KR20150125631A - Thermococcus mutant having improved hydrogen production from formate and methods of hydrogen production by using thereof - Google Patents

Abstract

The present invention relates to a Thermococcus onnurineus NA1 mutant with enhanced capability of producing hydrogen from formate, and to a hydrogen production method using the same. The Thermococcus onnurineus NA1 mutated by the present invention shows more increased capability of producing hydrogen in a formate-containing culture medium and increased growth rate compared to a wild type, thereby making it possible to produce hydrogen with high efficiency from formic acids, if using a strain according to the present invention.

Description

TECHNICAL FIELD The present invention relates to a thermococase mutant having increased hydrogen production ability from a formate and a hydrogen production method using the same.

The present invention relates to a thermococase mutant having increased hydrogen production ability from a formate and a method for producing hydrogen using the same.

Currently, the use of hydrogen in industry is increasing over the years, and the use of hydrogen as a clean energy source for fuel cell vehicles and hydrogen power plants is gradually becoming commercialized, and hydrogen supply is expected to increase exponentially. As the value of hydrogen as clean energy is increased and a method of supplying a large amount of hydrogen constantly is considered, researches on a method of producing hydrogen are actively carried out.

Hydrogen energy has been attracting much attention as a substitute for fossil energy in the future because it does not emit substances that may harm the environment such as carbon dioxide, NOx, SOx and the like, even though the calorific value per weight is three times higher than that of petroleum.

Hydrogen production methods conventionally used include electrolysis of water, thermal cracking of natural gas or naphtha, or steam reforming. However, these methods have a problem of making high temperature and high pressure conditions by using fossil fuel again and generate a mixed gas including carbon monoxide, which causes a difficult problem to remove carbon monoxide from such gas.

 On the other hand, the biological hydrogen production method using microorganisms does not require the addition of extra energy to make high-temperature and high-pressure conditions, and does not include carbon monoxide in the generated gas. These biological hydrogen production methods can be roughly divided into the use of photosynthetic microorganisms and the use of non-photosynthetic microorganisms (mainly anaerobic microorganisms). An example belonging to the former is Korean Patent No. 10-0680624 entitled " Hydrogen Production Method Using Photosynthetic Bacterium Staphylococcus sp. Rhododes Strain with High Hydrogen Production Capability at High Salt Concentration ".

 However, the technology of high concentration culture of photosynthetic bacteria using light as an energy source has not been sufficiently developed yet, and conventional photosynthetic bacteria have a disadvantage that substrate inhibition is severe when a high partial pressure substrate is present. In addition, they have a problem that the hydrogen generating ability can be maintained only when light is present.

Therefore, attempts have been made to produce hydrogen using microorganisms capable of producing hydrogen using organic carbon. For example, Korea Patent No. 10-0315663 entitled " Korean Patent No. 10-0315662, " Rhodopseudomonas palustris P4 and hydrogen production by this method ", and the like.

In Korean Patent Application No. 10-2010-7013071 by the present inventors, FDH2-MFH2-MNH2 hydrogenase clusters have been disclosed and it has been reported that fdh2-mfh2-mnh2 is important for hydrogen production using formic acid. F420 Hydrogenase

Korean Patent Application No. 10-2011-0021390 by the present inventors discloses a method for producing hydrogen gas using a strain of the genus Thermocucus.

However, the role of F420-reduced hydrogenase in the formation of hydrogen in formic acid has not been established. The inventors of the present invention focused on the fact that F420-reducing hydrogenase (frh) is present just before the fdh2-mfh2-mnh2 cluster producing hydrogen using formic acid. Thus, F420-reducing hydrogenase (F420-reducing hydrogenase , frh), and found that the hydrogen production was increased in the strain of Thermococosus, and the present invention was completed.

An object of the present invention is to provide a strain having increased hydrogen production capacity.

It is still another object of the present invention to provide a method for efficiently producing hydrogen using the strain.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

For this purpose, a first embodiment of the present invention is a method for the treatment of an over-expressed F420-Reduced Hydrogenase in Thermococcus spp . Thereby providing a mutant strain. But are not limited to, the Thermococcus spp . Is Thermococcus onnurineus , and more preferably the microorganism deposit number KCTC 12356BP. It is a mutant strain to be gong.

The implementation of the second of the present invention Thermococcus spp . Mutant to increase the expression rate of the F420-reductive hydrogenase; And culturing the strain in a medium containing formic acid. It is now limiting the Ioannina Thermococcus spp . Thermococcus onnurineus , and even more preferably the microorganism deposit number KCTC 12356BP.

