KR20220052671A - Methanotrophs with increased ability of producing lactic acid - Google Patents

Abstract

The present invention relates to recombinant methanotrophs transformed with an expression vector comprising an inducible promoter and a lactate dehydrogenase gene operably linked to the inducible promoter. The recombinant strain of the present invention increased acid resistance and lactic acid production capacity by expressing the LDH gene using an inducible promoter in methanotrophs using methane as the sole carbon source. Therefore, the recombinant strain of the present invention can be usefully used to produce lactic acid using methane in a high concentration.

Description

METHANOTROPHS WITH INCREASED ABILITY OF PRODUCING LACTIC ACID

The present invention relates to a recombinant methanogenic strain having increased lactic acid-producing ability and a method for producing lactic acid using the same.

PLA (Polylactic Acid) is a biodegradable polymer made by converting lactic acid to lactide and ring-opening polymerization thereof, and its raw material, lactic acid, is produced through fermentation. Lactic acid is a kind of organic acid that is widely used in industries such as cosmetics, chemicals, metals, textiles, dyes, and pharmaceuticals as well as being generally used as a food additive.

Lactic acid has L-form and D-form optical isomers. Among these optical isomers, type D was mainly used for medical/drug delivery purposes. However, when applied to PLA, the crystallization rate due to D-type lactide increased and the thermal properties improved. In addition, pure L-type polymer and pure D When stereocomplex PLA is structurally formed according to the processing conditions in which the type polymer is mixed, a new polymer with higher heat resistance than PE/PP as well as existing PLA is discovered. Research and commercialization of methods for enhancing physical properties are progressing rapidly.

In the past, lactic acid was mainly produced using sugars and yeast, and research on a method for producing high-purity lactic acid using this method was conducted.

Meanwhile, methane is the main component of natural gas and biogas, and is the most abundant and cheapest carbon source. Recently, research on a lactic acid production method using a methane magnetized strain that can use methane as the only carbon and energy source is being actively researched. However, the methanogenic strain has low resistance to lactic acid to such an extent that it hardly grows in a medium to which 0.5 g/L of lactic acid is added. Therefore, there is a problem that the growth rate of the methanogenic strain decreases as the concentration of lactic acid increases because the methanogenic strain produces lactic acid.

The results of a study on the production of lactate from methane using M. buryatense 5GB1S have been reported, and the strain exhibited a maximum lactate production concentration of 0.8 g/L and a lactate productivity of 0.008 g/L/h ( CA Henard et al., Sci. Rep. , 6(2185):1-9, 2016). However, due to the slow growth rate of methane magnetized strains, there is a limit to increasing lactic acid production.

Recently, Methylomonas sp. with increased lactate tolerance by the present inventors. A strain with increased lactic acid production capacity was developed by introducing the lactate dehydrogenase (LDH) gene required for lactic acid production into the JHM80 strain derived from DH-1. However, even in the above strain, when the LDH gene is expressed using a strong promoter, cell growth is greatly inhibited due to the toxicity of lactic acid.

Therefore, there is a continuous need for the development of a methanogenic strain with enhanced lactate-producing ability through sophisticated regulation of LDH gene expression intensity.

(Non-Patent Document 1) Biotechnol Biofuels 12, 234 (2019)

(Patent Document 1) Registered Patent No. 211286

Accordingly, the present inventors studied to develop a methanogenic strain with increased lactic acid production, and developed a strain in which the expression intensity of the LDH gene was increased by using an inducible promoter. Specifically, when LDH was introduced, the methanogenic strain using the inducible promoter produced significantly higher lactate production per cell concentration when treated with IPTG (Isopropyl β-D-1-thiogalactopyranoside) compared to the methanogenic strain using the constitutive promoter. By confirming that, the present invention was completed.

In order to solve the above problems, one aspect of the present invention is an inducible promoter; And it provides a recombinant methanogenic strain transformed with an expression vector comprising a lactate dehydrogenase gene operably linked to the inducible promoter.

Another aspect of the present invention provides a method for producing lactic acid comprising culturing the methanogenic strain under conditions containing methane and an inducer.

The recombinant methanogenic strain of the present invention increased the expression of the lactate dehydrogenase gene in the methanogenic strain by using an inducible promoter instead of the conventional constitutive promoter. In addition, the expression of the lactate dehydrogenase gene is regulated in the recombinant methanogenic strain of the present invention by controlling the concentration of an inducer such as IPTG. Therefore, the recombinant methanogenic strain of the present invention can be usefully used to increase the production of lactic acid produced from methane to a desired level.

