KR102005664B1 - Transformed methanotrophs for producing aminolevulinic acid and uses thereof - Google Patents

Abstract

The present invention relates to a method for producing an aminolevulinic acid synthase gene in which methanotrophic bacteria in which a TCA metabolic pathway (TCA cycle) for biosynthesis of succinyl-CoA and a serine pathway for serine biosynthesis for glycine biosynthesis are simultaneously activated, A transformant for acid production, a composition for producing aminolevulinic acid containing the transformant, a kit for producing aminolevulinic acid containing the composition, and the transformant are cultured to produce aminolevulinic acid from methane . Using the transformants provided in the present invention, aminolevulinic acid can be produced by using atmospheric methane as a sole carbon source, so that it can be widely used for production of economical aminolevulinic acid.

Description

[0001] Transformed methanotrophs for producing aminolevulinic acid and uses thereof [0002]

The present invention relates to a transformant for the production of aminolevulinic acid and a use thereof. More specifically, the present invention relates to a TCA metabolic pathway (TCA cycle) for biosynthyl-CoA biosynthesis and a serine pathway for biosynthesis of glycine ), A transformant for producing aminolevulinic acid in which an aminolevulinic acid synthase gene is introduced into a methanotrophic bacterium activated simultaneously, a composition for producing aminolevulinic acid containing the transformant, an amino And a method for producing aminolevulinic acid from methane by culturing the transformant.

Methane is the main component of natural gas and shale gas, which occurs in the anaerobic digestion process of biomass or mainly in landfill gas. Each year, 2 million tonnes are generated domestically and 150 billion tonnes are generated globally. It is known to cause 21 times higher greenhouse gas effect than CO ₂ . These methane gases can produce various high-value-added end products such as biofuels or chemical raw materials through bioconversion using methane magnetism bacteria.

Aminolevulinic acid (ALA) is an essential precursor of the tetrapyrrole biosynthetic process involving chlorophyll and heme in organisms with five aliphatic compounds of carbon. Tetrapyrrole in vivo has undergone several steps Hemoglobin, chlorophyll, vitamin B12 and the like.

According to the characteristics of this biosynthetic pathway of ALA, it can be applied for agriculture such as plant growth promoter, herbicide, or insecticide. It can be used for medical applications such as cancer treatment or brain tumor diagnosis and production of enzymes including cosmetic additives and heme structure .

ALA is an environmentally friendly biochemical pesticide and plant promoter that promotes the growth and development of plants upon the treatment of plants. It has been reported that this promoting effect is due to increase of chlorophyll content, increase of photosynthesis activity and inhibition of respiration.

In the pharmaceutical sector, ALA has been widely used as a biologically active substance such as microbial resistance and skin cancer treatment, and is widely known as a photodynamic therapy (PDT) photodynamic therapy for the treatment of malignant tumors allowed by the FDA. ALA forms a precursor, protoporphyrin IX, through the heme biosynthetic pathway, which is rapidly metabolized after a brief period of action. When the protoporphyrin IX is activated as a photosensitizer, it exhibits fluorescence and cytotoxicity, and is used for diagnosing or treating cancer.

Although ALA can be synthesized through chemical processes, it has a complicated synthesis step and low yield. However, when using microorganisms, a large amount of ALA can be synthesized and the biological synthesis process is very simple.

The biological synthesis process of ALA is largely possible through two pathways. ALA can be produced via the C5 pathway (C5 pathway, Beale pathway) or the C4 pathway (Shemin pathway) in microorganisms. The C5 pathway is present in plants, algae and many microorganisms and synthesizes ALA from glutamate, wherein glutamyl-tRNA synthase (gltX), NADPH-dependent glutamyl-tRNA reductase (NADPH- dependent glutamyl-tRNA reductase, hemA) and glutamate-1-semialdehyde aminotransferase (hemL). The C4 pathway is known to synthesize ALA from succinyl-CoA and glycine using aminolevulinic acid synthase.

However, in order to produce ALA through such a biological synthesis process, since it is necessary to supply excess amount of glucose used as a carbon source for growth of microorganisms and ALA synthesis, there is a disadvantage that production cost of ALA is high, However, effective solutions have not been proposed yet.

As a result of efforts to develop a technology for producing ALA more economically through biological synthesis under these circumstances, it has been found that TCA metabolism pathway (TCA cycle) biosynthyl-CoA biosynthesis and serine pathway biosynthesizing glycine biosynthesis pathway ) Were simultaneously activated, methane was used as a sole carbon source to produce ALA economically, and the present invention was completed.

It is an object of the present invention to provide a method for the production of aminolevulinic acid (hereinafter abbreviated as " aminolevulinic acid ") in a methanotrophic bacterium which simultaneously activates the TCA metabolic pathway TCA cycle for biosynthesis of succinyl- CoA and the serine pathway for biosynthesis of glycine synthase, ALAS) gene into a transformant for producing aminolevulinic acid.