The increase in the expression ratio of the F420-reductive hydrogenase may be achieved by increasing the number of copies of the F420-reductase gene, increasing the transcription rate, increasing the translation rate, increasing the stability of the F420-reductase, have. As a method for increasing the number of gene copies, increasing the transcription rate, increasing the translation rate, or increasing the stability of the enzyme, well-known techniques can be used. The term "expression" refers to the transcription and translation of a gene sequence that results in the production of the corresponding protein, gene product. In a preferred aspect of the present invention, the gene encoding the F420-Reduced Hydrogenase is over-expressed by a microorganism. The terms "increased expression "," enhanced expression ", or "overexpression" are used interchangeably herein and have a similar meaning, that is, transcription and translation of a gene relative to a non-recombinant microorganism is increased, It is said to cause.

To increase the expression of genes, experts in the art know of other ways to manipulate gene expression. In particular, the gene may be expressed using a promoter having a different strength that can be induced. Such promoters may be homologous or heterologous. Those skilled in the art will know which promoter is most convenient, for example the promoter of glutamate dehydrogenase (Pgdh), the promoter Ptrc, Ptac, Plac or the lambda promoter cI.

In one embodiment of the invention, the gene may be expressed by a plasmid or vector introduced into the microorganism. The microorganism is then referred to as a "host microorganism" and may be an exogenous or heterologous gene or an extra duplicate of its own gene

Refers to a microorganism capable of accepting binding and capable of expressing such a gene to produce an active protein product.

The term "transformation" refers to the introduction of a new gene or an extra copy of an existing gene into the host organism.

The term "transformation vector" refers to any carrier used to introduce polynucleotides into a host organism. Such carriers may be, for example, plasmids, phage or other components known to those skilled in the art, depending on the organism used. The transformation vector may generally contain, in addition to the polynucleotide or expression cassette, other components that facilitate transformation of a particular host cell. The expression vector comprises a cassette and an expression cassette that allows for proper expression of the gene delivered by the additional components enabling replication of the vector into the host organism. The expression vector may be present as a single replicate or multiple replicates in the host organism. Those skilled in the art know of other types of plasmids whose replication origins and thus the number of intracellular copies are different.

Another means of achieving overexpression of the gene is to improve the expression or regulation of the component that stabilizes the corresponding messenger RNA if translation of the mRNA is optimized (Carrier et al. Biotechnol Bioeng. 59: 666- 72, 1998]) to increase the amount of available enzyme.

The mutant strain according to the present invention has a growth rate about 31% faster than that of the conventional thermococorticus strain, and the hydrogen production rate per unit time is increased by about 20%. Unlike the conventional chemical production method, the hydrogen production method using the mutant strain of the present invention does not require high temperature and high pressure conditions, and can generate hydrogen at room temperature and normal pressure, and does not generate harmful by-products. In addition, even when compared with conventional technologies for producing hydrogen using microorganisms, high purity hydrogen can be produced with high efficiency, and hydrogen can be produced even under high temperature conditions.

Figure 1a shows a comparative analysis of the gene structure of the frh-fdh2-mfh2-mnh2 cluster of Thermococcus onnurineus NA1.
FIG. 1B is a schematic diagram showing a production strategy of a mutant strain. _{Pgdh hmg Pfu} cassette was inserted into the alpha subunit gene (Ton_1559) of F420-reducing hydrogenase (F420-reducing hydrogenase, frh) through gene recombination. The location of the primers used for confirmation is indicated by black arrows below the corresponding genes.
Figure 2 shows Western blot analysis of F420-reducing hydrogenase (frh) and protein levels. SDS-PAGE stained with Coomassie Brilliant Blue R-250 was shown under Western blot.
FIG. 3 shows changes in hydrogen production of wild type and mutant in CSTR fermentation using formate as a substrate. (a), growth curve; (b), the cumulative hydrogen production curve.
Figure 4 shows kinetic analysis of hydrogen production of wild type and mutant in CSTR fermentation using formate as substrate.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Methods and Materials

1) Strain and medium

As a general culture condition, yeast extract (4), NaCl (35), KCl (0.7), MgSO ₄ in g / l in 80 anaerobic environments (3.9), CaCl ₂ .H ₂ O (0.4), NH ₄ Cl (0.3), Na ₂ HPO ₄ (0.15), NaSiO ₃ (0.03), NaHCO ₃ (Modified-MI; MMI) consisting of cysteine · HCl (0.5) and sodium bicarbonate (0.5 mM) was used. After sterilization, a 1.0 ml trace element mixture and 1 ml / l Fe-EDTA, and 1 ml / l Balch's vitamin solution were added to the medium. The initial pH of the medium was set at 6.5 at atmospheric pressure.