1 is a schematic diagram of the mutation site in the genome of the JHM80 strain.
2 is a view showing a plasmid included in the JHM87 strain, which is an embodiment of the present invention.
3 is a view showing the cell growth and lactate production (top) according to the anhydrous tetracycline (aTC) concentration of the JHM88 strain and the cell growth and the lactate production (bottom) by the IPTG concentration of the JHM87 strain.
4 is a view showing the cell growth and lactate production by IPTG concentration of the JHM87 strain.
5 is a view showing the lactate production per cell concentration in JHM86 strain and JHM87 strain.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention is an inducible promoter; And it provides a recombinant methanogenic strain transformed with an expression vector comprising a lactate dehydrogenase gene operably linked to the inducible promoter.

The term "methanotrophs" used in the present invention, also called methanotrophs, refers to prokaryotes that grow using methane as the sole carbon source. Most methanogenic strains use methane in the atmosphere as a carbon source, which is usually converted into methanol and then used for in vivo metabolism.

The methanogenic strain is Methylomonas sp., Methylovulum sp., Methylocaldum sp., Methylomicrobium sp. or Methylococcus sp. It may be a genus strain. Preferably, the methanogenic strain is Methylomonas sp . DH-1 , Methylomonas koyamae, Methylomonas denitrificans, Methylomonas methanica, Methylovulum psychrotolerans, Methylocaldum marinum, Methylomicrobium alcaliphilum or Methylococcus capsulatus , but is not limited thereto.

The term " Methylomonas sp. DH-1 strain" used in the present invention means a strain belonging to the genus Methylomonas sp., which is derived from sewage sludge and has not been reported previously. The DH-1 strain refers to the strain deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center as of April 8, 2016 and given the deposit number KCTC13004BP. The strain was deposited at the Korea Institute of Biotechnology and Biotechnology Biological Resources Center on August 27, 2015, and the strain given the deposit number KCTC18400P was converted to the deposit under the Budapest Treaty. It is obvious that they are identical.

The term "lactate dehydrogenase" used in the present invention is an enzyme that converts hydrogen from L-lactic acid or D-lactic acid to pyruvic acid by using NAD+. When catalyzing the final step of the reverse reaction, NADH is It is an enzyme that produces L-lactic acid or D-lactic acid by reducing pyruvic acid using That is, the lactate dehydrogenase of the present invention may be L-lactate dehydrogenase or D-lactate dehydrogenase, specifically D-lactate dehydrogenase.

The lactate dehydrogenase gene has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology thereto as well as wild-type lactate dehydrogenase. It may contain the gene of the enzyme having. The lactate dehydrogenase gene can be confirmed through GenBank of NCBI, etc., and Leuconostoc mesenteroides subsp. It may be a gene encoding lactate dehydrogenase from mesenteroides ATCC 8293. Specifically, the lactate dehydrogenase gene may be a nucleotide sequence encoding the amino acid sequence of GenBank: ABJ62843.1. Preferably, the lactate dehydrogenase gene may be the nucleotide sequence of SEQ ID NO: 1.

In this case, the methanogenic strain may be a strain in which the glgA (glycogen synthase) gene is deleted. The recombinant strain into which the lactate dehydrogenase gene is introduced may be a strain capable of producing lactic acid by introducing the lactate dehydrogenase gene.

In addition, the methanogenic strain into which the lactate dehydrogenase gene will be introduced may be a methanogenic strain into which an expression vector including a nucleotide sequence encoding a LysR family transcriptional regulator is introduced. Specifically, the methanogenic strain may be a recombinant strain transformed to increase the expression of the AYM39_21120 gene by deleting some bases of the promoter of the AYM39_21120 gene, which is a gene contributing to lactic acid resistance.

In addition, the methanogenic strain into which the lactate dehydrogenase gene is to be introduced may further include a lacI promoter, a lacI and a lac operator. In this case, the LacI promoter may be the nucleotide sequence of SEQ ID NO: 2. In addition, the lacI may be the nucleotide sequence of SEQ ID NO: 3, and the lac operator may be the nucleotide sequence of SEQ ID NO: 4.

As used herein, the term "promoter" refers to a DNA nucleotide sequence to which transcriptional regulators can bind. The promoter binds to RNA polymerase through a transcriptional regulator as a mediator, thereby transcribing a gene located downstream thereof. can induce

As used herein, the term “inducible promoter” refers to a promoter that induces expression in response to a specific condition, and is distinct from a constitutive promoter that is constitutively expressed. The inducible promoter can respond to changes in the environment, such as temperature, concentration, and other physical/chemical changes, or to the addition or concentration of inducers such as IPTG, and can exhibit various aspects in terms of the amount of expression. there is.

As inducible promoters suitable for the present invention, metalloprotein promoters such as tetracycline regulated promoter, doxycycline inducible (TRE) promoter, cAMP inducible promoter, glucocorticoid activated promoter system, IPTG inducible promoter, Cd2+ or Zn2+ inducible promoter, interferon conditional such as dependent promoter (such as murine MX promoter), HIV LTR promoter (Tat), DMSO inducible promoter, globin promoter globin LCR, hormone regulated promoter (GLVP/TAXI, ecdysone), and rapamycin inducible promoter (CID) Promoters selected from inducible promoters selected from activating promoters and promoter systems exist, but are not limited to the promoters exemplified above.

