KR101326255B1 - Recombinant Microorganism Having Enhanced Productivity of 5-Aminolevulinic Acid and Method of Producing 5-aminolevulinic Acid Using the Same - Google Patents

Abstract

The present invention relates to a recombinant microorganism with improved production capacity of 5-aminolevulinic acid and its use. More particularly, 5-aminolevulinic acid synthase derived from Zymomonas mobilis in Bacillus microorganisms. Recombinant microorganism having improved production capacity of 5-aminolevulinic acid (hereinafter referred to as 'ALA') by introducing a gene encoding (5-aminolevulinic acid synthase, hereinafter referred to as 'ALAS') The present invention relates to a method for producing 5-aminolevulinic acid (ALA) with improved efficiency by culturing specific conditions. Recombinant microorganism according to the present invention can be transformed by introducing the ALAS of Zymomonas mobilis to Bacillus sp. Microorganisms to increase the yield of ALA compared to the wild type. In addition, by culturing the transformed recombinant microorganism in a culture solution to which glycine and succinic acid are added at an optimized concentration, it is expected to be a very useful invention because the production of ALA is further increased. In addition, since the recombinant microorganism according to the present invention directly uses a gene of a bacterium rather than a eukaryotic gene, it is possible to produce stable ALA and has the advantage of using the prepared microorganism itself as a microbial pesticide.

Description

5-Recombinant Microorganism Having Enhanced Productivity of 5-Aminolevulinic Acid and Method of Producing 5-aminolevulinic Acid Using the Same}

The present invention relates to a recombinant microorganism with improved production capacity of 5-aminolevulinic acid and its use. More particularly, 5-aminolevulinic acid synthase derived from Zymomonas mobilis in Bacillus microorganisms. Recombinant microorganism having improved production capacity of 5-aminolevulinic acid (hereinafter referred to as 'ALA') by introducing a gene encoding (5-aminolevulinic acid synthase, hereinafter referred to as 'ALAS') It relates to a method for producing 5-aminolevulinic acid (ALA) with improved efficiency by culturing under specific conditions.

The rapid growth of the global population has resulted in many problems, including the destruction of derived ecosystems, pollution of the environment, depletion of resources and lack of food. With the advance of science, the food supply has increased, but it has not been able to increase the population, and it is still difficult to supply sufficient food. As a way of overcoming these problems, humans research and develop many pesticides to increase food production.

Herbicides account for the largest proportion of pesticides (39.2% herbicides, 32.7% insecticides, 22% fungicides, 6.1% growth regulators) and are currently highly toxic diphenyl ethers (DPEs), and oxiadia. Synthetic photodynamic herbicides such as oxadiazol are produced and used through chemical synthesis, but environmental pollution due to the slow decomposition rate and harmfulness to animals and humans has been raised. .

Therefore, the development of biologically-derived pesticides using microbial metabolites with low residual toxicity and easy to produce has been actively studied. To date, practically available biologically-derived herbicides include bialaphos and herbicidine. (herbicidin), herbimycin (herbimycin), cycloheximide (cycloheximide) can be concentrated in about 30 species. However, these substances are also reported to exhibit strong toxicity to the human body, and therefore, an herbicidal active substance which is excellent in selectivity, non-toxic to the human body and also biodegradable is inevitably required.

In general, ALA is a major precursor of tetrapyrrole compounds, such as heme, bacteriochlorophyll, and corinoid, in all living systems. Is formed, and a series of oxidation reactions occur by this substance, which selectively destroys and kills phospholipids of leaves only to dicotyledonous plants. Therefore, ALA can be used as an environmentally friendly herbicide that selectively kills weeds without harming humans, animals, and crops (Rebeiz CA, Montazer-Zouhoor A., Hopen H., and Wu SM). Enzyme Microb.Technol., 6: 390 (1984).

In addition, ALA has the effect of promoting growth by promoting plant photosynthesis, inhibition of respiration and assimilation of carbon dioxide when used at low concentrations as well as these herbicides (Hua Z., et al., Cancer Reasearch). 55: 1723 (1995); Matsumoto TH, et al., Weed Research, 37:60 (1992); Matsumoto TH, et al., Pesticide Biochemistry, 48: 214 (1994); Rebeiz N., et al., Photochem.Photobiol., 55: 431 (1995)), after soaking rice seed in 1 to 3 ppm of ALA solution for 1 to 48 hours, it was found that seeding promoted growth of size, weight and roots.