Another object of the present invention is to provide a composition for producing aminolevulinic acid comprising the transformant.

Yet another object of the present invention is to provide a kit for producing aminolevulinic acid comprising the above composition.

It is still another object of the present invention to provide a method for producing aminolevulinic acid from methane by culturing the transformant.

The present inventors have focused on methane magnetism bacteria while carrying out various studies to develop a technology for producing ALA through a biological synthesis process more economically. Since methane magnetism bacteria use atmospheric methane as a carbon source, it is expected that when ALA is produced through a biological synthesis process using methane magnetism bacteria, it is possible to produce ALA more economically by reducing the amount of glucose supplied to the medium Respectively.

However, when methane magnetism was used, ALA could be produced through the C5 pathway using atmospheric methane as a carbon source, but it was confirmed that the C4 pathway requiring succinyl-CoA and glycine could not be used, It was confirmed that the TCA metabolic pathway supplying succinyl - CoA and the serine metabolic pathway supplying glycine are not activated simultaneously in general methanotrophic bacteria.

As a result, various studies have been conducted to discover methanotrophic bacteria that simultaneously activate the TCA metabolic pathway and the serine metabolic pathway. As a result, the present inventors have found that the novel methane magnetization Pseudomonas genus DH-1 to gyunin methyl (Methylomonas sp. DH-1) strain (KCTC18400P) that (hereinafter, "DH-1 strain" abbreviated as LA) the methane bacteria magnetization that TCA pathway and serine pathway activated at the same time Respectively. However, in the DH-1 strain, ALAS, which plays an important role in the C4 pathway, was not expressed and it was confirmed that ALA could not be produced through the C4 pathway.

Thus, a transformant in which the ALAS gene involved in the C4 pathway was introduced into strain DH-1 was prepared, and the transformant was cultured in a medium containing succinic acid and glycine under an atmospheric condition containing methane , It was confirmed that the ALA productivity of the transformant was increased to about 1.8 times as compared with the ALA productivity of the DH-1 strain.

As described above, the transformant into which the ALAS gene was introduced into DH-1 strain, in which the TCA metabolic pathway and the serine metabolism pathway were simultaneously activated under the condition that succinic acid and glycine were not supplied to the medium, Thus, the technology for producing ALA has not been known at all and has been developed for the first time by the present inventors.

In order to achieve the above-mentioned object, in one embodiment, Succino - CoA Biosynthetic TCA  Metabolic pathway ( TCA  cycle) and glycine Biosynthetic  Serine metabolism pathway serine  pathway) are activated simultaneously In methane magnetism Aminolevulinic acid  Synthetic enzyme (aminolevulinic acid synthase , ALAS) gene introduced Aminolevulinic acid  A transformant for production is provided.

The term " methanotrophs "of the present invention is also referred to as methanotrophic bacteria, and refers to prokaryotes that grow using methane as a unique carbon source. Most methane magnetisms use atmospheric methane as a carbon source, usually methane converted to methanol and then used for in vivo metabolism.

In the present invention, it can be interpreted that the methanotrophic bacterium is a methanotrophic bacillus which simultaneously activates the TCA metabolic pathway for succinyl-CoA biosynthesis and the serine metabolic pathway for biosynthesis of glycine. In the embodiment of the present invention, the methanotrophic bacteria in which the TCA metabolic pathway (TCA cycle) biosynthyl-CoA biosynthesis and the serine pathway biosynthesizing glycine biosynthesis are simultaneously activated are methyl methanas sp . DH-1) was used.

The term "DH-1 " ( Methylomonas sp . DH-1) strain "refers to a strain belonging to the genus Methylomonas sp . Derived from sewage sludge, which has not been reported in the past, and deposited on August 27, 2015 at the Korea Biotechnology Research Institute , Strain No. KCTC18400P.

In the present invention, the above-mentioned DH-1 ( Methylomonas sp . DH-1) strain (hereinafter abbreviated as "DH-1 strain") exhibits the following characteristics.

(a) soluble methane monooxygenase (sMMO) gene is not present.

(b) a gene encoding a PQQ-dependent methanol dehydrogenase (mxaFJGIRSACKLDEK) gene, a PQQ biosynthesis gene cluster (pqqBCDE) gene, a pyruvic acid A glutamyl-tRNA synthetase (gltX) gene, a NADPH-dependent glutamyl-tRNA reductase (hemA) gene, a glutamate- 1-semialdehyde aminotransferase (hemL) gene, squalene hopene cyclase (shc) gene, and the like are expressed.

(c) the RuMP monophosphate cycle, the Entner-Doudoroff pathway, the EMP metabolism pathway, the PP metabolic pathway, the pentose phosphate pathway, and the H ₄ Tetrahydromethanopterim pathway, H ₄ F tetrahydrofolaste pathway, serine pathway, TCA metabolic pathway, C30 carotenoid synthesis pathway, hapanoid biosynthetic pathway, hopanoid biosynthesis pathway, and the C40 carotenoid synthesis pathway.