Meanwhile, the inducible promoter suitable for the present invention may be preferably selected from the tac promoter, the trc promoter, the lac promoter or the T3 promoter.

As used herein, the term "inducing factor" refers to sugars, metal ions, antibiotics, dyes, flavonoids, non-saccharide metabolites esterases, oleates, c-d-AMPs, cholates, salicylates, steroid hormones, etc. It refers to all factors that can affect the induction of promoters. Preferably, the inducer is one selected from sugar, metal ion, antibiotic, dye, flavonoid, non-saccharide metabolite esterase, oleate, c-d-AMP, cholate, salicylate and steroid hormone. may be more than In particular, preferably, as the inducer, lactose, IPTG, L-arabinose, maltose, trehalose, glucose-6P, glycerol-P, glucitol, fucose, L-ascorbate, deoxyribonucleoside, One or more saccharides selected from inositol and fructose may be used, and more preferably IPTG.

As used herein, the term "operably linked" refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function. . For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. The operative linkage with the expression vector can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can use enzymes generally known in the art.

As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a target protein in a host cell, and refers to a genetic construct including essential regulatory elements operably linked to express an inserted gene. The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator, and the start codon and stop codon are generally considered to be part of a nucleotide sequence encoding a polypeptide, and when the gene construct is administered, the individual It must exhibit an action in the sequence and must be in frame with the coding sequence.

The expression vector may be any one selected from the group consisting of a plasmid vector or a cosmid vector.

The plasmid vector may be a commercially developed pUC18 plasmid, pBAD plasmid, pIDTSAMRT-AMP plasmid, pJK001 plasmid, and the like, but is not limited thereto.

Methods for introducing the expression vector include a method using a liposome containing an expression vector, a method for introducing a recombinant expression vector using PEG, a direct injection method of the recombinant expression vector, a microparticle bombardment method, a gene gun, and an electroporation method ( electroporation), a transformation method using a virus, a vacuum infiltration method, a floral meristem dipping method, and the like can be used. Preferably, the method for introducing the expression vector may be electroporation.

The expression vector may express a gene through episomal DNA form or insertion into the genome of the strain, preferably gene expression through insertion into the genome of a methanogenic strain.

The expression vector may include a promoter consisting of the nucleotide sequence of SEQ ID NO: 5 at an upstream position of the nucleotide sequence encoding the lactate dehydrogenase gene.

The methanogenic strain may be a strain in which lactic acid-producing ability is increased when cultured in the presence of an inducer by introducing an expression vector comprising the nucleotide sequence of SEQ ID NO: 5.

On the other hand, as a result of research to develop a methanogenic strain with improved resistance to lactic acid, the present inventors selected a methanogenic strain with significantly improved resistance to lactic acid through adaptive evolution in a medium containing high concentration of lactic acid. . The methanogenic strain is characterized in that the 456th and 457th bases (TT) of the promoter of the AYM39_21120 gene represented by the nucleotide sequence of SEQ ID NO: 6 are deleted.

On the other hand, the present inventors have discovered that even if the LDH gene is introduced into the methanogenic strain with greatly improved resistance to lactic acid to produce lactic acid, the lactic acid production ability is still limited. After research and development to solve the above problem, it was confirmed that the lactate production of the methanogenic strain could be increased when an inducible promoter using IPTG as an inducer was used in the methanogenic strain (see FIG. 4 ). Therefore, the recombinant methanogenic strain of the present invention has an increased ability to produce lactic acid while having high resistance to lactic acid, and thus can be usefully used for the production of lactic acid using methane.

Another aspect of the present invention is to provide a recombinant methanogenic strain in which the inducible promoter is induced by IPTG. The inducible promoter of the present invention may be induced by IPTG to increase the expression of genes located downstream of the inducible promoter.

Another aspect of the present invention provides a recombinant methanogenic strain in which expression of lactate dehydrogenase gene is regulated according to the concentration of IPTG. The recombinant methanogenic strain of the present invention has an increased lactate production when cultured in a medium containing IPTG than in a medium containing no IPTG. The increase in lactate production may be a result of an increase in LDH gene expression and/or an increase in acid resistance to lactic acid present in the medium. By adjusting the concentration of IPTG contained in the medium, it is possible to control the lactate production produced by the recombinant methanogenic strain.