In addition, ALA is expanding its use as a pesticide of pests such as Trichopusia ni. In particular, the pesticidal effect using ALA is complicated by its action stage, unlike the existing pesticides that participate in specific metabolic stages, and thus, it is difficult for pests to develop resistance to it, and thus it can be used as an environmentally friendly insecticide. In addition, ALA has been reported to be used as a bioactive material in medical fields such as skin cancer drugs and microbial resistance drugs. In particular, photosensitizer of photodynamic theraphy (PDT) for the treatment of various malignancies It has been found that it can be widely used as an active research. The administration of ALA to malignant tumors has been shown to increase the concentration of intracellular porphyrin, especially protoporphyrin, the final precursor of the heme biosynthetic pathway, damaging the irradiation of visible light and killing tumors. On the other hand, normal cells have been found to have low damage due to light irradiation because ALA uptake is lower than tumor cells and due to the slow growth rate, the concentration of protoporphyrin that accumulates is relatively low.

Currently, ALA is produced using a complex organic synthesis method (Beale SI, et al., Phytochemistry, 18: 441 (1979)), but due to its high production cost, it is not profitable, so Rhodobacter sphaeroides ), Clostridium thermoacetium thermoaceticum ), Methanobacterium thermoautotropicum , Agnemellum Cuadrupricom ( Agnemellum) quadruplicum ), Anacystis marina and Chlorella studies on the production of ALA using fermentation of microorganisms such as vulgaris and the use thereof (Sasaki K., et al., J. Ferment. Technol., 65: 511 (1987); Sasaki K. , et al., Biotechnol. Lett., 15: 859 (1993); Tanaka T., et al., Biotechnol. Lett., 13: 589 (1991); Janschen R., et al., FEMS Microb. 12: 167 (1981); Kipe-Not JA and Steven SE, Plant Physiol., 65: 126 (1980); Beale SI, and Castelfranco PA, Plant Physiol., 53: 297 (1974).

ALA is a precursor of heme and is known to be biosynthesized by two biosynthetic systems (C4 and C5 pathways). The C4 systems found in animals, fungi and aerobic bacteria are glycine and succinyl- It is produced by the condensation of succinyl-CoA (CoA), which is catalyzed by ALA synthase, a pyridoxal phosphate dependent enzyme. Another pathway, C5, is found in plants, algae, and Escherichia coli, and this pathway is derived from glutamate, glutamyl-tRNA synthetase, and glutamic-tRNA hydrogen release enzymes. It is converted into ALA by a series of reactions by dehydrogenase and glutamate-1-semialdehyde (GSA) aminotransaminase.

The molecular biological ALA biosynthetic pathway was revealed through the isolation of ALA auxotrophy, whereas the ALA synthase gene in the C4 pathway was found to have two isozymes hemA and hemT, whereas C5 The pathway is composed of hemA, hemL and hemM genes.

In order to increase the synthesis of ALA by these microorganisms, there have been studies on reinforcing the precursors in the culture medium or separating and adding lower fatty acids from organic waste resources, and controlling the pH, temperature, supply of oxygen, and photosynthetic bacteria. Investigations on the increase in the amount of ALA produced by the investigation have also been reported.

Therefore, ALA can be obtained in high yield, can be industrialized through pure purification, and there is an urgent need for improvement of new strains using genetic engineering techniques for practical use as a practical herbicide.

Accordingly, the present inventors have diligently tried to provide a new strain capable of biosynthesizing ALA more stably with a high yield. As a result, the recombinant strain transformed by introducing the ALAS gene of Zymonas mobilis into a bacterium of Bacillus yields a high yield of ALA. It was found that it can be produced, the present invention was completed.

The information described in the Background section is intended only to improve the understanding of the background of the present invention and thus does not include information forming a prior art already known to those skilled in the art .

It is an object of the present invention to provide a recombinant microorganism that is improved to allow 5-aminolevulinic acid (ALA) production to be increased.

Still another object of the present invention is to provide a method for producing 5-aminolevulinic acid using the recombinant microorganism.

In order to achieve the above object, the present invention is a 5-amino, in which a recombinant vector containing a 5-aminolevulinic acid synthase gene of Zymonas mobilis is introduced into a Bacillus sp. It provides a recombinant microorganism with improved levulinic acid production capacity.

The present invention also provides an improved recombinant ability of 5-aminolevulinic acid, in which a 5-aminolevulinic acid synthase gene of Zymonas mobilis is inserted into a chromosome of a Bacillus microorganism. Provide microorganisms.

The present invention also comprises the steps of culturing the recombinant microorganism in the culture medium to which 8 to 12 g / L succinic acid or 8 to 10 g / L glycine is added; And it provides a method for producing 5-aminolevulinic acid comprising the step of recovering 5-aminolevulinic acid from the culture medium.