(d) Carbon dioxide is absorbed and fixed by the combined activity of a pyruvate carboxylase gene, a phosphoenolpyruvate carboxylase gene, and a pyruvate decarboxylase (PDC) gene .

The term "transformant" of the present invention is intended to mean a mutant gene that is introduced into a host microorganism and expressed, or inhibits the expression of an inherent gene, so as to newly exhibit a genetic characteristic different from that of the host microorganism ≪ / RTI >

In the present invention, the transformant is prepared by introducing the ALAS gene involved in the C4 pathway into a methanotrophic bacillus which simultaneously activates the TCA metabolic pathway for succinyl-CoA biosynthesis and the serine metabolic pathway for biosynthesis of glycine, It can be interpreted as meaning a mutant strain capable of producing ALA from gaseous methane.

The methanotrophic bacteria activated simultaneously with the TCA metabolic pathway biosynthesizing succinyl-CoA and the serine metabolic pathway biosynthesizing glycine are activated by TCA pathway and glutamyl-tRNA synthase, gltX ) Gene, a combination of genes encoding NADPH-dependent glutamyl-tRNA reductase (hemA) gene and glutamate-1-semialdehyde aminotransferase (hemL) And expression of ALA from gaseous methane by the C5 pathway.

Therefore, the transformant can be used as a glutamyl-tRNA synthase (gltX) gene, a NADPH-dependent glutamyl-tRNA reductase (hemA) gene and a NADPH- It may be a mutant strain in which the activity of the C5 pathway is increased by introducing a gene encoding glutamate-1-semialdehyde aminotransferase (hemL) alone or in combination.

The additional introduction of each of the above genes may be carried out singly or in combination to produce a transformant.

The ALAS gene to be introduced into the transformant is not particularly limited, but it may be a homologous gene derived from methanotrophic bacteria, or may be a heterologous gene derived from another microorganism. In the example of the present invention, a transformant obtained by introducing an ALAS gene (SEQ ID NO: 1) derived from type 2 methanotrophic bacteria or an ALAS gene (SEQ ID NO: 2) derived from Rhodobacteria microorganism into DH-1 strain was prepared.

The TCA metabolic pathway activated by both the TCA metabolic pathway for biosynthesis of succinyl-CoA and the serine metabolic pathway for biosynthesis of glycine provided in the present invention is capable of intrinsically synthesizing succinyl-CoA , The serine metabolism pathway is activated and glycine can be intrinsically synthesized. Therefore, the transformant into which the ALAS gene has been introduced can be transformed into ALA through C4 pathway using methane as a single carbon source without supplying glycine or succinic acid externally, Can be biosynthesized.

The characteristics of these transformants are that the serine metabolism pathway and the TCA metabolic pathway are not activated at the same time, and in order to biosynthesize ALA through the C4 pathway, it is necessary to distinguish the succinic acid or glycine from the conventional methanotrophic bacteria It can be an advantage.

In addition, the transformant can also produce ALA through the normal C5 pathway.

Therefore, unlike the conventional methanotrophic bacteria, the transformant can produce ALA through the C4 pathway and the C5 pathway using methane in the atmosphere as a single carbon source. Therefore, the transformant is more economical than conventional methanotrophic bacteria ALA can be produced.

In another embodiment, the present invention provides a method for producing Aminolevulinic acid  Thereby providing a composition for production.

As described above, in the transformant into which the ALAS gene is introduced, ALA can be produced from methane by the activated C4 pathway. Therefore, ALA can be produced from methane with high yield by using the transformant microorganism or culture thereof .

In the present invention, the culture product of the transformant is not particularly limited as long as it can be used for producing ALA from methane. However, for example, the culture product, the culture supernatant, the crushed product, As another example, the culture supernatant obtained by centrifuging the culture of the transformant, the crushed product obtained by physically or ultrasonically treating the transformant, the culture, the culture supernatant, the crushed product, etc. May be a fraction obtained by applying centrifugation, chromatography or the like.

In yet another embodiment, Aminolevulinic acid  There is provided a kit for producing aminolevulinic acid comprising a composition for production.

The kit for producing ALA of the present invention can be used to produce ALA including the composition for producing ALA, but may include one or more other component compositions, solutions or devices suitable for the reaction, including, but not limited to, A buffer used for ALA production, a reaction vessel for performing ALA production, a shaking incubator for performing ALA production, a timer for performing the ALA production reaction, and the like.

In another embodiment, the present invention provides a method for producing aminolevulinic acid, comprising culturing the transformant under conditions comprising methane.

At this time, the transformant is the same as that described above.

The method of culturing the transformant is not particularly limited, but can be carried out by inoculating and culturing in a medium containing no succinic acid and glycine under atmospheric conditions containing methane.