The amount of IPTG used for inducing the inducible promoter of the present invention and regulating the expression of lactate dehydrogenase gene may be 1 μM to 10 mM or 1 μM to 1 mM. Preferably, the amount of IPTG added is 1 μM to 900 μM, 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 400 μM, 1 μM to 300 μM, 1 μM to 200 μM, 1 μM to 100 μM, 1 μM to 90 μM, 1 μM to 80 μM, 1 μM to 70 μM, 1 μM to 60 μM, 1 μM to 50 μM, 1 μM to 40 μM, 1 μM to 30 μM, 1 μM to 20 μM, or 1 μM to 10 μM. More preferably, 5 to 10 μM of IPTG may be used. In addition, as the pH in the culture process of the strain increases, the amount of IPTG added may be increased.

Another aspect of the present invention is to provide a recombinant methanogenic strain transformed so that the LysR family transcriptional regulator is overexpressed.

The term "LysR family transcriptional regulator" used in the present invention is a protein encoded by the AYM39_21120 gene and refers to a LysR family transcriptional regulator. The LysR family transcriptional regulator may be a protein having an amino acid sequence represented by SEQ ID NO: 7. In addition, the nucleotide sequence encoding the LysR family transcriptional regulator may be a nucleotide sequence represented by SEQ ID NO: 8.

The expression vector used in the present invention may include a promoter composed of the nucleotide sequence of SEQ ID NO: 5 at an upstream position of the nucleotide sequence encoding the LysR family transcriptional regulator.

Another aspect of the present invention provides a method for producing lactic acid, comprising the steps of culturing the recombinant methanogenic strain under conditions containing methane and an inducer to produce lactic acid and obtaining the produced lactic acid. .

The method for culturing the recombinant methanogenic strain is not particularly limited, but may be performed by inoculating and culturing a medium under atmospheric conditions containing methane.

The concentration of methane in the atmosphere may be 0.01 to 50% (v/v), specifically, the concentration of methane may be 10 to 40% (v/v) or 20% (v/v).

The culture temperature is not particularly limited, but may be 20°C to 40°C. Preferably, it may be 25 °C to 35 °C. As an example of the incubation temperature, it may be 30°C. The culture may be performed under stirring conditions. Specifically, the stirring condition may be 100 rpm to 300 rpm, preferably 150 rpm to 250 rpm or 170 rpm. Incubation time may be incubated for 20 hours to 200 hours, specifically, may be cultured for 20 hours to 140 hours. Specifically, it can be cultured for 24 hours to 120 hours.

In the present invention, the method for culturing the recombinant methanogenic strain can be performed using a method widely known in the art. Specifically, the culture is not particularly limited as long as it can produce lactic acid from the methanogenic strain. In addition, the culture may be continuously cultured in a batch process or in a fed batch or repeated fed batch process.

The medium used for the culture may be a nitrate mineral salts (NMS) medium known to be used for the culture of methanogenic strains. Depending on the methanogenic strain, the components contained in the medium or a medium in which the content thereof is adjusted may be used (http://methanotroph.org/wiki/culturing-tips/). In particular, when salts such as NaCl and KCl are included in the medium, the proliferation of the DH-1 strain or its transformant or the productivity of lactic acid can be improved. In this case, the concentration of salts contained in the medium may be 0.1% (w/w) to 3.0% (w/w), preferably 1.0% (w/w) to 2.0% (w/w) or 1.5% (w/w).

In addition, precursors suitable for the culture medium may be used. The above-mentioned raw materials may be added in a batch, fed-batch or continuous manner by an appropriate method to the culture during the culturing process, but is not particularly limited thereto. A basic compound such as sodium hydroxide, potassium hydroxide, ammonia, or an acid compound such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture. In addition, an antifoaming agent such as a fatty acid polyglycol ester may be used to inhibit the formation of bubbles.

The step of obtaining lactic acid may be performed by methods known in the art such as dialysis, centrifugation, filtration, solvent extraction, chromatography, crystallization, and the like. For example, a method of recovering lactic acid by centrifuging the transformant culture to remove the recombinant strain and applying the obtained supernatant to a solvent extraction method may be used. In addition, any method capable of recovering the lactic acid by combining known experimental methods according to the characteristics of the lactic acid may be used without particular limitation.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Preparation of Transformed Recombinant Methanogenic Strain

Example 1.1. Selection of strains with increased resistance to lactic acid through adaptive evolution

In order to select methanogenic strains that can grow even in high concentrations of lactic acid, adaptive evolution on a laboratory scale was performed. The genus Metyhlomonas sp . DH-1 strain was sold at Kyunghee University.

In order to induce the adaptive evolution of the DH-1 strain to lactic acid, the DH-1 strain was started with a medium supplemented with lactic acid at a concentration of 0.5 g/L, and the concentration of lactic acid was increased by 0.2 g/L to 8 g/L. It was subcultured in a medium supplemented with lactic acid. Through a total of 35 consecutive passages, a DH-1 strain capable of growing in a medium treated with lactic acid at a concentration of 8 g/L was selected. By comparing the growth of the selected strain with the wild-type strain in the medium to which lactic acid was added, it was confirmed that the resistance to lactic acid was improved. The selected strain was named “JHM 80”.

