorganisms were found to produce formate (FIG. 10).

(30) Example 3: Confirmation of FDH1 Expression Level

(31) To analyze an FDH1 expression level of F1A-P1, polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed. The target bands for FDH1α and FDH1β were difficult to distinguish by Coomassie blue staining due to their relatively weak expression in SDS-PAGE, despite molecular weight estimates of 108 kDa and 62 kDa, respectively (FIG. 3A). For this reason, Western blotting was further performed (FIG. 3B).

(32) In detail, Western blotting was performed as follows. Microorganisms were lysed with a urea buffer (6 M urea, 200 mM NaCl, 20 mM Tris, pH 8.0), and an extract thereof was separated on SDS-PAGE (10% Tris/glycine). Thereafter, the resultant was transferred to a PVDF membrane (Cat. No. KDM20, 10 cm×10 cm, KOMA BIOTECH) through a semi-dry transfer (AE-8130, ATTA) with a transfer buffer (24.9 mM Tris, 2.5 M methanol, 191, 8 mM glycine, pH 8.4). Thereafter, the membrane was put in a blocking buffer (PBST; 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 1% (w/v) Tween 20) (2% (w/v) skim milk) and was incubated with gently shaking for 1 hour. The membrane was washed with a PBST buffer four times for 20 min and then transferred to a blocking buffer mixed with a primary antibody, and incubated with gently shaking for 1 hour. Next, the membrane was washed with a PBST buffer four times for 20 min, and then transferred to a blocking buffer mixed with a secondary antibody, and incubated with gently shaking for 1 hour. Finally, the membrane was washed with a BST buffer four times for 20 min, and then stained with a BCIP/NBT liquid substrate solution (B1911, SIGMA). For FDH1α, the primary antibody was Anti-6x His tag antibody (ab18184, ABCAM) (1:1000 dilution), and the secondary antibody was rabbit anti-mouse antibody (ab6729, ABCAM) (1:2000 dilution). For FDH1β, a customized primary antibody was used (ABFRONTIER) (1:1000 dilution), and the secondary antibody was goat anti-rabbit antibody (ab6722, ABCAM) (1:2000 dilution).

(33) As a result, expression of FDH1α appeared decreased after incubation for 41 hours, even though the recovered expression level was observed repeatedly after incubation of F1A-P1 under basic culture conditions for 48 hours. These observations imply that a substantial proportion of homologously expressed recombinant FDH1 may be degraded through endogenous metabolism [21].

(34) Example 4: Optimized Culture Conditions for Recombinant Microorganism

(35) To obtain optimal conditions for producing formate from the recombinant microorganism, optimal culture conditions for recombinant microorganism were explored in the electrochemical carbon dioxide reduction system.

(36) Microorganisms were cultured at 26° C. in a 200-rpm shaking incubator, and dissolved oxygen was maintained at about 1 mg/L. A culture medium for the microorganism was the basic culture medium used in Example 1, which was supplemented with trace elements (15 mg/L Na.sub.2EDTA.sub.2HO, 4.5 mg/L ZnSO.sub.4.7H.sub.2O, 0.3 mg/L CoCl.sub.2.6H.sub.2O, 1 mg/L MnCl.sub.24H.sub.2O, 1 mg/L H.sub.3BO.sub.3, 2.5 mg/L CaCl.sub.2, 0.4 mg/L Na.sub.2MoO.sub.4.2H.sub.2O, 3 mg/L FeSO.sub.4.7H.sub.2O, and 0.3 mg/L CuSO.sub.4.5H.sub.2O) and sodium tungstate, as needed. When methanol is added as an inducer, the culture medium was incubated for 19 hours, and then methanol was added thereto at indicated concentrations. Methanol may be used as an expression inducer and a carbon source for the recombinant microorganism F1A-P1, since expression of FDH1 is controlled by the methanol-inducible promoter PmxaF [20].

(37) As a result, it was observed that the methanol concentration in the medium affects FDH1 expression in F1A-P1, especially, after 48-hr incubation, the higher methanol concentration produces greater FDH1 expression in F1A-P1 (FIG. 4A). As predicted, the recombinant microorganism F1A-P1 cultured in the medium with methanol at an initial concentration of 2.0 v/v % based on the volume of the medium showed 2.11 mM-formate/hr/g-wet cell as the highest formate production rate in the electrochemical carbon dioxide reduction system (FIG. 4B).

(38) It was also observed that tungstate concentrations affect FDH1 expression in F1A-P1. According to FIG. 5, F1A-P1 cultured in 2x tungstate (60 μM) resulted in increased FDH1 expression and the highest formate productivity (FIG. 5B). However, F1A-P1 cultured in 4x tungstate (120 μM) showed no increase in the MeFDH1 expression (FIG. 5A). Interestingly, the optimum concentration of tungstate seems to repress FDH1 degradation (FIG. 5A), which implies that when tungstate is deficient, FDH1 apo-enzyme may be more vulnerable to endogenous degradation.

(39) Various combinations of methanol and tungstate conditions were used to compare formate productivity between the wild-type and F1A-P1. As a result, F1A-P1 produced over 30 mM of formate from carbon dioxide within 24 hours. This was three times greater than the production of the wild-type cultured at optimal methanol and tungstate concentrations (FIG. 6). It was also observed that optimal conditions did not greatly affect formate productivity of the wild-type Methylobacterium extorquens AM1. Furthermore, even though FDH1 was simply overexpressed in the wild-type, which was cultured in the optimal conditions of F1A-P1 (MeOH 2.0 v/v %, W(tungstate) 60 μM), the wild-type did not achieve the formate productivity of F1A-P1 (FIG. 9). This result demonstrates that although a genetic material including other promoters of the gene encoding FDH1 is used in the wild-type, it is difficult to obtain the same effect as in F1A-P1. Consequently, a promoter such as PmxaF was one of core factors for regulation of formate production of the recombinant microorganism F1A-P1 and for homologous expression of FDH1.

(40) An artificial electron mediator is suitable for electron transfer from the copper plate cathode to FDH1 in the electrochemical carbon dioxide reduction system. Therefore, to determine whether F1A-P1 prefers a particular electron mediator, formate production was measured in environments in which many different electron mediators are present. As a result, it was confirmed that F1A-P1 could produce formate from carbon dioxide only when methyl viologen (MV) and ethyl viologen (EV) were employed as electron mediator (FIG. 7).

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(66) Depository institution: Korea Research Institute of Bioscience and Biotechnology

(67) Accession No: KCTC13388BP

(68) Date of deposit: 20171110