In this case, the concentration of methane in the atmosphere is not particularly limited, but may be 0.01 to 50% (v / v) as an example, and 10 to 40% (v / v) as another example , And as another example, 30% (v / v).

In addition, the incubation temperature is not particularly limited, but may be, for example, 20 to 40 ° C, and as another example, it may be 25 to 35 ° C, and as another example, 30 ° C.

In addition, the culture may be performed under agitation conditions. The agitation conditions are not particularly limited. For example, the agitation conditions may be 200 to 300 rpm. As another example, the agitation conditions may be 210 to 250 rpm. , Which can be 230 rpm.

Finally, the incubation time is not particularly limited, but can be, for example, 30 to 200 hours, as another example, 60 to 140 hours, and as another example, 120 hours.

In the present invention, the method for culturing the transformant may be carried out by a method well known in the art. Specifically, the culture can be continuously cultured in a batch process or an injection batch or a repeated fed batch process, as long as it can produce ALA from the transformant.

NMS (nitrate mineral salts) medium known to be used for the culture of methanotrophic bacteria can be used as the medium used for the culture, and a medium in which the components contained in the medium or the content thereof is adjusted according to the methanotrophic bacteria can be used (http://methanotroph.org/wiki/culturing-tips/).

Particularly, when the medium contains salts such as NaCl and KCl, the productivity of the DH-1 strain or its transformant or the productivity of ALA can be improved. In this case, the concentration of the salt contained in the culture medium is not particularly limited, but may be, for example, 0.1 to 3.0% (w / w), and as another example, 1.0 to 2.0% (w / As another example, it can be 1.5% (w / w).

In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in the culture process in a batch manner, in an oil-feeding manner or in a continuous manner by an appropriate method, but it is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture.

In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester.

In addition, the step of recovering ALA can be carried out by methods known in the art such as dialysis, centrifugation, filtration, solvent extraction, chromatography, crystallization and the like. For example, the culture of the transformant may be centrifuged to remove the transformant, and the resultant supernatant may be applied to a solvent extraction method to recover ALA. In addition, in accordance with the characteristics of the ALA, Any method capable of recovering the ALA by combining experimental methods can be used without any particular limitation.

Using the transformants provided in the present invention, aminolevulinic acid can be produced by using atmospheric methane as a sole carbon source, so that it can be widely used for production of economical aminolevulinic acid.

1A is a schematic diagram illustrating various metabolic pathways activated in the DH-1 strain of the present invention.
FIG. 1B is a schematic diagram showing a process of biosynthesis of aminolevulinic acid through the C4 pathway (Shemin pathway) and the C5 pathway (Beale pathway) present in general methanotrophic bacteria.
Figure 2a shows the vector map of DH-1 shuttle vector_OB3bALAS recombination vector.
Figure 2b shows the vector map of the DH-1 shuttle vector_RsALAS recombination vector.
FIG. 2C is an electrophoresis image showing the transcription of a gene introduced through a recombinant vector in each transformed DH-1 strain. FIG.
FIG. 3 is a graph showing the results of comparing the productivity of aminolevulinic acid from two DH-1 strains and two transformed DH-1 strains into which aminolevulinic acid synthase was introduced.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Methylromonas  genus DH -One( Methylomonas sp . DH -1), the genome and transcript analysis of the strain

The inventors of the present invention have found that a genome of a novel methane magnetism strain Methylomonas sp . DH-1 (KCTC18400P) (hereinafter abbreviated as "DH-1 strain & Analysis was performed to newly identify the specific characteristics of the DH-1 strain.

First, the genome of DH-1 strain (Genbank NZ_CP014360, NZ_CP014361) was analyzed by DDH (DNA-DNA hybridization), and it contained 4.86 Mb of genome chromosome and 278 kb of plasmid and classified as type 1 methanotroph Methylomonas It was confirmed that 73.9% of the total genes were similar to koyamae Fw12E-YT, and the average nucleotide identity (ANI) between the two strains was 97.76%.

As a result of analyzing the metabolism pathway using methane using the transcription product of the DH-1 strain, it was confirmed that various metabolic pathways different from the conventional type 1 methanotrophs were activated (FIG. 1A).

1A is a schematic diagram illustrating various metabolic pathways activated in the DH-1 strain of the present invention.

As shown in FIG. 1A, in the case of a gene related to C1 metabolism existing in a general methanotrophic bacterium, the DH-1 strain does not have a soluble methane monooxygenase (sMMO) gene, whereas two copies of particulate methane oxidation (PQQ) -dependent methanol dehydrogenase (mxaFJGIRSACKLDEK) gene, a PQQ biosynthesis gene cluster (pQqBCDE) gene, a RuMP metabolite metabolizing formaldehyde path (ribulose monophosphate cycle), ED pathway (Entner-Doudoroff pathway), EMP pathway (Embden-Meyerhof-Parnas pathway) , PP pathway (pentose phosphate pathway), H ₄ MPT pathway (Tetrahydromethanopterim pathway) and H ₄ F < / RTI > metabolic pathway (tetrahydrofolate pathway) were all activated.