Example 1.2. Genome analysis and gene expression confirmation of JHM80 strain

In order to find a gene contributing to the improvement of resistance to lactic acid of the JHM80 strain selected in Example 1, whole genome sequencing of the JHM80 strain was commissioned to Macrogen. As a result, a mutation in which two nucleotide sequences (TT) were deleted in the promoter between the AYM39_21115 gene and the AYM39_21120 gene in the JHM80 strain was confirmed (FIG. 1). The promoter is represented by the nucleotide sequence of SEQ ID NO: 6, which corresponds to the upper rank of the LysR family transcriptional regulator.

Example 1.3. Preparation of Transformed Recombinant Methanogenic Strain

A plasmid containing an inducible promoter together with the LDH gene cassette was constructed using a homologous recombination system. DNA of JHM80 strain was used as a template for the plasmid. The homology region allowing insertion into the AYM39_03770 ( glgA ) locus of the methanogenic strain was 1 kb upstream and 1 kb downstream of the glgA gene. Through PCR, the upper 1 kb and lower 1 kb of the AYM39_03770 ( glgA ) gene were inserted into the pInsUD-LDH-Kan vector using Not1/MauB1 and Apa1/Sac1 restriction enzymes, respectively. The above vector was named "pglgAUD-LDH-Kan" vector.

A study on lactic acid production using the Tet promoter, an anhydrous tetracycline-inducible promoter, has been reported in the Methylomicrobium buryatense 5GB1S strain, a type of methanogenic strain. Accordingly, the inventors selected the Tet promoter as an inducible promoter for introduction into the JHM80 strain selected in Example 1.1 in order to compare the LDH expression ability with the Tac promoter widely used in other strains such as E. coli .

For the introduction of the inducible promoter, the Tet promoter and the Tac promoter represented by the nucleotide sequence of SEQ ID NO: 5 were inserted into the pglgAUD-LDH Kan vector using MauB1/BamH1 restriction enzymes, respectively. In addition, the LacI promoter represented by the nucleotide sequence of SEQ ID NO: 2 and lacI represented by the nucleotide sequence of SEQ ID NO: 3 were inserted above the Tac promoter, and the lacO operator represented by the nucleotide sequence of SEQ ID NO: 4 was inserted below the Tac promoter. The vectors thus obtained were named "pglgAud-P.tet-LDH" and "pglgAud-P.tac-LDH", respectively.

Additionally, a MauB1/BamH1 restriction enzyme recognition site was provided so that various inducible promoters could be used, and a BamH1/Pst1 restriction enzyme recognition site was inserted into the LDH gene to facilitate cloning of other genes ( FIG. 2 ).

For transformation of JHM80 strain, a transformation method using electroporation was used using Micropulser ^TM Electroportor (Biorad). The strains and plasmids used in this experiment are shown in Table 1 below.

strain name genotype source DH-1 KCTC18400P JHM80 Evolved strain from DH-1 - JHM86 JHM80 △glgA::LDH-KanR Biotechnol Biofuels 12, 234 (2019) JHM87 JHM80 △glgA::P.tac-LDH-KanR - JHM88 JHM80 △glgA::P.tet-LDH-KanR - plasmid characteristic source pInsUD-LDH-Kan Plasmid containing KanR marker with LDH - pglgAUD-LDH-Kan Plasmid containing LDH-KanR cassette with glgA integration site Biotechnol Biofuels 12, 234 (2019) pglgAUD-P.tet-LDH-Kan Plasmid containing P _tet -LDH-KanR cassette with glgA integration site - PglgAUD-P.tac-LDH-Kan Plasmid containing P _tac -LDH-KanR cassette with glgA integration site -

Plasmids were respectively introduced into the genome of the JHM80 strain through electroporation. The JHM80 strain into which the pglgAUD-LDH-Kan plasmid was introduced was named "JHM86" strain. The JHM80 strain into which the PglgAUD-P.tac-LDH-Kan plasmid containing the Tac promoter-LDH cassette was introduced was named "JHM87" strain, and the pglgAUD-P.tet-LDH-Kan plasmid containing the Tet promoter-LDH cassette The strain into which was introduced was named "JHM88" strain.