The present invention also comprises the steps of culturing the recombinant microorganism in a culture medium to which 1 to 2 g / L glycine and 3 to 9 g / L succinic acid is added; And it provides a method for producing 5-aminolevulinic acid comprising the step of recovering 5-aminolevulinic acid from the culture medium.

Recombinant microorganism according to the present invention can be transformed by introducing the ALAS of Zymomonas mobilis to Bacillus sp. Microorganisms to increase the yield of ALA compared to the wild type. In the present invention, a recombinant vector having a gene for overexpressing ALAS from Z. mobilis is introduced into a B. subtilis strain, and then the transformed Bacillus subtilis. ( B. subtilis ) confirmed that the yield of ALA was higher than that of the wild type. It is expected to be a very useful invention because a large amount of ALA can be produced using a strain engineered to produce a large amount of ALA according to the present invention. In addition, by culturing this transformed B. subtilis in a culture solution to which glycine and succinic acid are added at an optimized concentration, the production of ALA is expected to be a very useful invention. In addition, since the recombinant microorganism according to the present invention directly uses a gene of a bacterium rather than a eukaryotic gene, it is possible to produce stable ALA and has the advantage of using the prepared microorganism itself as a microbial pesticide.

Figure 1 shows a schematic diagram showing the recombination process of the gene insertion of the recombinant vector pHT43-ALAS inserted ALAS gene presented in the present invention.
Figure 2 is a graph comparing the ALA production between the ALA synthase expression Bacillus subtilis ( B. subtilis ) and the ALA synthase non-introduced Bacillus subtilis strains of the present invention.
Figure 3 is a graph showing the ALA production according to the culture temperature when culturing the ALA synthase expression Bacillus subtilis of the present invention.
4 is a graph showing ALA production amount according to the amount of precursor (succinic acid) added when culturing ALA synthase-expressing Bacillus subtilis of the present invention.
5 is a graph showing the ALA production amount according to the amount of precursor (glycine) added when culturing the ALA synthase expression Bacillus subtilis of the present invention.
Figure 6 is a graph showing the ALA production amount according to the amount of precursors (glycine and succinic acid) when culturing ALA synthase expression B. subtilis of the present invention.
7 is a graph showing the results of ALA production according to the incubation time under the optimized conditions of ALA by the present study (triangle: ALA production; square: growth).
Figure 8 is a graph comparing the ALA synthase expression of Bacillus subtilis (B. subtilis) and ALA synthase US introduced Bacillus subtilis bacteria week ALA production in the optimized conditions of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, when the 5-aminolevulinic acid synthase gene of Zymonas mobilis is introduced into a Bacillus microorganism, 5-aminolevulinic acid of the Bacillus microorganism to which the gene is introduced is introduced. It was found that the improvement of the production ability.

Therefore, in one aspect, the present invention provides a 5-aminore, wherein a recombinant vector containing a 5-aminolevulinic acid synthase gene of Zymonas mobilis is introduced into a bacterium of the genus Bacillus. The present invention relates to a recombinant microorganism having improved blenic acid production capacity.

As used herein, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication, including several to hundreds of plasmid vectors per host cell, and (b) host cells transformed with plasmid vectors. It has a structure that includes an antibiotic resistance gene that allows it to be used and a restriction enzyme cleavage site (c) into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA. After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using calcium chloride methods or electroporation (Neumann, et al., EMBO J. , 1: 841, 1982) and the like.

As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used.

Sequences are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As is well known in the art, to raise the expression level of a transgene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the chosen expression host. Preferably, the expression control sequence and the corresponding gene will be included in one recombinant vector containing the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the recombinant vector must further comprise an expression marker useful in the eukaryotic expression host.

Host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term “transformation” means introducing DNA into a host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration.

Of course, it should be understood that not all vectors function equally well to express the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.

In addition, the 5-aminolevulinic acid synthase gene of Zymomonas mobilis may be introduced into the genome of the bacterium of Bacillus sp., Which is a host cell, and exist as a chromosome. It will be apparent to those skilled in the art that the present invention may have the same effect as when the recombinant vector is introduced into the host cell even when the gene is inserted into the genomic chromosome of the host cell.

Therefore, in another aspect, the present invention provides 5-aminolevulinic acid in which a 5-aminolevulinic acid synthase gene of Zymonas mobilis is inserted into a chromosome of a Bacillus microorganism. The present invention relates to a recombinant microorganism with improved ability.

As the method for inserting the gene on the chromosome of the host cell in the present invention can be used a commonly known genetic engineering method, for example, retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector , Poxvirus vectors, lentiviral vectors or non-viral vectors.

