WO2021060923A1 - Method and composition for producing compound having four or more carbon atoms from methane - Google Patents

Abstract

The present invention relates to: a composition for producing a compound having four or more carbon atoms from methane (CH₄), the composition containing methanotroph and mutant E. coli; and a method for producing a compound having four or more carbon atoms by co-culturing the strains. In addition, the present invention relates to a method for co-culturing methanotroph and E. coli.

Description

Method and composition for producing a compound having 4 or more carbon atoms from methane

The present invention relates to a composition and method for producing a compound having 4 or more carbon atoms by using methanogens and mutated E. coli.

Methane (CH ₄ ) is a hydrocarbon gas with a simple structure composed of one carbon. It is relatively cheaper than a liquid hydrocarbon such as petroleum, but it is difficult to attempt to convert it to a high value-added compound. In addition, since it is known as a gas that can induce a greenhouse effect, attempts to industrially apply it while reducing methane by converting methane into other compounds are continuing.

In order to convert methane using biological means, a method using methanotroph can be considered. Methanogens are bacteria that can use methane as the sole carbon source or energy source, and can convert methane to methanol using methane monooxygenase (MMO), and then convert methanol to methanol through various biosynthetic pathways. This is because it can be converted into a kind of organic compound.

In order to convert methane into a high value-added compound using a biological method, 1) a high-efficiency methane activation property, and 2) a high-energy cellular environment with high energy metabolism efficiency for carbon-carbon (CC) bonding is required. However, the methanogens that exist in nature have specialized properties to produce essential proteins or cell growth with minimal energy, so they have both of the above-described properties required to convert methane into a high value-added compound. It is difficult to find out. In addition, unlike other microorganisms that have already studied a lot of molecular biological manipulation methods, such as E. coli , methanogens are difficult to effectively apply essential molecular biology techniques such as mass expression and regulation of various enzymes required for metabolic engineering. have.

A study that attempted to produce lactic acid from methane by introducing a gene related to lactate production in the conventional methanotrophic bacterium Methylomicrobium buryatense (Bioconversion of methane to lactate by an obligate methanotrophic bacterium, Scientific Reports volume 6, Article number: 21585 (2016)) However, in order to introduce an enzyme gene derived from a microorganism other than the methanogens into the methanogens, it is necessary to find out which species of enzyme genes can be successfully expressed in the methanogens through repeated experiments. A complex introduction process is involved, such as having to undergo codon optimization in consideration of the characteristics of magnetophilus only. In addition, even if the introduction of a specific foreign gene through such a cumbersome process is successful, there is a drawback that if you want to introduce another kind of gene to produce another compound, you must study and manufacture a newly improved methanogen with another gene introduced therein. .

Considering the above problems, it gives the methane-magnetizing bacteria, which have a slow growth rate and relatively weak metabolic activity, 1) the methane solubilization step, and 2) the production steps of useful carbon compounds, giving them high added value from methane. Attempts to produce compounds have many drawbacks and limitations in terms of efficiency.

The inventors of the present invention use a microorganism such as E. coli, which has a high growth rate and a considerable level of research on the introduction method of external genes, as a co-culture partner microorganism of the methanogen, instead of directly improving and using the methanogens. , To solve the problems of the prior art as described above. Until now, there have been no research results that suggested a method of co-culturing methanogens with other types of microorganisms, such as E. coli, synergistically with each other, and high value-added compounds from methane, especially compounds with carbon number of 4 or more, using their co-culture system. There was also no attempt to produce it. Accordingly, the inventors of the present invention newly developed a microbial co-cultivation system capable of industrially producing high value-added compounds with high efficiency while being much more applicable than the method that directly improved the conventional methanogens by co-culturing methane-magnetizing bacteria and mutant Escherichia coli. Constructed to complete the present invention.

The present invention is a relatively inexpensive carbon number 1 gas, especially methane (CH ₄ ), while cultivating and using microorganisms to convert and produce various useful compounds, without directly improving and using microorganisms that can utilize methane. An object of the present invention is to provide a composition for use in producing a compound having 4 or more carbon atoms with high efficiency. In addition, it is an object of the present invention to provide a method for co-culture with methane magnetizing bacteria, such as E. coli, with methane magnetizing bacteria that are difficult to co-culture. In addition, it is an object of the present invention to provide a method for producing a compound having 4 or more carbon atoms with high productivity by utilizing the microorganism culture method as described above.

One aspect of the present invention, in order to achieve the above object, for producing a compound having a carbon number of 4 or more from _{methane (CH 4 ), including a mutant E. coli capable of metabolizing acetic acid and methanotroph.} The composition is provided.

Another aspect of the present invention, in order to achieve the above object, comprising the step of simultaneously culturing methanotroph, and mutant E. coli capable of metabolizing acetic acid, as a co-culture method of methanogen and Escherichia coli the mutant E. coli frdA (fumarate reductase flavoprotein subunit), ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase) and adhE (alcohol / acetaldehyde dehydrogenase) activity of the protein expressed from one or more genes selected from the group consisting of and patZ (peptidyl-lysine acetyltransferase) Provides a method for co-culture of methanza-degrading bacteria and E. coli, in which the activity of the protein expressed from the gene has been lost.

Another aspect of the present invention is a method for producing a compound having 4 or more carbon atoms, comprising simultaneously culturing methanotroph and mutant E. coli capable of metabolizing acetic acid in order to achieve the above object. to provide.

The present invention provides a composition and method capable of producing a compound having 4 or more carbon atoms with high efficiency by converting _{methane (CH 4) at a lower price than petroleum.} Methanogens, which are microorganisms that use methane, may be inhibited by the high concentration of organic acids produced thereby.In the present invention, partner microorganisms capable of producing useful compounds having 4 or more carbon atoms using organic acids produced by methanogens By co-culturing the methanogens and the two microbial strains, a system was developed in which both microbial strains can grow stably.

In particular, the general wild-type E. coli overcomes the disadvantage of not having the characteristic of not growing together with methanogens, and exhibits excellent growth rates even when co-cultured with methanogens, and has a characteristic capable of producing a compound having a carbon number of 4 or more. Methane Capital Using Mutants The present invention provides a composition and method capable of producing a compound having 4 or more carbon atoms with high efficiency by converting _{methane (CH 4) at a low price compared to petroleum.} Methanogens, which are microorganisms that use methane, may be inhibited by the high concentration of organic acids produced thereby.In the present invention, partner microorganisms capable of producing useful compounds having 4 or more carbon atoms using organic acids produced by methanogens By co-culturing the methanogens and the two microbial strains, a system was developed in which both microbial strains can grow stably.

In particular, the general wild-type E. coli overcomes the disadvantage of not having the characteristic of not growing together with methanogens, and exhibits excellent growth rates even when co-cultured with methanogens, and has a characteristic capable of producing a compound having a carbon number of 4 or more. Using the mutant, we succeeded in constructing a co-culture system with methanogens, and both the methanogens and partner microorganisms can exhibit a high growth rate through the optimal inoculation ratio.

In addition, in the case of a technology that has improved the methanogens to directly produce useful compounds having 4 or more carbon atoms using methane, the methanogens are regenerated through repetitive and continuous fermentation and cultivation processes. There is a disadvantage in that stability is poor because it may have characteristics, but in the present invention, it has the advantage of showing excellent stability by confirming that the same characteristics are maintained even during continuous fermentation while culturing the methane-magnetizing bacteria together with a partner microorganism. Accordingly, the composition and production method using the co-culture system of the present invention is expected to be useful for repetitive production on a larger scale for industrial purposes.

In the present invention, a new system can be constructed by changing the type of compound to be produced without difficulty, by using a partner microorganism having the characteristics of easy introduction of external genes and easy strain engineering, without directly improving the methanogens. In addition, there is an effect of producing a high value-added compound at a similar level of productivity when compared to the case where the methane magnetizing bacteria are directly improved in the related art.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a M. capsulatus bath classified as Type I among methanogens and M. trichosporium OB3b classified as Type II. 1B) is a graph comparing the cell growth rate and the concentrations of acetic acid (AA) and succinic acid (SA) in the medium by culturing by supplying methane under oxygen-restricted conditions.