Example 1.4. Cultivation of methane magnetized strains

DH-1, JHM80, JHM86, JHM87, JHM88 strains are NMS (0.488 g/L MgSO ₄ , 1 g/L KNO ₃ , 0.228 g/L CaCl ₂ -2H ₂ O, 3.8% Fe-EDTA, 0.1% NaMo- 4H ₂ O, Trace element solution) was cultured in a medium containing phosphoric acid solution, vitamins, and 10 μM CuSO ₄ . Trace element solution is 500 mg/L FeSO ₄ -7H ₂ O, 400 mg/L ZnSO ₄ -7H ₂ O, 15.71 mg/L MnCl ₂ -4H ₂ O, 50 mg/L CoCl ₂ -6H ₂ O, 10 mg Consists of /L Nicl ₂ -6H ₂ O, 15 mg/LH ₃ BO ₃ , 250 mg/L EDTA, phosphoric acid solution (26 g/L KH ₂ PO ₄ , 32.83 g/L Na ₂ HPO ₄ ) and vitamins (2.0 mg/L biotin, 2.0 mg/L folic acid, 5.0 mg/L Thiamine HCl, 5.0 mg/L Ca pantothenate, 0.1 mg/L Vitamin B ₁₂ , 5.0 mg/L Riboflavin, 5.0 mg/L Nicotinamide) Is as follows. Cell culture was performed at 30 °C and 170 rpm using a shaking incubator. As culture conditions, the initial inoculation cell concentration was fixed at OD ₆₀₀ = 0.2 or 0.5, and 12.5 ml medium was used in a 125 ml shake flask, and 20% methane was used using a gas syringe (Agilent 50 ml gas tight syringe 5190-1547). was supplied.

For gene induction, an appropriate concentration of anhydrous tetracycline (aTC) or IPTG was used.

Example 2. Comparison of LDH gene expression level according to the type of inducible promoter

In order to examine the difference in LDH gene expression level in the transformed recombinant methanogenic strain according to the type of inducible promoter, lactate production in JHM87 strain and JHM88 strain was compared.

Analysis of the concentration of lactate contained in the medium was performed by filtering 300 μL of the medium through a 0.45 μm filter and performing HPLC analysis. UtiMate 3000 HPLC system (Thermo fishers scientific), BioRad Aminex HPX-87H column and RI detector were used. As the mobile phase, 5 mM sulfuric acid was used, the flow rate was 0.6 ml/min, and the temperature was set to 60°C. Cell density was measured at a wavelength of 600 nm using Multiskan Go (Thermo fishers scientific).

After the JHM87 strain having an inducible promoter using IPTG as an inducer was treated with IPTG at concentrations of 0, 0.05, 0.1 and 0.2 mM, cell growth and lactate production were confirmed. Cell growth and lactate production were confirmed after the JHM88 strain having an inducible promoter using tetracycline as an inducer was treated with aTC at concentrations of 0, 0.1, 0.2 and 0.5 mg/L.

In the condition not treated with aTc, lactate production was not observed in the JHM88 strain. On the other hand, when aTc at concentrations of 0.1 mg/L, 0.2 mg/L and 0.5 mg/L was treated, the JHM88 strain produced lactate at concentrations of 10 mg/L, 15 mg/L, and 30 mg/L, respectively, for 42 hours. produced (Fig. 3 top).

On the other hand, when 0.05 mM, 0.1 mM, and 0.2 mM IPTG was treated, cell growth was not observed in the JHM87 strain, but it was shown that lactate at a concentration of 150 mg/L or more was produced for 70 hours in all groups treated with IPTG ( Fig. 3 bottom).

Example 3. Lactate-producing ability of recombinant transformed methanogenic strain upon low-concentration IPTG treatment

In order to check whether the lactate production capacity was improved according to the expression of the LDH gene using the Tac promoter, the JHM87 strain was cultured by treatment with a low concentration of 5, 10, or 25 μM IPTG.

Cell growth and lactate production were observed by culturing in a 125 mL shake flask with an additional supply of 20% methane every 24 hours. Compared with the control group not treated with IPTG, lactate production was increased when IPTG was treated. When 5 μM IPTG was treated, the highest lactate was produced and lactate at a concentration of 862 mg/L was produced for 96 hours ( FIG. 4 ).

On the other hand, since the growth of cells was significantly inhibited as the concentration of IPTG increased, the amount of lactate produced per cell concentration was compared. In the absence of IPTG, the lactate production of the JHM87 strain was lower than that of the JHM86 strain using the constitutive promoter under the same culture conditions. However, when a low concentration of 5, 10, or 25 μM IPTG was treated, the lactate production per cell concentration was increased in the JHM87 strain than in the JHM86 strain ( FIG. 5 ). In particular, when 25 μM IPTG was treated, the lactate production per cell concentration in the JHM87 strain was about 4 times higher than that in the JHM86 strain as of 72 hours.