Specifically, it contains all the serine pathway related genes known to be mainly present in the type 2 methanotrophic bacteria, and can express ppc (Phosphoenolpyruvate carboxylase), so that carbon dioxide can be released through the ppc and serine metabolism pathways While absorbing PEP (Phosphoenolpyruvate) into OAA (oxaloacetate). In addition, it was confirmed that the TCA metabolic pathway (TCA cycle), which is known to be not functioning normally in conventional methanotrophs, works normally.

It is known that ppc is not expressed in a general type 1 methanotrophic bacterium and the serine metabolism pathway does not function normally. However, the DH-1 strain has not only ppc but also pyruvate carboxylase, , Acetyl-CoA carboxylase, and PEP carboxylase, which can absorb carbon dioxide at a significantly higher level than previously known type 1 methanotrophs. Respectively.

On the other hand, it is generally known that aminolevulinic acid can be biosynthesized through the C4 pathway (Shemin pathway) or the C5 pathway (Fig. 1B).

FIG. 1B is a schematic diagram showing a process of biosynthesis of aminolevulinic acid through the C4 pathway (Shemin pathway) and the C5 pathway (Beale pathway).

As shown in FIG. 1B, aminolevulinic acid can be biosynthesized through the reaction of aminolevulinic acid synthetase using succinyl-CoA and glycine synthesized through C4 pathway as a substrate; Glutamyl-tRNA synthetase (gltX), NADPH-dependent glutamyl-tRNA reductase (hemA), and glutamate-1 - Aminolevulinic acid can be biosynthesized by continuous reaction of glutamate-1-semialdehyde aminotransferase (hemL).

However, in general E. coli and Corynebacterium, mainly aminolevulinic acid is biosynthesized through the C5 pathway, but the type 1 or type 2 methanotrophic bacteria has a limitation in the biosynthesis of aminolevulinic acid due to inactivation of some genes in the TCA pathway do. Also, in the case of glycine and succinyl-CoA used as substrates in the C4 pathway, there is no report on the production of aminolevulinic acid in the conventional type II methanotrophic bacteria, and as long as glycine or succinic acid is not supplied externally, The biosynthesis of aminolevulinic acid through the pathway is limited.

Although the restriction of the biosynthesis of aminolevulinic acid via the C4 pathway does not occur in the DH-1 strain, the aminolevulinic acid synthase is not expressed in the DH-1 strain, and consequently the DH- The levulinic acid can not be biosynthesized.

However, since gltX, hemA and hemL involved in C5 pathway are expressed in DH-1 strain, DH-1 strain was able to biosynthesize aminolevulinic acid through C5 pathway.

Example  2: Methylromonas  genus DH -One( Methylomonas sp . DH -1) strain Aminolevulinic acid Biological performance  Verification

In Example 1, DH-1 strain was able to biosynthesize aminolevulinic acid through the C5 pathway, so that it was experimentally confirmed.

Approximately, DH-1 strain, type 1 methanotrophic bacteria ( Methylomicrobium alcaliphilum 20Z) or type 2 methanotrophs ( Methylosinus trichosporium OB3b) each NMS medium _{(1 g / ℓ MgSO 4 ·} 7H 2 O, 1 g / ℓ KNO 3, 0.2 g / ℓ CaCl 2 · H 2 O, 0.0038 g / ℓ Fe-EDTA and 0.0005 g / ℓ NaMo · 4H ₂ O), and cultured at 230 rpm for 120 hours in an atmospheric condition containing 30% (v / v) methane and at a temperature of 30 ° C.

After completion of the culture, a culture supernatant of each strain was obtained, and the concentration of aminolevulinic acid contained in the culture supernatant thus obtained was measured. At this time, the concentration of aminolevulinic acid was measured as follows: 2 ml of the culture supernatant and 1 ml of 1.0 M sodium acetate (pH 4.6) were mixed and then 0.5 ml of acetylacetone was added to obtain a mixture. The mixture was then heated at 100 < 0 > C for 15 minutes and then cooled for 15 minutes. 2 ml of the cooled mixture and 2 ml of Ehrlich's reagent were mixed and reacted for 30 minutes and then the absorbance at 554 nm was measured.

As a result, about 2.19 mg / L of aminolevulinic acid was detected in the culture supernatant obtained by culturing strain DH-1 for 120 hours, while the amount of aminolevulinic acid detected in culture supernatant of type 1 methane magnetism ( Methylomicrobium alcaliphilum 20Z) ( Methylosinus trichosporium OB3b) the distraction called a trace amount of amino LES was detected in each culture supernatant obtained by cultivation for 120 hours.

Therefore, DH-1 strain can aminolevulinic acid biosynthesis through C5 pathway, and its efficiency is superior to conventional methanotrophic bacteria.