Figure 2 is a M. capsulatus bath (M. capsulatus bath) NMS medium (with copper, Figure 2A) to which 10 μM of CuCl _{2 is added and a medium to which CuCl 2} is not added (without copper, Figure 2) After culturing by supplying methane under oxygen-restricted conditions in B), the cell growth rate and the concentration of acetic acid (AA) and succinic acid (SA) in the medium were measured and compared.

Figure 3 is a methylococcus capsulatus bath (M. capsulatus bath) oxygen restriction condition (reduced from 14.7% to 3.5%), sulfur restriction condition (reduced to 1/10 level), nitrogen restriction condition (1/10 level) After cultivation in each cell mass), creativity 1 per cell mass is methyllococcus capsulatus bath, classified as Type I, and methyl, classified as Type II, among methanogens. Sinus trichosporium OB3b ( M. trichosporium OB3b, Fig. 1B) was cultured by supplying methane under oxygen-limited conditions, and their cell growth rate and the concentration of acetic acid (AA) and succinic acid (SA) in the medium were measured. This is a comparison graph.

Figure 2 is a M. capsulatus bath (M. capsulatus bath) NMS medium (with copper, Figure 2A) to which 10 μM of CuCl _{2 is added and a medium to which CuCl 2} is not added (without copper, Figure 2) After culturing by supplying methane under oxygen-restricted conditions in B), the cell growth rate and the concentration of acetic acid (AA) and succinic acid (SA) in the medium were measured and compared.

Figure 3 is a methylococcus capsulatus bath (M. capsulatus bath) oxygen restriction condition (reduced from 14.7% to 3.5%), sulfur restriction condition (reduced to 1/10 level), nitrogen restriction condition (1/10 level) Is a graph comparing by measuring the concentration of acetic acid produced per cell mass after culturing each in (reduced to ).

Figure 4 is a graph comparing the concentration of acetic acid measured by culturing M. capsulatus Bath in a medium containing limited nitrogen source under various conditions. As a control (Normal (NO ₃ )), a medium containing nitric acid was used as a nitrogen source, and a medium containing nitric acid was limited to 1/10 level (1/10 NO ₃ ), and ammonia was limited to 1/10 level. A medium containing (1/10 NH ₄ ), a medium containing a mixture of nitric acid and ammonia was limited to a level of 1/10, and the containing medium (1/10 NO ₃ & NH ₄ ) was used, respectively.

5 is a diagram showing a fermentation system of methane magnetizing bacteria.

Figure 6 is a methylococcus capsulatus bath (M. capsulatus Bath) after culturing in two different medium nitrogen sources, cell growth rate and concentrations of acetic acid (AA), succinic acid (SA) and malic acid (MA) in the medium. This is a graph that was measured and compared. 6A shows the results of culturing in 3xNMS medium, and FIG. 6B shows the results of culturing in 3xAMS medium.

Figure 7 shows the amount of acetic acid (acetate) production measured after culturing M. capsulatus Bath in 3xNMS medium and 3xAMS medium for 48 hours and succinate production measured after incubating for 44 hours. This is a comparison graph.

Figure 8 is after co-culturing each of its partner microorganism candidate strains with M. capsulatus Bath, cell growth rate and acetic acid (AA), succinic acid (SA), and mevalonic acid (MVA) in the medium. It is a graph comparing by measuring the concentration of. FIG. 8A is a wild-type E. coli MG1655 strain as a partner microorganism, FIG. 8B is an E. coli DSM01 strain as a partner microorganism, and FIG. 8C is a result of using the E. coli SBA01 strain as a partner microorganism.

9 is an E. coli SBA01 strain transformed with a plasmid vector constructed using a gene related to the mevalonic acid production pathway ("SBA01-MVA"), an Escherichia coli SBA01 strain without introducing the plasmid ("SBA01-no plasmid") and meval After culturing the E. coli SBA01 strain ("SBA01-control plasmid") transformed with a plasmid irrelevant to the production pathway of ronic acid, the cell growth rate and the concentration of acetic acid and mevalonic acid in the medium were measured and compared.

FIG. 10 is a graph comparing M. capsulatus Bath and E. coli SBA01 strains by co-culture at different inoculation ratios, and then measuring and comparing their cell growth rates and concentrations of acetic acid and succinic acid in the medium. . A to C of Figure 10 shows the cell growth and acetic acid, succinic acid concentration change of the methane-magnetizing bacteria, and Figure 10 D, E shows the cell growth of the E. coli SBA01 strain. A is the result of inoculating the strains at a ratio of 1:1, B and D at a ratio of 5:1, and C and E at a ratio of 10:1, respectively.

Figure 11 is after co-culture with M. capsulatus Bath and Escherichia coli SBA01 strain under optimized conditions using methane as the sole carbon source, and then their cell growth rate and acetic acid (AA) and succinic acid in the medium ( SA), malic acid (MA) and mevalonic acid (MVA) concentrations were measured and compared. FIG. 11A shows the cell growth rate of Methylococcus capsulartus bath, and FIG. 11B shows the cell growth rate of E. coli SBA01.

Figure 12 is not co-cultured with methanogens, and after culturing the E. coli SBA01 strain introduced with the mevalonic acid production pathway alone while supplying methane, the cell growth rate of the E. coli SBA01 strain and the concentration of succinic acid and mevalonic acid in the medium are measured. It is a graph that was compared.

13 is a M. capsulatus Bath (M. capsulatus Bath) and E. coli SBA01 strains were first cultured and then continuously secondary cultured in a new medium, and then their cell growth rate and acetic acid (AA) and succinic acid in the medium It is a graph comparing concentrations of (SA), malic acid (MA) and mevalonic acid (MVA). FIG. 13A shows the cell growth rate of Methylococcus capsulartus bath, and FIG. 13B shows the cell growth rate of E. coli SBA01.

FIG. 14 is a diagram showing a process of converting mevalonic acid from methane by co-culture by introducing a gene related to mevalonic acid biosynthesis to a mutant Escherichia coli, which is a methane magnetizing bacterium and its partner microorganism, a mutant E. coli.

Hereinafter, the present invention will be described in detail.

1. Composition for production of compounds having 4 or more carbon atoms

One aspect of the present invention provides a composition for producing a compound having 4 or more carbon atoms.

The composition for production of a compound having 4 or more carbon atoms includes a mutant E. coli capable of metabolizing methanotroph and acetic acid, and the mutant E. coli is a frdA (fumarate reductase flavoprotein subunit) gene, hereinafter, the present invention in detail. Explain.

1. A composition for producing a compound having 4 or more carbon atoms

One aspect of the present invention provides a composition for producing a compound having 4 or more carbon atoms.

The composition for producing the compound having 4 or more carbon atoms includes methanotroph, and mutant E. coli capable of metabolizing acetic acid. The composition may be for producing a compound having 4 or more carbon atoms from methane (CH _{4 ).}

The mutant E. coli is active in the protein expressed from one or more genes selected from the group consisting of frdA (fumarate reductase flavoprotein subunit), ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase) and adhE (alcohol / acetaldehyde dehydrogenase) and patZ ( The activity of a protein expressed from the peptidyl-lysine acetyltransferase) gene may be lost, and a biosynthesis-related gene of the compound having 4 or more carbon atoms may be introduced.

_{In the composition for producing a compound having 4 or more carbon atoms from methane (CH 4} ) of the present invention, two microbial strains can construct a co-culture system as the methanogens and mutant E. coli are included together. have. Methanogens produce and secrete organic acids (acetic acid, succinic acid, etc.) using compounds with 1 carbon number such as methane (CH _{4 ), and mutant Escherichia coli uses organic acids produced by methanogens to produce compounds with 4 or more carbon atoms.} Since it can be produced, the composition of the present invention can be used for the production of a compound having 4 or more carbon atoms.

In the present invention, the term "co-culture system" refers to a system for culturing two or more kinds of strains together under the same culture conditions, and a culture in which two or more strains are cultivated together, a culture method, the activity of each of the strains and It is a concept that includes all of the interactions between strains and the resulting products.