2) Cell growth and mutagenesis

An anaerobic chamber (Coy) was used for cell inoculation. Cells were cultured in MM1-sodium formate (MM1-sodium formate, sodium fomate) medium. Cell growth was assessed using a DC protein assay kit (DCW) or a UV-vis spectrometer (DCW) according to the assumption that the amount of cellular protein in the dry cell weight (DCW) is 50% (Kengen & Stams, Eppendorf) was measured by measuring the amount of protein in 1 ml of the cell culture at an optical density (OD600) at 600 nm. One unit of OD600 corresponds to 0.361 g-DCW / L. Transformation and disruption of Thermococcus was carried out according to kodakarensis (Matsumi et al., 2007). Briefly, approximately 1 kb DNA region with the target gene linked is inserted on both sides of the _{Pgdh hmg Pfu} cassette cloned into the pUC118 vector. Cells grown in the ASW-YT medium were transformed with these vectors (2 to 6 μg) and cultured with simvastin as the selective pressure. Mutant candidates were identified by PCR amplification. The developed strain was deposited with KCTC 12356BP at the Korea Microorganism Resource Center on January 23, 2013.

3) Gas analysis

Gas composition analysis was performed using a YL6000 GC gas chromatograph (YL6000 GC gas chromatograph) equipped with a thermal conductivity detector and a flame ionization detector with a Molesieve 5A column (Supelco, Bellefont, PA) and a Porapak N column (Supelco) And YL Instrument Co.). Argon was used as a gas carrier. A gas calibration standard (Supleco) containing 1% (w / w) of each component (CO, CO ₂ , H ₂ , CH ₄ and O ₂ ) in nitrogen was used to quantify the hydrogen gas.

4) Western blotting

Antibodies were obtained from overexpressed protein (frh α subunit) in E. coli BL21 and purified by Ni-NTA column. Exponentially growing cells in MM1-formate were harvested by centrifugation and sonicated. Cell debris was removed by centrifugation, and the protein concentration of the crude extract was quantitated with a Bio-Rad protein assay solution. A 5 ㎍ crude extract of each strain was redissolved in 10% SDS-PAGE and transferred to a PVDF membrane (Trans-Blot Turbo ^TM transfer pak) with Trans-BlotTurbo ^™ . The membranes were soaked in Tris-buffered saline buffer containing 0.1% Triton X-100 (TBST) supplemented with 0.5% BSA. The membrane was incubated with TBS-T buffer solution after the antibody diluted 1: 5000 was added. Horse raddish peroxidase-conjugated anti-rabbit antibody (Ab Frontier) was used as the secondary antibody and Immun-Star ^TM A signal generated by a fluorescent chemical HRP kit (Bio-Rad) ChemiDoc ^TM MP imaging system (Bio-Rad).

5) Kinetic analysis of hydrogen production in wild type and mutant strains

For kinetic analysis of hydrogen production, a continuous stirred tank reactor (CSTR) with an autoclave mode at 80 DEG C and a volume of 2 l-, 3 l of fine sparger (hole diameter of 5 diameters) ). The stirring speed was 150 rpm and the pH was adjusted to 6.2 +/- 0.1 using 4M formic acid containing 3.5% NaCl. The seed culture was cultured at 80 캜 until it became an exponential growth period. 5 ml of the seed culture was inoculated into the CSTR using a 10 ml hypodermic syringe. MM1 medium containing 400 mM sodium formate was used. The hydrogen gas was measured using a gas chromatograph YL6000 GC equipped with a TCD detector on a Molesieve 5A column (Supelco, Bellefont, PA) and a Porapak N column (Supelco). Argon was used as a gas carrier. The oven temperature was 40. Ten gas samples for analysis were taken out through a gas-tight syringe through the butyl rubber of the culture bottle. The measurement of the detected hydrogen gas was calculated by comparing the peak area with the calibration curve performed by regression analysis with a standard gas containing 40% hydrogen in nitrogen.