Example  3: Aminolevulinic acid  Synthetic enzyme ( aminolevulinic  acid synthase , ALAS) gene-transfected DH -1 strain Aminolevulinic acid  Biosynthesis

As shown in Example 1, the DH-1 strain can not biosynthesize aminolevulinic acid through the C4 pathway because it does not express the aminolevulinic acid synthase involved in the C4 pathway. However, when the aminolevulinic acid synthase gene is introduced into the DH-1 strain and the aminolevulinic acid synthase is expressed, aminolevulinic acid is biosynthesized through the C4 pathway by the aminolevulinic acid synthase .

Example  3-1: Transformation DH -1 strain

First, as an aminolevulinic acid synthase gene to be introduced, aminolevulinic acid synthase gene (OB-ALAS, SEQ ID NO: 1) derived from type 2 methanotrophic bacteria or amino resin derived from Rhodobacter sp . (Rs_ALAS, SEQ ID NO: 2) were synthesized.

Next, a pDH shuttle vector was prepared by inserting a nucleotide sequence encoding a replication origin and a DNA binding protein derived from the strain DH-1 into the pAWP78 vector.

Using the prepared pDH shuttle vector as a template, inverse PCR amplification was performed using the following primers.

DH_inverse_FP: 5'-CAAtaaggatccccaggcatc-3 '(SEQ ID NO: 3)

DH_inverse_RP: 5'-CCATATGAGATctcctttaacag-3 '(SEQ ID NO: 4)

Next, using the synthesized OB-ALAS as a template, PCR amplification was performed using the following primers.

OB3bALAS_FP: 5'-gttaaaggagATCTCATATGGATTACCGGCGCTTTTTCGAGACGG-3 '(SEQ ID NO: 5)

OB3bALAS_RP: 5'-gatgcctgggatccttaTTGCGCGGCGAGCTTGATCTCGGGATAG-3 '(SEQ ID NO: 6)

In addition, PCR amplification was performed using the synthesized Rs_ALAS as a template and the following primers.

RsALAS_FP: 5'-cacatacactgttaaaggagATCTCATATGGACTACAACTTGGCCTTGGACACCGC-3 '(SEQ ID NO: 7)

RsALAS_RP: 5'-cgttttattggatgcctggggatccTCAGGCGACGACTTCGGCGCGGTTCAAG-3 '(SEQ ID NO: 8)

DH-1 shuttle vector_OB3bALAS recombination vector was constructed by introducing OB-ALAS into the amplified pDH shuttle vector, and Rs_ALAS was introduced into the amplified pDH shuttle vector to construct a DH-1 shuttle vector_RsALAS recombinant vector (FIGS. 2A and 2B ).

FIG. 2A shows the vector map of the DH-1 shuttle vector_OB3bALAS recombination vector, and FIG. 2B shows the vector map of the DH-1 shuttle vector_RsALAS recombination vector.

The prepared DH-1 shuttle vector_OB3bALAS recombination vector or DH-1 shuttle vector_RsALAS recombination vector was introduced into DH-1 strain by electrotransformation transformation and stabilized by culturing in NMS medium for 16 hours, and added with kanamycin DH-1 (OB3bALAS) or DH-1 (RsALAS), which is a transformed strain of DH-1, was prepared by inducing double cross hybrid recombination by culturing on NMS solid medium for one week.

In order to verify the transformation of the DH-1 (OB3bALAS) or DH-1 (RsALAS) thus produced, first, colony PCR was performed to verify the transformation.

Also, cDNAs were obtained by performing reverse transcription to obtain the total RNAs from the respective transformed DH-1 strains, and the cDNAs were subjected to PCR amplification to determine whether transcription of the aminolevulinic acid synthase gene (Fig. 2C).

FIG. 2C is an electrophoresis image showing the transcription of a gene introduced through a recombinant vector in each transformed DH-1 strain. FIG.

As shown in FIG. 2C, it was confirmed that the respective genes for aminolevulinic acid synthase were transcribed by the introduced genes in each transformed DH-1 strain.

Example  3-2: Transformation DH -1 < / RTI > Aminolevulinic acid  Productivity Analysis

After incubation in NMS medium, DH-1 strain, DH-1 (OB3bALAS) or DH-1 (RsALAS) were inoculated at 120 rpm in an atmospheric condition containing 30% (v / v) Lt; / RTI > After completion of the culture, a culture supernatant of each strain was obtained, and the concentration of aminolevulinic acid contained in the culture supernatant thus obtained was measured (Fig. 3).

FIG. 3 is a graph showing the results of comparing the productivity of aminolevulinic acid from two transformed DH-1 strains into which the DH-1 strain and the aminolevulinic acid synthase gene are introduced.