The composition may not contain a carbon source other than methane (CH _{4 ).} The methane may be dissolved and included in the composition, or may be provided in a form in which methane gas is supplied to the composition. Other carbon sources other than methane may be, for example, methanol, glucose, starch, or sugar, but are not limited thereto. In the absence of a carbon source other than methane, the concentration of the other carbon source in the composition is 0, or the methanogens and mutant E. coli in the composition contain a carbon source at a level that cannot be continuously grown using a carbon source other than methane. It may have been. However, the initial composition does not contain other carbon sources other than methane, but as the methanogens and mutant Escherichia coli in the composition progress through growth and metabolism using the methane as a carbon source, organic acids produced using methane, such as Acetic acid, succinic acid, malic acid, and the like may be included in the composition, and a compound having 4 or more carbon atoms produced by mutant E. coli using the organic acid (eg, acetic acid) may be included in the composition.

The methanotroph is a bacterium that can metabolize methane by using methane as a carbon source and an energy source, and any strain having such properties may be used. Methanobacteria can be classified into two types: Type I (γ-proteobacteria) and Type II (α-proteobacteria), depending on which pathway is used in the process of producing organic compounds from methane. The methanogens of the present invention may be those that perform carbon assimilation using the RuMP (ribulose monophosphate) pathway, or those that perform carbon assimilation using the serine pathway, for example, using the RuMP pathway. Thus, it may be a methanogens capable of producing organic acids from methane. The methanogens may be strains classified as Methylococcus capsulatus , Methylosinus trichosporium , or Methylomonas sp., for example, methylococcus capsulatus. It may be a Lococcus capsulatus Bath (Methylococcus capsulatus Bath) strain.

The mutant E. coli is a strain capable of metabolizing and growing even under a condition where an organic acid is the only carbon source, and specifically, the organic acid may be acetic acid, succinic acid, and/or malic acid, and more specifically, acetic acid. The mutant E. coli can be used for production, growth (increase of biomass), or energy metabolism of a compound having 4 or more carbon atoms by using the organic acid as a carbon source or an energy source.

Proteins expressed from the frdA (fumarate reductase flavoprotein subunit) gene, ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase), and adhE (alcohol/acetaldehyde dehydrogenase), respectively, are involved in the metabolic circuit of succinic acid, lactic acid, acetic acid and alcohol production. It is a protein, and when the activity of a protein expressed from one or more genes selected from the group consisting of the above genes is lost, the production of by-products is suppressed, and the characteristic of producing acetyl-CoA from a carbon source such as glucose may be increased. Accordingly, in the mutant E. coli of the present invention, its acetyl-CoA productivity may be increased than that of E. coli in which the activity of the protein is not lost. The mutant E. coli may have lost all of the activities of proteins expressed from frdA (fumarate reductase flavoprotein subunit), ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase), and adhE (alcohol/acetaldehyde dehydrogenase).

In addition, the mutant Escherichia coli is an Escherichia coli strain characterized in that the activity of the protein expressed from the patZ gene is essentially lost, cspC (cold shock domain-containing protein), mukB (Mukaku), lomR (lambda outer membrane protein) And yhjE (putative major facilitator superfamily transporter). The activity of a protein expressed from at least one gene selected from the group consisting of yhjE (putative major facilitator superfamily transporter) may be additionally lost. In addition, the mutant E. coli may have lost the activity of all proteins expressed from the genes of cspC , mukB , lomR, and yhjE.

The term "lost activity of a protein expressed from a gene" of the present invention means that the protein fails to perform the function indicated by the protein in a wild-type individual or has a reduced function. This means that a mutation occurs in the gene, that the expression of the protein (such as RNA transcription or protein translation) is inhibited, that an inhibitor of the activity of the protein is present, that the protein is moved or released to the outside of the cell, the The concept includes, but is not limited to, that the decomposition of the protein is accelerated, that the substrate of the reaction catalyzed by the protein cannot bind to the protein, and that the protein cannot bind to its receptor.

When the patZ , cspC , mukB , lomR and yhjE genes are mutated, the mutated patZ , cspC , mukB , lomR and yhjE genes may each include a nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 5, but are limited thereto. It does not become.

The mutation of the patZ gene may be that Trp, the 501st amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 6, is mutated with a stop codon (Trp501Stop), and the mutation of the cspC gene is of SEQ ID NO: 7 Gln, the 58th amino acid of the wild-type enzyme consisting of an amino acid sequence, may be mutated with a stop codon (Gln58Stop), and the mutation of the mukB gene is the 54th amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 8. Phosphorus Asp may be mutated to Glu (Asp54Glu), and the mutation of the lomR gene may be that Pro, which is the 114th amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 9, is mutated to Leu (Pro114Leu), The mutation of the yhjE gene may be that Ile, which is the 210th amino acid of the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 10, is mutated to Met (Ile210Met).

In the present invention, the term "mutation" means that the DNA molecule on which the genetic information is recorded differs from the original DNA by various factors. When a mutation occurs, a change occurs in the protein produced by the gene, which is genotype. It brings about a change in quality, which can change the biological characteristics of the individual.

The mutant E. coli may be a mutant microorganism described in Korean Patent Publication No. 2018-0034280, or a gene related to the biosynthesis of a compound having 4 or more carbon atoms is introduced into the mutant E. coli prepared through the method described in the Korean Patent Application Publication No. 2018-0034280. Can be.

The mutant E. coli may have its acetyl-CoA productivity higher than that of wild-type E. coli, and specifically, the frdA (fumarate reductase flavoprotein subunit), ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase) and adhE. The activity of the protein expressed from (alcohol/acetaldehyde dehydrogenase) may be increased compared to the acetyl-CoA productivity of E. coli that has not been lost.

The mutant E. coli of the present invention is a strain capable of growth and metabolism using acetic acid even under conditions in which acetic acid is the only carbon source as the activity of proteins expressed from the above genes is lost.

The mutant E. coli may be a gene introduced into the E. coli SBA01 strain deposited with the accession number KCTC13040BP, and a gene related to the biosynthesis of the compound having a carbon number of 4 or more was introduced, and the E. coli SBA01 strain was assigned the accession number KCTC13040BP to the Microbial Resource Center of the Korea Institute of Bioscience and Biotechnology in 2016 Since it was deposited on June 10, it is a microorganism that can be easily obtained by a person skilled in the art pre-sale from the depositing institution.

The compound having 4 or more carbon atoms may be a compound having 5 or more carbon atoms, a compound having 6 or more carbon atoms, a compound having 8 or more carbon atoms, a compound having 10 or more carbon atoms, or a compound having 12 or more carbon atoms, but is not limited thereto. In addition, the compound having 4 or more carbon atoms may be mevalonate, butanol, isobutanol, pentanol, isopentanol, bisabolen, bisabolol, isoprene, lycopene, or himachalene, but is not limited thereto. The composition of the invention may be a composition for use in producing mevalonic acid. In order to produce a compound having 4 or more carbon atoms as described above, a gene related to biosynthesis of the compound having 4 or more carbon atoms is introduced into the mutant E. For example, when the compound having 4 or more carbon atoms is mevalonic acid, the mutant E. coli may be transformed by introducing a polynucleotide encoding an MvaE enzyme and an MvaS enzyme. The MvaE enzyme has acetoacetyl-CoA thiolase activity and 3-hydroxy-3-methylglutaryl-CoA reductase activity. In addition, the MvaS enzyme may have a 3-hydroxy-3-methylglutaryl-CoA synthase activity. The polynucleotide encoding the MvaE enzyme and the MvaS enzyme may be derived from Enterococcus faecalis , and the polynucleotide may be included in a vector such as a plasmid and transformed into the mutant E. coli.

In a specific embodiment of the present invention, the introduction of genes related to biosynthesis thereof into mevalonic acid in relation to the compound having 4 or more carbon atoms is described, but this is only one representative example, so the compound having 4 or more carbon atoms is mebalan The present invention is not limited to ronic acid, and any known gene or metabolic pathway for biosynthesis of compounds having 4 or more carbon atoms can be applied to the present invention. In addition, a person skilled in the art in the same technical field as the present invention can appropriately select and apply any introduction method for introducing the biosynthesis-related gene or related metabolic pathway into the mutant E. coli.