On the other hand, ALAS can be obtained by centrifuging the cells or supernatants from the cultures obtained, and by individually or suitably combining cell disruption, extraction, affinity chromatography, cation or anion exchange chromatography, gel filtration, and the like. I can do it. Confirmation that the obtained purified substance is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis, western blotting or the like.

In the present invention, the Zymomonas mobilis ( Zymomonas Gene encoding the 5-aminolevulinic acid synthase (hereinafter referred to as 'ALAS') derived from mobilis ) may be characterized in that the nucleic acid sequence represented by SEQ ID NO: 2. Considering the degeneracy of the genetic code, 80% homology, preferably 85% homology, more preferably 90% homology, most preferably 95% Genes having homology are also included in the ALAS gene of the present invention. In addition, the ALAS preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but also a mutant having ALAS activity in which one or more substitutions, deletions, additions, etc. of the protein described in SEQ ID NO: 1 is induced in the ALAS of the present invention. do.

In the present invention, unlike the use of eukaryotic-derived enzymes by using ALAS derived from a bacterium other than eukaryotic cells, it does not require a construction process through synthesis of cDNA after total RNA extraction, and immediately uses a bacterial gene. Therefore, there is an advantage that it is easy to search for coding genes directly in the genomic sequence and easier gene construction through them. In addition, in the case of the present invention, by constructing a gene of about 1.2kb has the advantage of obtaining a higher expression rate and transformation rate.

The bacterium of the genus Bacillus to be used as a host cell in the present invention is preferably derived from the bacterium of the genus Bacillus subtilis ( B. subtilis ), but is not limited thereto. In the present invention, a bacterium of the genus Bacillus, which is a representative plant growth promoting bacterium, has a number of useful functions for plant growth and pest control, such as antibiotic production, nitrogen fixation, disease resistance-induced volatile growth accumulation, and phosphoric acid solubilization. . Therefore, the recombinant microorganism according to the present invention has the advantage that not only can use its own ALA produced, but also the strain itself as a plant disease control agent. That is, in another aspect, the present invention provides a plant disease control agent comprising the recombinant microorganism as an active ingredient.

In one embodiment of the present invention, a recombinant strain is prepared by transforming the ALAS gene derived from Zymonas mobilis having the nucleic acid sequence represented by SEQ ID NO: 2 to a Bacillus subtilis using a recombinant vector (FIG. 1). ALA production capacity has been improved, and this is why B. subtilis (pHT43-ALAS) and named it to the Korea Microorganism Conservation Center (KCCM) in Yurim Building, Hongje 1-dong, Seodaemun-gu, Seoul, Korea The deposit was made on November 18, 2011 under the accession number KCCM11226P.

In another embodiment of the present invention, 5-aminolevulinic acid (ALA) was produced by culturing the prepared recombinant strain.

As a method of culturing the transformant of the present invention, a conventional method used for culturing a host may be used. As the culture method, any method used for culturing ordinary microorganisms, such as a batch type, a flow batch type, a continuous culture, or a reactor type, can be used. As a medium for culturing the transformant obtained by using bacteria such as E. coli as a host, a complete medium or a synthetic medium such as LB medium, NB medium and the like can be given. In addition, the incubation temperature is accumulated in the temperature range, preferably about 30 degrees, ALAS is accumulated in the cells and recovered.

The carbon source is required for the growth of microorganisms, and for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol and butanol; Polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid and gluconic acid; Fatty acids such as propionic acid, butanoic acid, pentanic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid can be used.

Examples of the nitrogen source include natural products derived from natural products such as peptone, meat juice, yeast extract, malt extract, caseinate and corn steep liquor, in addition to ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate. Moreover, as an inorganic substance, 1 potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, etc. are mentioned, for example. You may add antibiotics, such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin, to a culture liquid.

In addition, when the promoter cultures the microorganism transformed using an inducible expression vector, an inducer suitable for the type of promoter may be added to the medium. For example, isopropyl-D-thiogalactopyranoside (IPTG), tetracycline, indole acrylic acid (IAA), etc. are mentioned as an inducer.

In the present invention, the optimum conditions for culturing the recombinant microorganisms have been determined. Therefore, in another aspect, the present invention provides the recombinant microorganisms in a culture medium to which 8 to 12 g / L of succinic acid or 8 to 10 g / L of glycine is added. Culturing; And it relates to a method for producing 5-aminolevulinic acid comprising the step of recovering 5-aminolevulinic acid from the culture medium.