<110> Seoul National University R&DB Foundation <120> METHANOTROPHS WITH INCREASED ABILITY OF PRODUCING LACTIC ACID <130> 202009-0028 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 996 <212> DNA <213> Leuconostoc mesenteroides <400> 1 atgaagattt ttgcttacgg cattcgtgat gatgaaaagc catcacttga agaatggaaa 60 gcggctaacc cagagattga agtggactac acacaagaat tattgacacc tgaaacagct 120 aagttggctg agggatcaga ttcagctgtt gtttatcaac aattggacta tacacgtgaa 180 acattgacag ctttagctaa cgttggtgtt actaacttgt cattgcgtaa cgttggtaca 240 gataacattg attttgatgc agcacgtgaa tttaacttta acatttcaaa tgttcctgtt 300 tattcaccaa atgctattgc agaacactca atgattcaat tatctcgttt gctacgtcgc 360 acgaaagcat tggatgccaa aattgctaag cacgacttgc gttgggcacc aacaattgga 420 cgtgaaatgc gtatgcaaac agttggtgtt attggtacag gtcatattgg ccgtgttgct 480 attaacattt tgaaaggctt tggggccaag gttattgctt atgacaagta cccaaatgct 540 gaattacaag cagaaggttt gtacgttgac acattagacg aattatatgc acaagctgat 600 gcaatttcat tgtatgttcc tggtgtacct gaaaaccatc atctaatcaa tgcagatgct 660 attgctaaga tgaaggatgg tgtggttatc atgaacgctg cgcgtggtaa tttgatggac 720 attgacgcta ttattgatgg tttgaattct ggtaagattt cagacttcgg tatggacgtt 780 tatgaaaatg aagttggctt gttcaatgaa gattggtctg gtaaagaatt cccagatgct 840 aagattgctg acttgattgc acgcgaaaat gtattggtta cgccacacac ggctttctat 900 acaactaaag ctgttctaga aatggttcac caatcatttg atgcagcagt tgctttcgcc 960 aagggtgaga agccagctat tgctgttgaa tattaa 996 <210> 2 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> LacI promoter <400> 2 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt 60 caattcaggg tggtgaat 78 <210> 3 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> LacI <400> 3 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt 60 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg 120 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag 180 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc 240 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa 300 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt 360 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc 420 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt 480 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag 540 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc 600 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg 660 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact 720 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc 780 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca 840 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc 900 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 960 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc 1020 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 1080 tga 1083 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Lac operator <400> 4 ttgtgagcgg ataacaa 17 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> tac promoter <400> 5 ttgacaatta atcatcggct cgtataatg 29 <210> 6 <211> 524 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of promoter (Methylomonas sp. DH-1) <400> 6 gactattaat ctcgatttcg aacgggctga atcatagcat gacgcccaaa ctgccggaaa 60 aactgcgggc aaaaaaaacc tcccaatgct cgcgcagagg gaggcaagaa aaccagcgga 120 gtgcttggaa ggaggttacc gctcgttaat tacaacacca ggaggtagtt acaaattctg 180 gggatcagcg gctcgcttgg gtattgagcg agatattgct gaacattgag gtcgctgcaa 240 acaggacagt ggaaattacg aacaggccgg aaagaaaatt caaaatgccg gcaataaaca 300 aagtaccgac cagtacatct tttttaaatt cattcattgt gctcaaatcc tctgtttaat 360 cttgcatttg gcaagatttt ctgtgctttc tcgttaagtt gccgcacact ataccggaga 420 taaaaaagtt tacaattagg cgtttattaa ataaattgta tgaaattaag aaacaataaa 480 aatgtcaacg ttttttagca acttccaacg ccaccggagg caga 524 <210> 7 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of LysR family transcriptional regulator (Methylomonas sp. DH-1) <400> 7 Met Asp Lys Leu Thr Ser Met Asn Val Phe Val Arg Val Ala Lys Ala 1 5 10 15 Gly Ser Phe Ala Gly Ala Ala Lys Asp Leu Asp Ile Ser Arg Ala Met 20 25 30 Ala Thr Lys His Ile Met Gln Leu Glu Ser Glu Leu Asn Thr Arg Leu 35 40 45 Phe Asn Arg Thr Thr Arg Ser Leu Ser Leu Thr Glu Ala Gly Glu Ala 50 55 60 Tyr Leu Glu Arg Cys Gln Gln Val Leu Leu Asp Val Ala Glu Met Glu 65 70 75 80 Ser Ala Ile Thr His Leu Gln Thr Glu Pro Arg Gly Thr Leu Lys Ile 85 90 95 Leu Ala Pro Pro Val Ile Gly Ala Ser His Ile Ser Pro Gly Leu Thr 100 105 110 Glu Tyr Leu Lys Asn Tyr Pro Asp Leu Ser Val Glu Met Val Leu Lys 115 120 125 Gly Gly Gln Val Asp Leu Ile Asp Glu Gly Val Asp Leu Ala Ile Tyr 130 135 140 Leu Gly Gln Leu Asn Asp Thr Ser Leu Val Ala Arg Lys Leu Ala Ser 145 150 155 160 Ser Ser Leu Val Val Cys Ala Ser Pro Glu Tyr Leu Lys Asn His Gly 165 170 175 Ile Pro Gln Asp Pro Glu Asp Leu Glu Asp His Ser Cys Leu Ile Asn 180 185 190 Trp Ala Ile Pro Pro Arg Asn Lys Trp Arg Phe Lys Gly Ile Leu Gly 195 200 205 Glu Arg Thr Val Thr Val Thr Gly Arg Met Gln Ala Asn Met Ala Asp 210 215 220 Pro Ile Arg Asn Ala Ala Val Asn Gly Leu Gly Leu Ile Met Leu Pro 225 230 235 240 Arg Tyr Ile Val Gly Arg Asp Ile Glu Gln Gly Arg Leu Gln Val Val 245 250 255 Met Glu Gln Tyr Gly Ile Ala Pro Leu Glu Ile Tyr Ala Val Tyr Pro 260 265 270 His Arg Lys Tyr Leu Ser Ala Lys Val Arg Ser Phe Leu Glu Phe Ile 275 280 285 Gln Ala Trp Leu Pro His Arg Ile Gly Met Asn Pro 290 295 300 <210> 8 <211> 903 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of LysR family transcriptional regulator (Methylomonas sp. DH-1) <400> 8 atggacaaac taaccagcat gaacgttttt gtccgcgtcg ccaaggccgg cagcttcgcc 60 ggggccgcca aggatttgga tatctctcga gctatggcca ccaagcacat catgcaattg 120 gagagcgagc tgaatacccg cctgttcaac cgtacgaccc gcagcttaag cctgactgaa 180 gccggcgagg cttatctgga acgctgccag caggtgttgc tggacgtcgc ggaaatggaa 240 tcggccatca cccatctgca aaccgaaccg cgcggcactt taaaaattct ggcaccaccg 300 gtaatcggcg ccagccacat ctcgcccggc ctgaccgagt atctgaaaaa ctacccggat 360 ctttcggtgg agatggtgct gaaaggcggt caggtggatt tgatcgacga aggcgtcgac 420 ttagcgattt atctcggcca gctcaacgac accagcctgg tcgcccgcaa actggccagc 480 tcctcgctgg tagtctgcgc gtcgccggaa tacctgaaaa accacggcat cccgcaagac 540 ccggaagact tggaagacca tagctgcctg attaactggg ccatcccgcc gcgcaacaaa 600 tggcgcttca aaggcatcct cggcgaacgc accgtcactg tgaccggcag gatgcaagcc 660 aacatggccg acccgatccg caatgcggcc gttaacggcc taggcttgat tatgttgccg 720 cgctacatcg tcggccgtga catcgaacaa ggccgtctgc aagtggtgat ggaacaatac 780 ggcatcgccc cgttggagat ttacgcggtt tacccacacc gcaagtatct ttccgctaaa 840 gtccgttcct ttctggaatt tatccaagcc tggctaccgc accggatcgg catgaaccca 900 tga 903