_{The composition for producing a compound having 4 or more carbon atoms from methane (CH 4} ) of the present invention may include the methanogens and the mutant Escherichia coli in a ratio of 3:1 to 15:1. Specifically, the ratio of the methanogens and the mutant E. coli is, 4:1 to 13:1, 5:1 to 12:1, 8:1 to 11:1, 9:1 to 11:1 or 10:1 May be, but is not limited thereto. When the methanogens and mutant E. coli are included in the composition in the ratio of the above range, the two strains may not negatively affect each other's growth, and the growth rate of cells may be improved. For example, in the case of inoculating and culturing methanogens and mutant Escherichia coli at a ratio of the above range, the number of cells may increase by 20 times or more for methanogens and 10 times or more for mutant Escherichia coli within 48 hours. The ratio of the methanogens and mutant E. coli may be the ratio of the number of microbial cells measured through optical density (OD), and may be, for example, an optical density of 600 nm wavelength. Specifically, the ratio may be calculated by comparing the measured optical density value of the mutant E. coli, based on the measured optical density value 1 of the methane magnetizing bacteria.

The composition for producing a compound having 4 or more carbon atoms from _{methane (CH 4} ) of the present invention may further include copper ions. The copper ions _{may be included in the form of CuCl 2} , for example, 5 μM to 15 μM, 7 μM to 13 μM, or 9 to 11 μM. The copper ions can promote the activity of methane monooxygenase (MMO) or electron transport, and accordingly, the process of producing organic acids from methane in the composition of the present invention is promoted, resulting in production efficiency. Can be increased.

The composition for producing a compound having 4 or more carbon atoms from _{methane (CH 4} ) of the present invention _{may include nitric acid (NO 3} ⁻ ) as a nitrogen source. The "nitrogen source" refers to an external nitrogen compound accepted by the methanogens or mutant E. coli contained in the composition of the present invention to synthesize a nitrogen compound. The nitrogen source may be included in the composition in the form of a mixture of nitric acid and ammonia (NH ₄ ^{+ ), but the composition may not contain ammonia.} When nitric acid is used as the nitrogen source in the composition of the present invention, the growth rate of methanogens or the production of organic acids may be increased compared to the case of using ammonia. When the amount of organic acid produced by the methanogens increases, the mutant E. coli can use the organic acid to produce a compound having 4 or more carbon atoms, thereby improving the productivity of a compound having 4 or more carbon atoms.

The nitrogen source may be included in the composition at a concentration of 5% to 15% of the nitrogen source concentration contained in a conventional NMS (nitrate mineral salts) medium, and the nitrogen source concentration contained in the NMS medium may be, for example, 0.5 mM to 2 mM. However, it is not limited thereto.

In addition, the nitrogen source may be included in a concentration of 0.1 mM to 50 mM in the composition of the present invention. Specifically, the nitrogen source may be included in a concentration of 0.1 mM to 45 mM, 0.2 mM to 45 mM, 0.5 mM to 40 mM, or 0.8 mM to 35 mM in the composition of the present invention. More specifically, for an excellent organic acid production efficiency effect, the nitrogen source may be included in the composition at a concentration of 0.1 mM to 3 mM, 0.5 mM to 2 mM, or 0.8 mM to 1.5 mM, while securing a sufficient cell mass. In order to achieve excellent organic acid production efficiency, the nitrogen source may be included in the composition at a concentration of 15 mM to 45 mM, 20 mM to 40 mM, or 25 mM to 35 mM.

The composition of the present invention may further include an additive. Any of the additives may be used as long as they do not inhibit the growth and metabolism of methanogens and mutant Escherichia coli contained in the composition of the present invention. For example, the additive may be a protective agent, a buffer, or a carrier. The protective agent protects the methanogens and mutant Escherichia coli in the process of storing, storing, and distributing the composition of the present invention without affecting the growth and metabolism of the methanogens and mutant Escherichia coli, and their damage Or anything that functions to prevent death. When the composition of the present invention is stored, stored, distributed or used in a lyophilized state, the protective agent may be a cryoprotectant. The cryoprotectant may be one or more selected from the group consisting of skim milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclodextrin, glycerol, chicory, potassium phosphate, and honey, but is not limited thereto, and freezing of the composition Any agent that can prevent the methanogens and mutant E. coli from being damaged or killed during the drying process may be included. When the cryoprotectant is included in the composition of the present invention, the composition may be used in the form of a lyophilized product, such as a powder, through a lyophilization process. The lyophilized composition has advantageous advantages in formulation, packaging, storage, etc., and has an effect of preserving the methanogens and mutant E. coli contained therein for a long time.

2. Co-cultivation method of Methanobacteria and Escherichia coli

Another aspect of the present invention provides a method of co-culturing methanogens and E. coli.

The co-culture method of the methanogens and E. coli includes culturing methanotroph and mutant E. coli capable of metabolizing acetic acid together in the same medium, and the mutant E. coli is frdA (fumarate reductase flavoprotein subunit). ), the protein expressed from the ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase) and adhE (alcohol / acetaldehyde dehydrogenase) activity and patZ (peptidyl-lysine acetyltransferase protein expressed from one or more genes selected from the group consisting of a) gene The activity of is lost.

For the explanation of the above methane magnetizing bacteria, refer to '1. It is the same as that described for the methanogens in'Composition for producing a compound having 4 or more carbon atoms'.

The mutant E. coli is a strain capable of metabolizing and growing even under a condition where an organic acid is the only carbon source, and specifically, the organic acid may be acetic acid, succinic acid, and/or malic acid, and more specifically, acetic acid. The mutant E. coli can be used for the production, growth (increase of biomass), or energy metabolism of useful compounds by using the organic acid as a carbon source or an energy source.

In addition, the mutant Escherichia coli is an Escherichia coli strain characterized in that the activity of the protein expressed from the patZ gene is essentially lost, cspC (cold shock domain-containing protein), mukB (Mukaku), lomR (lambda outer membrane protein) And yhjE (putative major facilitator superfamily transporter). The activity of a protein expressed from at least one gene selected from the group consisting of yhjE (putative major facilitator superfamily transporter) may be additionally lost. In addition, the mutant E. coli may have lost the activity of all proteins expressed from the genes of cspC , mukB , lomR, and yhjE.

The loss of the activity of the protein expressed from the gene and the mutation of the genes are the same as those described in 1. Composition for producing a compound having 4 or more carbon atoms.

The mutant E. coli may be a mutant microorganism described in Korean Patent Publication No. 2018-0034280, or a mutant E. coli produced through the method described in the Korean Patent Application Publication.

The mutant Escherichia coli may be an Escherichia coli SBA01 strain deposited with the accession number KCTC13040BP, and the Escherichia coli SBA01 strain was deposited on June 10, 2016 under the accession number KCTC13040BP at the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center, Is a microorganism that can be easily obtained by pre-sale from the depository institution.

The mutant E. coli may be transformed by introducing a specific gene according to the purpose of use thereof, and for example, may be introduced a gene related to biosynthesis of a compound having 4 or more carbon atoms.

Methanogens can metabolize methane by using methane as a carbon source, but the efficiency of reusing organic acids produced by methanogens is low, and growth may be inhibited by accumulation of organic acids such as succinic acid. Therefore, when co-cultured with a partner microorganism capable of using an organic acid produced by methanogens, there is a more advantageous advantage compared to culturing the methanogens alone. In addition, when only methane is supplied as the sole carbon source during the cultivation of methanogens, microorganisms that cannot use methane as a carbon source must have the property of using organic acids produced by the methanogens as the sole carbon source. The mutant E. coli of the present invention can use an organic acid as the sole carbon source and, in particular, can be grown using acetic acid, so that the growth of both methanogens and mutant E. coli can be increased through the co-culture method of the present invention.