As shown in FIG. 3, the transformed strain DH-1 showed relatively good aminolevulinic acid productivity as compared with the strain DH-1, and the difference in the productivity of aminolevulinic acid was further increased with the passage of time . The DH-1 strain produces about 2.19 mg / L of aminolevulinic acid when cultured for 120 hours, whereas the transformed strain DH-1 produces about 3.78 to 3.94 mg / L of aminolevulinic acid Thus, it was confirmed that the transformed strain DH-1 exhibited an increased amount of aminolevulinic acid by 1.8 times as compared with the strain DH-1.

As described above, in order to biosynthesize aminolevulinic acid through the C4 pathway, succinyl-CoA and glycine are required as substrates, and serine metabolism pathway supplying glycine and succinyl-CoA are supplied In the conventional methanotrophic bacteria in which the TCA metabolic pathway is not activated simultaneously, one of the substrates must be supplied externally, so that aminolevulinic acid can be biosynthesized through the C4 pathway.

However, since the transformed DH-1 strain into which the aminolevulinic acid synthase gene is introduced into the DH-1 strain provided in the present invention is activated simultaneously with the serine metabolism pathway and the TCA metabolism pathway, succinic acid or glycine is externally added It is possible to biosynthesize aminolevulinic acid by using methane as a single carbon source, and this advantage is confirmed to be a novel feature of the transgenic DH-1 strain which is not present in conventional methanotrophs.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Korea Biotechnology Research Institute KCTC18400P 20150827

<110> University-Industry Cooperation Group of Kyung Hee University <120> Transformed methanotrophs for producing aminolevulinic acid and          uses thereof <130> KPA161711-KR-P1 <150> KR 10-2017-0003302 <151> 2017-01-10 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 1284 <212> DNA <213> Artificial Sequence <220> <223> OB-ALAS <400> 1 atggattacc ggcgcttttt cgagacggcg atcacacgtc tccaggacga gcgccgttac 60 cgagtcttcg cccatctcga acgctgcgtc gaatgcttcc cgcgcgcgct ctggcaccgg 120 gacgacggca cgacgcagga tgtgacgatc tggtgctcca atgattatct cggcatgggc 180 cagcaccccg aggtgatcgc cgctatggtc gacaccgccg cgcgcgtcgg cgccggctcg 240 ggcggcacgc gcaacatctc cggcaccagc catgcgatcg tcgagctgga gagcgagctc 300 gccgacctgc acggcaagga ggcggcgctc gtcttcacct cgggatggat ttccaatctc 360 gcggcgatct ccacgatcgc cgatcttctg cccgactgtc tcatcctctc cgacgcctcc 420 aatcacaatt cgatgatcga gggcgtcaag cgctcgcgcg ccgagcgcaa gatcttccgc 480 cacaacgacc tcggccatct cgaggagctg ctcgccgcgg ccggcgcgcg gcccaagctc 540 atcgtcttcg agagcctcta ttcgatgaac ggcaatatcg cgccggtcgc cgagatcgcc 600 gcgctcgccg agcgttatgg cgcgatgacc tatatcgacg aggttcatgc ggtcggcatg 660 tatggcgcgc gcggcggcgg cgtttgcgag caggccggcg tgatggaccg tatcgatgtg 720 atcgagggca cgctggccaa gggcttcggc acgctcggcg gctatatcgc cggcgatcgc 780 gtcatcatcg acgcgatccg cagctatgcg gcgtccttca tcttcaccac agctctgccg 840 ccggcggtcg ccgcggcggc gaccgccgct gtgcgcctct tgaagacgcg ccccgatctg 900 cgcgccgcgc atcagcgcgc cacccatatc accaagcacg cgctcggcgc cgcgggcctg 960 ccggtgctgg agaacggctc gcatatcgtg ccggtgatgg tgcgcgaggc ggagctgtgc 1020 aaggccgcga gcgacatgct gctcgagcgg cacggcatct atattcagcc gatcaattat 1080 ccgacggtcg cacgcgggac ggagcgtctg cgcatcacgc cgacgccctg ccatacgggc 1140 gagcatatcg tcacgctggt cgaagcgatg gtcgatgtct ggaacacgct cggcatcgcc 1200 ttcgtcgagc cgccgcagca tctccacgtc gatcccgaga gccgcgagcg ctgtacctat 1260 cccgagatca agctcgccgc gcaa 1284 <210> 2 <211> 1224 <212> DNA <213> Artificial Sequence <220> <223> Rs_ALAS <400> 2 atggactaca acttggcctt ggacaccgcc ttgaaccgct tgcacaccga aggccgctac 60 cgcaccttca tcgacatcga acgccgcaaa ggcgccttcc cgaaagccat gtggcgcaaa 120 ccggacggca gcgaaaaaga aatcaccgtc tggtgcggca acgactactt gggcatgggc 180 caacacccgg tcgtcttggg cgccatgcac gaagccttgg acagcaccgg cgccggcagc 240 ggcggcaccc gcaacatcag cggcaccacc ttgtaccaca aacgcttgga agccgaattg 300 gccgacttgc acggcaaaga agccgccttg gtcttcagca gcgcctacat cgccaacgac 360 gccaccttga gcaccttgcc gcaattgatc ccgggcttgg tcatcgtcag cgacaaattg 420 aaccacgcca gcatgatcga aggcatccgc cgcagcggca ccgaaaaaca catcttcaaa 480 cacaacgact tggacgactt gcgccgcatc ttgaccagca tcggcaaaga ccgcccgatc 540 ttggtcgcct tcgaaagcgt ctacagcatg gacggcgact tcggccgcat cgaagaaatc 600 tgcgacatcg ccgacgaatt cggcgccttg aaatacatcg acgaagtcca cgccgtcggc 660 atgtacggcc cgcgcggcgg cggcgtcgcc gaacgcgacg gcttgatgga ccgcatcgac 720 atcatcaacg gcaccttggg caaagcctac ggcgtcttcg gcggctacat cgccgccagc 780 agcaaaatgt gcgacgccgt ccgcagctac gccccgggct tcatcttcag caccagcttg 840 ccgccggtcg tcgccgccgg cgccgccgcc agcgtccgcc acttgaaagg cgacgtcgaa 900 ttgcgcgaaa aacaccaaac ccaagcccgc atcttgaaaa tgcgcttgaa aggcttgggc 960 ttgccgatca tcgaccacgg cagccacatc gtcccggtcc acgtcggcga cccggtccac 1020 tgcaaaatga tcagcgacat gttgttggaa cacttcggca tctacgtcca accgatcaac 1080 ttcccgaccg tcccgcgcgg caccgaacgc ttgcgcttca ccccgagccc ggtccacgac 1140 agcggcatga tcgaccactt ggtcaaagcc atggacgtct tgtggcaaca ctgcgccttg 1200 aaccgcgccg aagtcgtcgc ctaa 1224 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caataaggat ccccaggcat c 21 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ccatatgaga tctcctttaa cag 23 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gttaaaggag atctcatatg gattaccggc gctttttcga gacgg 45 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gatgcctggg gatccttatt gcgcggcgag cttgatctcg ggatag 46 <210> 7 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cacatacact gttaaaggag atctcatatg gactacaact tggccttgga caccgc 56 <210> 8 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cgttttattt gatgcctggg gatcctcagg cgacgacttc ggcgcggttc aag 53