Claims (11)

inducible promoter; and
Recombinant methanotrophs transformed with an expression vector containing a lactate dehydrogenase gene operably linked to the inducible promoter. The method of claim 1,
The inducible promoter is induced by IPTG (isopropyl-β-D-thiogalactopyranoside), a recombinant methanogenic strain. 3. The method of claim 2,
The expression of the lactate dehydrogenase gene is regulated according to the IPTG concentration, a recombinant methanogenic strain. The method of claim 1,
The inducible promoter is a tac promoter, a trc promoter, a lac promoter, or a T3 promoter. The method of claim 1,
The lactate dehydrogenase gene consists of the nucleotide sequence of SEQ ID NO: 1, a recombinant methanogenic strain. The method of claim 1,
The methanogenic strain is Methylomonas sp. DH-1 , Methylomonas koyamae, Methylomonas denitrificans, Methylomonas methanica, Methylovulum psychrotolerans, Methylocaldum marinum, Methylomicrobium alcaliphilum or Methylococcus capsulatus , a recombinant methanogenic strain. The method of claim 1,
The methanogenic strain is a recombinant methanogenic strain transformed so that the LysR family transcriptional regulator is overexpressed. The method of claim 1,
The strain is a recombinant methanogenic strain having the ability to produce lactic acid. The method of any one of claims 1 to 8, comprising: culturing the recombinant methanogenic strain of any one of claims 1 to 8 under conditions containing methane and an inducer; and
A method for producing lactic acid, comprising the step of obtaining lactic acid produced from the methanogenic strain. 10. The method of claim 9,
The inducing factor in the culturing step is IPTG, a method for producing lactic acid. 11. The method of claim 10,
In the culturing step, inducing a promoter with IPTG at a concentration of 1 μM to 100 μM, a method for producing lactic acid.

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