The step of culturing the methanotroph and mutant E. coli capable of metabolizing acetic acid together in the same medium _{may be culturing under conditions in which methane (CH 4} ) is supplied as a carbon source. Methanophilic bacteria produce organic acids (such as acetic acid) using methane, and mutant E. coli can grow and metabolize using the organic acid as a carbon source.

When the methane (CH ₄ ) is cultivated under a condition in which it is supplied as a carbon source, it may be supplied to contain methane in a ratio of 20% to 40% of air supplied during cultivation, for example, 25% to 35%, Alternatively, it may be supplied to contain methane in a ratio of 28% to 32%. In this case, oxygen may be included in the supplied air in a ratio of 2% to 5%, 2.5% to 4.5%, or 3% to 4%.

In addition, the step of culturing the methanotroph and mutant Escherichia coli capable of metabolizing acetic acid together in the same medium includes inoculating the methanogenic bacteria and the mutant Escherichia coli in a ratio of 3:1 to 15:1. It can be. Specifically, the ratio of the methanogens and the mutant E. coli is, 4:1 to 13:1, 5:1 to 12:1, 8:1 to 11:1, 9:1 to 11:1 or 10:1 May be, but is not limited thereto. When the methanogens and mutant E. coli are inoculated at the ratio of the above range, the two strains may not negatively affect each other's growth, and the growth rate of cells may be improved. For example, in the case of inoculating and culturing methanogens and mutant Escherichia coli at a ratio of the above range, the number of cells may increase by 20 times or more for methanogens and 10 times or more for mutant Escherichia coli within 48 hours. The ratio of the methanogens and mutant E. coli may be the ratio of the number of microbial cells measured through optical density (OD), and may be, for example, an optical density of 600 nm wavelength. Specifically, the ratio may be calculated by comparing the measured optical density value of the mutant E. coli, based on the measured optical density value 1 of the methane magnetizing bacteria.

In addition, the step of culturing the methanotroph and mutant E. coli capable of metabolizing acetic acid together in the same medium may be culturing in the presence of copper ions. The copper ions _{may be included in the form of CuCl 2} , for example, 5 μM to 15 μM, 7 μM to 13 μM, or 9 to 11 μM. The copper ions can promote the activity or electron transport of methane monooxygenase (MMO), thereby increasing the methane conversion rate of the methane magnetizing bacteria to promote their growth and increase the production of organic acids by the methanogens to increase the production of mutant Escherichia coli. Growth can also be promoted.

The step of culturing the methanotroph and mutant Escherichia coli capable of metabolizing acetic acid together in the same medium may be culturing under conditions in which _{nitric acid (NO 3} ^{−) is supplied as a nitrogen source.} The "nitrogen source" refers to an external nitrogen compound that is accepted by the methane magnetizing bacteria or mutant Escherichia coli contained in the composition of the present invention to synthesize the nitrogen compound. The nitrogen source may be supplied in the form of a mixture of nitric acid and ammonia (NH ₄ ⁺ ), but the culturing step may be culturing under a condition in which ammonia is not supplied. When nitric acid is used as the nitrogen source, the growth rate or the production of organic acids of methanogens may be increased compared to the case of using ammonia, and the growth rate of mutant E. coli using the organic acid may also be increased.

The nitrogen source may be included in the composition at a concentration of 5% to 15% of the nitrogen source concentration contained in a conventional NMS (nitrate mineral salts) medium, and the nitrogen source concentration contained in the NMS medium may be, for example, 0.5 mM to 2 mM. However, it is not limited thereto.

In addition, the nitrogen source may be included in a concentration of 0.1 mM to 50 mM in the composition of the present invention. Specifically, the nitrogen source may be included in a concentration of 0.1 mM to 45 mM, 0.2 mM to 45 mM, 0.5 mM to 40 mM, or 0.8 mM to 35 mM in the composition of the present invention. More specifically, for an excellent organic acid production efficiency effect, the nitrogen source may be included in the composition at a concentration of 0.1 mM to 3 mM, 0.5 mM to 2 mM, or 0.8 mM to 1.5 mM, while securing a sufficient cell mass. In order to achieve excellent organic acid production efficiency, the nitrogen source may be included in the composition at a concentration of 15 mM to 45 mM, 20 mM to 40 mM, or 25 mM to 35 mM.

3. Method for producing compounds having 4 or more carbon atoms

Another aspect of the present invention provides a method for producing a compound having 4 or more carbon atoms.

The method for producing a compound having 4 or more carbon atoms includes culturing a methanotroph and a mutant E. coli capable of metabolizing acetic acid at the same time.

The mutant E. coli is a protein expressed from one or more genes selected from the group consisting of frdA (fumarate reductase flavoprotein subunit) gene, ldhA (D-lactate dehydrogenase) gene, pta (phosphotransacetylase) gene, and adhE (alcohol/acetaldehyde dehydrogenase) gene. Activity and activity of a protein expressed from the patZ (peptidyl-lysine acetyltransferase) gene may be lost, and a biosynthesis-related gene of the compound having 4 or more carbon atoms may be introduced.

The method may be to produce a compound having 4 or more carbon atoms from methane (CH _{4 ).}

The methanogens, mutant Escherichia coli, a compound having 4 or more carbon atoms, and a description of their production, refer to '1. It is the same as described for these in'Composition for producing a compound having 4 or more carbon atoms.

The mutant E. coli may be a biosynthesis-related gene of the compound having 4 or more carbon atoms introduced into the E. coli SBA01 strain deposited under the accession number KCTC13040BP.

For a description of the step of simultaneously culturing the methanotroph and mutant E. coli capable of metabolizing acetic acid, see '2. It is the same as described in'Co-cultivation method of methanogen and E. coli.'

The method may be cultured under conditions of supplying methane (CH _{4) as a carbon source.} The methane (CH ₄ ) may be supplied to a mass flow controller (MFC). When methane is supplied by the mass flow controller, the supply rate of methane can be controlled, and the ratio of methane when supplied with other gases such as oxygen and nitrogen can be controlled. The methane may be supplied to be included in a proportion of 20% to 40% of air supplied during cultivation, for example, may be supplied to be included in a proportion of 25% to 35%, or 28% to 32%. . In this case, oxygen may be included in the supplied air in a ratio of 2% to 5%, 2.5% to 4.5%, or 3% to 4%.

When the compound having 4 or more carbon atoms is mevalonate, the method for producing a compound having 4 or more carbon atoms of the present invention includes 10 hours or more, specifically 15 hours or more, 20 hours or more , When co-cultured for 30 hours or more, 40 hours or more, 48 hours or more, 50 hours or more, or 60 hours or more, mevalonic acid is 15 mg/L or more, specifically 20 mg/L or more, 30 mg/L or more, 40 mg It may be produced at a concentration of /L or more, 50 mg/L or more, 55 mg/L or more, 60 mg/L or more, or 61 mg/L or more.

The productivity of a compound having 4 or more carbon atoms according to the production method of a compound having 4 or more carbon atoms of the present invention is similar to the case of culturing the methanogens after improving the methanogens by having the property of producing a compound having 4 or more carbon atoms from methane. In comparison, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 105% or more, 110% or more, 115% or more of the productivity of the compound having 4 or more carbon atoms of the improved methanogens, Or 120% or more.

Another aspect of the present invention provides a composition comprising the methanogenic bacteria and mutant Escherichia coli capable of metabolizing the acetic acid for use in producing a compound having 4 or more carbon atoms from _{methane (CH 4 ).}

Another aspect of the present invention provides a use for producing a compound having 4 or more carbon atoms from _{methane (CH 4} ) of a composition comprising the methanogens and mutant Escherichia coli capable of metabolizing acetic acid.

Another aspect of the present invention provides the use of the methanogens and mutant E. coli capable of metabolizing the acetic acid for producing a composition for producing a compound having 4 or more carbon atoms from _{methane (CH 4 ).}

Hereinafter, the present invention will be described in detail by experimental examples.

However, the following experimental examples specifically illustrate the present invention, and the contents of the present invention are not limited by the following examples.

[Experimental Example 1]

Establishment of acetic acid production conditions using methanotroph

In order to construct a system for producing a compound having 4 or more carbon atoms from methane using methanotroph and its co-culture partner microorganism, first, organic acids, especially acetic acid (acetate) can be produced from methane with high efficiency. The conditions that can be checked were confirmed.