Claims (16)

The aminolevulinic acid synthase (ALAS) gene was introduced into the methanotrophic bacillus, which simultaneously activates the TCA metabolic pathway (TCA cycle) and the serine pathway that biosynthesizes the succinyl-CoA. Transformant for producing aminolevulinic acid.
The method according to claim 1,
Wherein the methanotrophic bacterium is a strain of Methylomonas sp. DH-1 deposited with KCTC18400P.
The method according to claim 1,
Wherein the aminolevulinic acid synthase gene is a homologous or heterologous gene.
The method according to claim 1,
Wherein the aminolevulinic acid synthase gene comprises the nucleotide sequence of SEQ ID NO: 1 or 2.
The method according to claim 1,
The transformant may be selected from the group consisting of glutamyl-tRNA synthase (gltX) gene, NADPH-dependent glutamyl-tRNA reductase (hemA) gene, glutamate-1-semialdehyde amino Wherein a gene selected from the group consisting of glutamate-1-semialdehyde aminotransferase (hemL) gene and a combination thereof is further introduced.
A composition for producing aminolevulinic acid comprising a transformant for producing aminolevulinic acid according to any one of claims 1 to 5.
A kit for producing aminolevulinic acid comprising the composition for producing aminolevulinic acid according to claim 6.
A method for producing aminolevulinic acid, comprising culturing a transformant for producing aminolevulinic acid according to any one of claims 1 to 5 under conditions comprising methane.
9. The method of claim 8,
Wherein the transformant is cultured in a medium not containing succinic acid and glycine.
9. The method of claim 8,
Wherein the culture is carried out under atmospheric conditions comprising gaseous methane.
11. The method of claim 10,
Wherein the content of gaseous methane contained in the atmosphere is 0.01 to 50% (v / v).
9. The method of claim 8,
Wherein the cultivation is performed at a temperature of 20 to 40 캜.
9. The method of claim 8,
Wherein the culture is carried out under stirring conditions of 200 to 300 rpm.
9. The method of claim 8,
Wherein the culture is performed for 30 to 200 hours.
9. The method of claim 8,
Further comprising the step of recovering aminolevulinic acid from the culture after said cultivation is terminated.
16. The method of claim 15,
The culture is preferably a culture of a strain of the genus Methylomonas DH-1 or a transformant thereof, a culture supernatant of the culture, a lysate of the strain Methylomonas DH-1 or a transformant thereof, fractions thereof, &Lt; / RTI &gt; wherein the aminolevulinic acid is selected from the group consisting of combinations thereof.

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