< Example 1 > Thermococcus onnurineus  Increased Expression of F420-Reduced Hydrogenase of NA1

We found that the ratio of hydrogenase and related proteins in the genome of T. onnurineus NA1 was high (5.5%), indicating that the oxidation and reduction reactions involving CO dehydrogenase and formate dehydrogenase Indicating an increase in the conservation or recycling of reducing power associated with enzymes. Among them, the F420-reducing hydrogenase (frh) of T. onnurineus NA1 was obtained from Vignais et al. (2000), Enzymes of hydrogen metabolism (2000), which belongs to group 3 according to the classification system of Enzymes, PJ, Van den Ban, EC, Wassink, H., Haaker, H., de Castro, B., Robb, FT and Hagen in Pyrococcus furiosus Eur. J. Biochem. 267, 6541-6551).

As shown in FIG. 1A, the F420-reducing hydrogenase (TON_1559-1561) containing α / β / γ subunits is located in front of fdh2-mfh2-mnh2 to make one cluster. The α / β / γ subunit of the F420-reductase has a specific primary sequence, which is the Pyrococcus yayanosii CH1 coenzyme F420-reducing hydrogenase (YP_004624049) (81%), Thermococcus coenzyme of gammatolerans EJ3 F420-reducing hydrogenase (YP_002958434 ) (77%), Thermococcus sp. 4557 of the coenzyme F420-reducing hydrogenase (YP_004762049) (72%), the coenzyme F420-reducing hydrogenase (YP_001097176) of Methanococcus maripaludis (33%) and Methanococcus (33%) of the coenzyme F420-reducing hydrogenase (NP_987940) of maripaludis S2.

< Example 2 > Formate  Expression of Hydrogenase Gene in Growth Conditions

It has been reported that T. onnurineus NA1 can grow with an external formate as a substrate and that the growth is closely correlated with hydrogen production. MRNA expression levels of the 10 ORFs before the fdh2-mfh2-mnh2 cluster were up-regulated more than two-fold compared to YPS when formate was used as a substrate, but the ORF expression levels of the other hydrogenase gene clusters were relatively high (Mbh, sulf I, and mfhl) at a constant level (mbx, frh and mch). These results suggest that the fdh2-mfh2-mnh2 cluster may react to the formate. Since F420-reducing hydrogenase genes are located immediately before the fdh2-mfh2-mnh2 cluster, F420-reducing hydrogenase may affect growth and hydrogen production when formate is used as a substrate. .

MFO01 was prepared by introducing a strong promoter of glutamate dehydrogenase (P _gdh ) from P. furiosus with HMG-CoA reductase gene to overexpress F420-reductase (Fig. 1B). The expression of the F420-reductase gene was successfully enhanced by the insertion of a strong promoter, and the protein expression by western blotting was 5 times higher than that of the wild type.

Cell growth and hydrogen productivity of the mutant were cultured in CSTR fermenter using formic acid as a substrate. The mutant showed a faster growth than the wild type after 2 hours of incubation, while the wild type doubling time was 80 minutes, whereas the mutant had a rapid growth rate of 61 minutes (FIG. 3A). These results indicate that cell growth was promoted by overexpression of F420-reducing hydrogenase (F420-reduced hydrogenase, frh). The cumulative hydrogen production was also increased compared to the wild type (Fig. 3B). The maximum specific H ₂ production rate per unit cell was 384 mmol / g / h for the wild type and 472 mmol / g / h for the mutant, Hydrogen production (Fig. 4). In conclusion, overexpression of F420-reducing hydrogenase of T. onnurineus NA1 could increase cell growth and hydrogen productivity.

Korea Biotechnology Research Institute KCTC12356BP 20130123

Claims (9)

F420-Reduced Hydrogenase overexpressed Hydrogen-enhanced Thermococcus spp . Mutant strain. The method of claim 1, wherein the Thermococcus spp . Thermococcus A mutant strain characterized by being onnurineus . The method of claim 1, wherein the Thermococcus spp . Is a microorganism deposit number KCTC 12356BP. Thermococcus spp . Mutant to increase the expression rate of the F420-reductive hydrogenase; And
And culturing the strain in a medium containing formic acid. 5. The method of claim 4, wherein the Thermococcus spp . Thermococcus onnurineus . &lt; / RTI &gt; 5. The method of claim 4, wherein the Thermococcus spp . Is the microorganism deposit number KCTC 12356BP. 5. The method according to claim 4, wherein the increase in the expression level of the F420-reductase is increased by increasing the number of replications of the F420-reductase gene, increasing the transcription rate, increasing the translation rate, increasing the stability of the F420- Hydrogen production process. 8. The method according to claim 7, wherein the increase of the F420-reductive hydrogenase transfer rate is achieved by using a strong promoter having a high expression rate operably linked to the gene. 9. The method according to claim 8, wherein the promoter is a promoter of glutamate dehydrogenase (Pgdh).

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