[1-1] Comparison of organic acid production between Type I and Type II methanogens

Methanobacteria can be classified into two types, depending on which pathway is used to produce organic compounds from methane. Type I (γ-proteobacteria) and serine using the ribulose monophosphate (RumP) pathway. It is classified as Type II (α-proteobacteria) using the pathway. Methylococcus capsulatus Bath (hereinafter referred to as'Bath') belonging to Type I and Methylococcus Tricot belonging to Type II in order to use the methanogens, which are more efficient in producing organic acids, among the methanogens. Sporium OB3b ( Methylosinus trichosporium OB3b, hereinafter'OB3b') strains _{were cultured under oxygen-restricted conditions in an environment where methane (CH 4} ) was given, and their organic acid production was checked and compared. Specifically, first, Bath and OB3b strains NMS medium (Nitrate Mineral Salts medium) Was inoculated to a concentration of OD _{600 1.7.} The composition of the NMS medium is as described in Table 1 below. After incubation for 72 hours at 37° C. or 30° C. under oxygen-restricted state, the amounts of acetic acid and succinate were measured by liquid chromatography (LC).

Composition of NMS medium	Content (per liter)
MgSO 4 7H 2 O	1.0 g
KNO 3	1.0 g
CaCl 2 2H 2 O	0.2 g
3.8% (w/v) solution Fe-EDTA	0.1 ml
0.1% (w/v) NaMo 4H 2 O	0.5 ml
FeSO 4 7H 2 O	0.5 mg
ZnSO 4 7H 2 O	0.4 mg
MnCl 2 7H 2 O	0.02 mg
CoCl 2 6H 2 O	0.05 mg
NiCl 2 6H 2 O	0.01 mg
H 3 BO 3	0.015 mg
EDTA	0.25 mg

As a result, the Bath strain showed a high cell growth rate for the same time compared to the OB3b strain, and the Bath strain produced 6.2 mg/L of acetic acid and 2.1 mg/L of succinic acid at 72 hours, compared to the acetic acid and succinic acid production of OB3b. It was measured to be produced in an amount more than twice as large (Fig. 1). From the above results, the methanogens have the ability to produce organic acids such as acetic acid and succinic acid using methane under oxygen-limited conditions. Among the two types of methanogenic bacteria, it was confirmed that the organic acid productivity was more excellent because the methane magnetizing bacteria classified as Type I had a better carbon utilization rate compared to the Type II strain.

[1-2] Confirmation of Methanophilic Bacteria Culture Conditions to Improve Organic Acid Productivity

Through the Experimental Example 1-1, it was confirmed that the methanogens classified as Type I have excellent organic acid production ability, so that the Methylococcus capsulartus bath (hereinafter referred to as'Bath') strain was cultured under various conditions while culturing organic acid production efficiency. Was confirmed.

First, it was confirmed whether or not the organic acid productivity increases when copper ions are contained in the medium. _{10 μM of CuCl 2} was added to the NMS medium of the composition shown in Table 1, and methane was supplied to the Bath strain, and the production of organic acid was measured through LC by culturing under oxygen-restricted conditions, and in the NMS medium to which copper ions were not added. The production of organic acids was compared with the case of the cultured control group.

As a result, the Bath strain cultured in NMS medium to which copper ions were added showed higher production of acetic acid and succinic acid as compared to the control group to which copper ions were not added. It was confirmed that the production of acetic acid was significantly higher than that of the control group (18.7 mg/L) by producing at a concentration of L (FIG. 2). Through the above experimental results, it was found that it is more advantageous to cultivate methane magnetizing bacteria for the purpose of producing organic acids in an environment in which copper ions are present, and copper ions are methane monooxygenase (MMO , methane monooxygenase) It is thought that it can improve the efficiency of converting methane into organic acids by controlling gene expression related to the regulation of enzyme production and helping electron transport.

Next, the methanogens were cultured under conditions in which oxygen, sulfur, and nitrogen were respectively restricted, and their organic acid conversion rate was confirmed. Specifically, 30% of the air was removed from the vial inoculated with the methanogens and replaced by supplying methane. And in order to set the oxygen limit condition, 14.7% of oxygen in the remaining 70% of air was replaced with nitrogen and reduced to 3.5%. In the case of the sulfur limiting condition and the nitrogen limiting condition, the Bath strain was cultured in a medium in which sulfur and nitrogen were reduced to 10% levels in the NMS medium of Table 1, respectively. After culturing the Bath strain under the oxygen, sulfur, and nitrogen conditions as described above, the production amount of acetic acid and succinic acid was confirmed through LC.

As a result, when the Bath strain was cultivated in a nitrogen source-restricted medium, the acetic acid production was 1,884 μmol-acetate/g-DCW at the time point of 24 hours. It was confirmed that it could be produced, and it was newly confirmed that the nitrogen limiting condition allows the production of a much higher concentration of organic acid than the oxygen limiting condition, which was known as an advantageous condition for producing organic acids from the methane magnetizing bacteria (Fig. 3). .

Thus, the nitrogen source to nitrate in order to evaluate more closely the effect of nitrogen source on acid conversion targeting Bath strain (nitrate, NO ₃ ^-), the 10 mM culture medium (control), the nitrate-containing medium (10 mM) used to contain Restriction medium (containing 1 mM nitrate) in which the concentration of nitrate was reduced to 10% level, and _{ammonia-containing medium containing ammonia (NH 4} ⁺ ) instead of nitrate at a concentration of 10 mM reduced the concentration of ammonia to 10% level. Restriction medium (containing 1 mM ammonia), and a restriction medium in which the concentration of nitrate/ammonia was reduced to 10% level in a medium in which nitrate and ammonia were mixed at a concentration of 2.5 mM and 5 mM, respectively (nitrate 0.25 mM, ammonia 0.5 mM containing) was used to compare the organic acid production.

As a result of culturing the Bath strain in the vial under the above three medium conditions and confirming the production amount of acetic acid, when compared with the results in the ammonia-containing medium and the ammonia and nitrate mixed medium, the concentration thereof was limited while using nitrate as a nitrogen source. It was confirmed that the amount of acetic acid production was measured to be the highest (FIG. 4).

[1-3] Optimization of fermentation conditions for methanogens

The fermentation conditions of the methanogens were established and optimized to produce organic acids with high efficiency while increasing the growth rate of the methanogens to secure sufficient cell mass. In order to control the ratio and supply rate of methane, oxygen, and nitrogen gas, gas was supplied at a ratio of 20% of methane and 80% of the remaining gas using a mass flow controller (MFC). A methanogen fermentation system was constructed using a micro-sparger (CNS) (FIG. 5). As a culture medium for methanogens, a medium modified by adding nitric acid (3X), phosphate (1.5X), and trace elements (3X) to the NMS medium containing the components according to Table 1 was used. Capsulartus bath (hereinafter referred to as'Bath') strain was fermented and cultured. In the case of using the medium under the conditions as described above, the growth rate of the methanogens is fast, but after sufficient cell growth occurs, the nitrogen source limiting conditions are set due to consumption of the nitrogen source, so that organic acids can be produced with high efficiency. As a result of culturing the Bath strain under the above conditions, ^{a growth rate of 0.16 h -1} was shown, and it was confirmed that 2.5 mM acetic acid and 2.5 mM succinic acid were produced at the end of fermentation (FIG. 6).

And, using the modified 3xNMS medium to which nitric acid was added, and 3xAMS medium to which ammonia was added instead of nitric acid, the Bath strain was cultured for 48 hours under two different nitrogen source conditions, and its growth rate and organic acid (acetic acid and succinic acid) production amount were measured. Compared. Compared with Bath cultured in 3xAMS medium containing ammonia as nitrogen source, Bath strain cultured in 3xNMS medium containing nitric acid as nitrogen source showed shorter growth delay and higher production of organic acids (especially acetic acid). There was (Fig. 7). Through the above results, it was found that the use of nitric acid as a nitrogen source rather than ammonia is more advantageous for the growth of Bath and the production of organic acids, which is thought to be because ammonia causes an imbalance in electron transport and affects cell physiology.

* [Experimental Example 2]

Confirmation of the characteristics of SBA01 strain as a co-culture partner for methanogens

In order to construct a system for producing a compound having 4 or more carbon atoms from methane, a co-culture partner microorganism having a characteristic capable of co-culture with methane magnetizing bacteria was selected. Methane by selecting co-culture partner microorganisms, which has the characteristics of being resistant to organic acids produced by methane-automated bacteria, and is capable of growing using them, and the characteristics of efficiently converting them into high value-added compounds using the organic acids. A platform for the production of derived useful substances was developed.

As described above, the E. coli SBA01 strain of the present invention was used as a microorganism having characteristics that can be used as a co-culture partner of methanogens. The SBA01 strain has high resistance to acetic acid and has an excellent growth rate using it. In addition, since the activity of acetyl-CoA synthetase (ACS) is 8 times higher than that of wild-type E. coli, acetic acid can be efficiently converted.

[2-1] Comparison of co-culture characteristics of E. coli SBA01 strain and other E. coli strains

In order to confirm that the E. coli SBA01 strain of the present invention has excellent properties that can be used as a co-culture partner of methanogens, wild-type E. coli MG1655, and E. coli DSM01 strain, which can inhibit the production of by-products, were used as a control. Was used. The DSM01 strain is characterized in that some genes are deleted by mutating the wild-type MG1655 strain (MG1655 β frdA β ldhA β pta β adhE ). The SBA01 strain of the present invention is an evolutionary improvement of the DSM01 strain to give new characteristics, and the activity of the patZ gene is lost. In addition, a mevalonate pathway was introduced to produce mevalonate in the E. coli strains. Specifically , an enzyme capable of synthesizing mevalonic acid by constructing a recombinant vector using the MvaE enzyme and MvaS enzyme gene derived from Enterococcus faecalis , and transforming it into the wild-type E. coli MG1655 strain, E. coli DSM01 strain, and E. coli SBA01 strain. E. coli strains expressing the were prepared.

While culturing the wild-type E. coli MG1655, E. coli DSM01 strain, and E. coli SBA01 strain in a minimal medium (M9) containing acetic acid and succinic acid, their cell growth was checked, and changes in the concentration of acetic acid and succinic acid in the medium, and mevalonic acid production were measured. . As a result, the E. coli DSM01 strain did not grow at all in a medium containing acetic acid and succinic acid, whereas the E. coli SBA01 strain showed a high cell growth rate without inhibition of growth by organic acids, and mevalonic acid at a concentration of 288 mg/L for 54 hours. As measured to be produced, it was confirmed that the mevalonic acid production effect was very excellent (FIG. 8). In addition, it was confirmed that the concentration of acetic acid and succinic acid in the medium of the E. coli SBA01 strain decreases with the passage of the culture time, so that the E. coli SBA01 strain can produce mevalonic acid, a compound having 5 carbon atoms, using an organic acid in the medium. It can be seen that it has a characteristic.

[2-2] Confirmation of mevalonic acid production depending on whether or not the mevalonic acid pathway is transformed

In addition to confirming that only Escherichia coli SBA01 strain can function as a co-culture partner of methanogens through Experimental Example 2-1, acetic acid was consumed only when the mevalonic acid pathway was transformed into E. coli SBA01 strain to obtain mevalonic acid. It was verified whether it was produced or not.

Specifically, as in Experimental Example 2-1 , a plasmid vector was constructed through recombination of Enterococcus faecalis- derived MvaE enzyme and MvaS enzyme gene, and the mevalonic acid-producing plasmid was transformed into E. coli SBA01 strain. In addition, an E. coli SBA01 strain that did not transform the mevalonic acid-producing plasmid and an E. coli SBA01 strain into which a plasmid unrelated to mevalonic acid production was introduced were prepared. The three types of E. coli SBA01 strains were pre-cultured three times by supplying glucose or acetic acid as a carbon source to M9 restriction medium, and then cultured again in M9 medium containing acetic acid. After supplying 0.1 mM IPTG to induce the expression of genes related to the mevalonic acid pathway, the concentration of acetic acid remaining in the medium and the amount of mevalonic acid produced were confirmed.

As a result, in the case of the E. coli SBA01 strain not transformed with the plasmid or the E. coli SBA01 strain transformed with the plasmid independent of mevalonic acid production, normal cell growth occurred, but no mevalonic acid was produced. In contrast, in the case of the E. coli SBA01 strain transformed with the mevalonic acid producing plasmid, the concentration of mevalonic acid also increased with cell growth (FIG. 9). Therefore, it was confirmed that the E. coli SBA01 strain had an activity capable of producing mevalonic acid by introducing the mevalonic acid pathway.

[Experimental Example 3]

Confirmation of co-culture of methanogen and SBA01 strain and optimization of inoculation ratio

In Example 2, the E. coli SBA01 strain, which has excellent acetic acid resistance and excellent acetic acid utilization activity, was selected as a candidate for co-culture partner microorganisms of methanogens. Thus, it was confirmed whether or not both strains were able to grow by co-culturing the methanogens and the SBA01 strain, and the co-culture conditions were optimized so that the two strains do not inhibit the growth of each other. To this end, a Methylococcus capsulatus Bath (hereinafter referred to as'Bath') strain was used as the methanogenic bacteria, and the growth rates of the two strains were confirmed by varying the inoculation ratio of the Bath strain and the SBA01 strain.

Specifically, the Bath strain and the SBA01 strain were inoculated into the 3xNMS medium of Experimental Example 1-3 at a ratio of 1:1, 5:1, and 10:1, respectively. As a result of confirming the growth rate by culturing the strains while supplying methane (CH ₄ ) as the sole carbon source, it was observed that both the Bath strain and the SBA01 strain were inoculated and cultured at a ratio of 5:1 and 10:1 It was confirmed that co-culture is possible (FIG. 10). In particular, when the ratio of the methanogen Bath strain and the partner microorganism SBA01 strain was 1:1, growth of both strains was not observed. As the initial inoculation amount of SBA01 increased, the growth of SBA01 and the methanogen Bath strain was negatively affected. It is thought to have an effect, and it was confirmed that inoculation at a ratio of 10:1 is the optimal ratio.

According to the results of previous studies, it is known that even when cultivated together with the methanogens and wild-type E. coli MG1655 strains, E. coli cannot proliferate and the methanogens cannot be continuously grown, so stable co-culture is not possible (Sascha MB Krause et al ., "Lanthanide-). dependent cross-feeding of methane-derived carbon is linked by microbial community interactions", PNAS, 2017 Jan 10;114(2):358-363). This means that any microbial species cannot be co-cultured with methanogens regardless of the type of microorganism. In particular, although it was known that common E. coli cannot be co-cultured with methanogens, in the present invention, SBA01, an E. coli mutant. It has been newly discovered that the strain can be effectively co-cultured with methanogens.

[Experimental Example 4]

Production of mevalonic acid (C5) from methane using the co-culture system of the present invention

[4-1] Confirmation of mevalonic acid productivity by co-culture of methanogens and SBA01 strains

The Methylococcus capsulatus Bath (hereinafter referred to as'Bath') strain was optimized according to the E. coli SBA01 strain and the mevalonic acid pathway-related enzyme introduced in the same manner as in Experimental Example 2. Co-fermentation culture was carried out under co-culture conditions (10:1 inoculation ratio). While co-culturing the methanogens and SBA01 strains for 48 hours, their growth rates were checked, and the concentrations of acetic acid, succinic acid, and mevalonic acid in the culture medium were measured.

As a result, as shown in A of FIG. 11, it can be confirmed that the methanogens grow normally even when cultured with the SBA01 strain, and in the case of succinic acid, its concentration increases similarly to the cell growth rate, but in the case of acetic acid, the medium It can be seen that it is hardly present within. And, as can be seen in B of Figure 11, the E. coli SBA01 strain gradually increased the number of cells according to the co-culture, and it can be confirmed that normal growth is possible. Mevalonic acid was produced by 61 mg/L after 48 hours of co-culture. Confirmed to exist.

In the conventional technology for producing compounds from methane using methanogens, Methylomicrobium alcaliphilum 20Z engineered to produce 2,3-butanediol (2,3-BDO) at a concentration of 68.8 mg/L or 86.2 mg/L. Research results (Anh Duc Nguyen et al ., "Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane", Metab Eng . 2018 May;47:323-333), crotonic acid up to 70 mg/L Methylomicrobium buryatense 5GB1C engineered to produce butyric acid at 40 mg/L (Shivani Garg et al ., "Bioconversion of methane to C-4 carboxylic acids using carbon flux through acetyl-CoA in engineered Methylomicrobium buryatense 5GB1C", Metab Eng . 2018 Jul;48:175-183.). In the present invention, unlike the prior art, although not directly engineering the methane-magnetizing bacteria, the productivity of the improved methane-automating bacteria was better or similar to that of the productivity. Accordingly, it was confirmed that the co-culture system of the present invention is a new technology having excellent effects that can produce compounds having 4 or more carbon atoms with high efficiency using methane as a raw material.

[4-2] Confirmation of mevalonic acid production according to methane supply when culturing E. coli SBA01 strain alone

Through the Experimental Example 2, it was confirmed that the E. coli SBA01 strain transformed with a gene related to the mevalonic acid pathway can produce mevalonic acid under the condition where acetic acid is given, and the SBA01 strain was methane magnetized through Experimental Example 4-1. When co-cultured with bacteria, it was confirmed that mevalonic acid could be successfully produced when methane was supplied. Thus, when methane is supplied while culturing the E. coli SBA01 strain alone without methanogens, it was confirmed whether or not mevalonic acid could be produced.

Specifically, the E. coli SBA01 strain transformed with the mevalonic acid-producing plasmid was cultured under the same conditions as the co-culture conditions in Experimental Example 4-1 (same medium, pH, temperature, etc.). While supplying methane as the sole carbon source, the growth of the E. coli SBA01 strain was confirmed, and the concentration of mevalonic acid was measured. As a result, it was determined that even though methane was continuously supplied, the SBA01 strain did not grow and mevalonic acid was not produced (FIG. 12).

From the above experimental results, E. coli SBA01 strain was transformed with a mevalonic acid producing plasmid, but there is no activity to use methane, so when only methane is supplied as the only carbon source, it cannot produce mevalonic acid. It was confirmed that only when successfully co-cultured, a compound with a carbon number of 4 or more, such as mevalonic acid, could be produced from methane and growth was possible.

[Experimental Example 5]

Confirmation of the stability of the co-culture system of methanogens and SBA01 strains

In order to confirm whether stable co-cultivation of the Methanobacteriaceae and E. coli SBA01 strains is possible , continuous fermentation of the Methylococcus capsulatus Bath (hereinafter'Bath') strain and the SBA01 strain was performed. The SBA01 strain was used in the same manner as in Experimental Example 3 to which the mevalonic acid pathway was introduced. The Bath strain and the SBA01 strain were subjected to primary co-fermentation culture under the optimized co-culture conditions (10:1 inoculation ratio) according to Experimental Example 3, and then 10% of the strain was again inoculated into a new fermentation medium under the same conditions. Secondly, continuous fermentation was performed.

As a result, even if continuous culture was performed, it was confirmed that both the Bath strain and the SBA01 strain continued to grow, confirming that stable co-culture was possible.In particular, the Bath strain in a steady-state even in the second inoculation stage. It was confirmed that the ratio of the strain and SBA01 was maintained at 10:1. In addition, in the second culture process, as in the first co-culture, the concentration of succinic acid in the medium increased, but the acetic acid concentration did not increase, and mevalonic acid was successfully synthesized and the concentration increased (FIG. 13). This means that even if fermentation through continuous culture is performed, succinic acid and acetic acid are produced by methanogens, and the SBA01 strain can normally produce mevalonic acid using acetic acid. Therefore, the system for co-culturing the SBA01 strain capable of using the methanogens and acetic acid of the present invention has the potential to be industrially applied for the purpose of producing useful compounds.

In the above, representative experimental examples of the present invention have been exemplarily described, but the scope of the present invention is not limited to the specific experimental examples as described above, and those of ordinary skill in the relevant field are within the scope of the claims of the present invention. It will be possible to change it accordingly.

Claims (17)

1. Methanotroph, and a composition for producing a compound having 4 or more carbon atoms from _{methane (CH 4} ), including mutant Escherichia coli capable of metabolizing acetic acid.
2. The method according to claim 1,

   The mutant E. coli is active in the protein expressed from one or more genes selected from the group consisting of frdA (fumarate reductase flavoprotein subunit), ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase) and adhE (alcohol / acetaldehyde dehydrogenase) and patZ ( peptidyl-lysine acetyltransferase) is that the activity of the protein expressed from the gene is lost, and the biosynthesis-related gene of the compound having 4 or more carbon atoms is introduced.
3. The method according to claim 1,

   The methanogens are to perform carbon assimilation using the RuMP (ribulose monophosphate) route, the composition.
4. The method of claim 3,

   The methanogens are methylococcus capsulatus bath (Methylococcus capsulatus Bath) will, the composition.
5. The method according to claim 1,

   The mutant E. coli has its acetyl-CoA productivity higher than that of wild-type E. coli acetyl-CoA, the composition.
6. The method according to claim 2,

   The mutant E. coli is the activity of a protein expressed from one or more genes selected from the group consisting of cspC (cold shock domain-containing protein), mukB (Mukaku), lomR (lambda outer membrane protein), and yhjE (putative major facilitator superfamily transporter). The composition is further lost.
7. The method according to claim 1,

   The compound having 4 or more carbon atoms is any one selected from the group consisting of mevalonate, butanol, isobutanol, pentanol, isopentanol, bisabolen, bisabolol, isoprene, lycopene, and himachalene. Composition.
8. The method of claim 7,

   The biosynthesis-related gene of mevalonic acid, the composition comprising a polynucleotide encoding the enzyme MvaE and MvaS enzyme.
9. The method according to any one of claims 1 to 8,

   The methanogen and the mutant Escherichia coli will be contained in a ratio of 3:1 to 15:1, the composition.
10. The method according to claim 1,

    The composition further comprises a copper ion, the composition.
11. The method according to claim 1,

    The composition will contain nitric acid (NO ₃ ^- ) as a nitrogen source, the composition.
12. The method of claim 11,

    The nitrogen source in the composition will be contained in a concentration of 0.1 mM to 45 mM, the composition.
13. As a co-culture method of methanogens and E. coli, comprising culturing methanotroph and mutant E. coli capable of metabolizing acetic acid together in the same medium,

    The mutant E. coli is active in the protein expressed from one or more genes selected from the group consisting of frdA (fumarate reductase flavoprotein subunit), ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase) and adhE (alcohol / acetaldehyde dehydrogenase) and patZ ( peptidyl-lysine acetyltransferase) is that the activity of the protein expressed from the gene is lost, the method of co-culture of methanogens and Escherichia coli.
14. Methanotroph, and a method for producing a compound having 4 or more carbon atoms comprising the step of simultaneously culturing mutant E. coli capable of using acetic acid.
15. The method of claim 14,

    The mutant E. coli is active in the protein expressed from one or more genes selected from the group consisting of frdA (fumarate reductase flavoprotein subunit), ldhA (D-lactate dehydrogenase), pta (phosphotransacetylase) and adhE (alcohol / acetaldehyde dehydrogenase) and patZ ( peptidyl-lysine acetyltransferase) is that the activity of the protein expressed from the gene is lost, and the biosynthesis-related gene of the compound having 4 or more carbon atoms is introduced, a method for producing a compound having 4 or more carbon atoms.
16. The method of claim 14,

    A method for producing a compound having 4 or more carbon atoms by culturing under conditions of supplying methane (CH _{4) as a carbon source.}
17. The method of claim 16,

    The methane (CH ₄ ) is supplied to a mass flow controller (MFC), a method for producing a compound having 4 or more carbon atoms.

Images