In addition, in one embodiment of the present invention was determined the optimum concentration conditions in the culture medium to which both succinic acid and glycine added, the present invention in another aspect, the recombinant microorganism glycine 1 to 2 g / L and succinic acid 3 Culturing in a culture solution to which 9 g / L is added; And it relates to a method for producing 5-aminolevulinic acid comprising the step of recovering 5-aminolevulinic acid from the culture medium.

In another aspect, the present invention provides a composition for producing ALA comprising the transformant of the present invention as an active ingredient. Compositions for producing ALA of the present invention include polypeptides, broths, cell lysates, purified or unenzymatic enzyme extracts or polypeptides, etc. suitable for producing ALA by culturing the transformant, for example.

On the other hand, the composition for producing ALA may be provided in the form of a kit for inhibiting gene expression. Kits for inhibiting gene expression may take the form of bottles, tubs, sachets, envelopes, tubes, ampoules, etc., which may be partially or wholly plastic, glass, paper, foil, It may be formed from a wax or the like. The container may be fitted with a cap which is initially part of the container or can be fully or partially detachable, which may be attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, which is accessible to the contents by the injection needle. The kit may include an external package, and the external package may include instructions for use of the components.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Bacillus Subtilis ( B. subtilis )of  5- Aminolevulinic acid  Synthetic enzyme ( ALAS Transformation of the gene encoding

Example  1-1: Zaimomonas  Mobilis ( Z. mobilis )of ALAS  Gene amplification

By over-expressing the gene of ALAS Xi thigh eggplant Mobilis (Z. mobilis) (KCTC 1534, Korea) for ALA production in order to increase production of ALA by them to first clone the gene, Xi thigh eggplant Mobilis (Z. mobilis BamH- F ( 5'-gga tcc atg gat tac aaa cat att ttc ag-3 ', SEQ ID NO: 3), XbaI -R (A) for the cloning of ALAS with reference to the nucleotide sequence of the peptide portion from the genomic DNA of 5'-tct aga gta tgc ggc aaa acg cat atc aag-3 ', SEQ ID NO. Then, PCR was performed using the synthesized primers.

The sequence of the synthesized primer is as follows.

SEQ ID NO: 5'-gga tcc atg gat tac aaa cat att ttc ag-3 '

SEQ ID NO: 5'-tct aga gta tgc ggc aaa acg cat atc aag-3 '

0.5 μl of Tag polymerase (Geneall, Korea), 5 μl of 10X PCR buffer, 4 μl of dNTP mix, 50 μl of primer sample, distilled water, and then 94 ℃ 5 min, 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 After repeating 30 minutes 1 ℃ 30 ℃, PCR was performed through 72 ℃ 10 minutes, once.

Example  1-2: Zaimomonas ALAS Of genes Cloning

The amplification product obtained in Example 1-1 was electrophoresed on 0.8% agarose gel and DNA fragments on agarose gel were recovered using a Biospin gel extraction kit (Bioflux).

Thereafter, the obtained ALAS was digested with BamH , XbaI , and then ligated to pHT43 (Mobitech GmbH, Germany), which is a Bacillus expression vector, to transform B. subtilis . The ligation of recombinant plasmid DNA was then isolated from the transformants. The recombinant vector was named pHT43-ALAS and is illustrated in FIG. 1.

At this time, in order to prepare a bacterium of the genus Bacillus expressing the pHT43-ALAS protein, according to the method of Ito And Nagane (Ito M. & Nagane M., Biosci . Biotechnol . Biochem ., 65: 2773, 2001) Tilis (Electrocompetent Bacillus subtilis ) was prepared. That is, Bacillus subtilis pre-incubated in LB agar plate at 30 ℃, and then cultured Bacillus subtilis in SpC medium (T base 20ml, 50% glucose 0.2ml, 1.2% MgSO ₄ H ₃ O 0.3ml, 10 ml of 10% Bacto yease extract 0.4ml, 1% casamino acid 0.5ml) was incubated at 37 ° C. at OD600 to 0.5, and 2 ml of the SpII medium (T base 200ml, 50% glucose 2ml, 1.2% MgSO ₄ .3H ₂ O 14ml, 10% Bacto yease extract 2ml, 1% casamino acid 2ml, 0.1M CaCl ₂ 1ml). After 90 minutes of incubation, the cells were collected through a centrifuge, 2 ml of glycerol was dispensed to release the cells well, and the cells were divided into 500ul and stored at -70 ° C.

2 μg of ALAS of pHT43-ALAS was transformed into Electrocompetent Bacillus subtilis ) was added to 100ul, transferred to Gene-Pulser cuvette, and transformed using Gene-Pulser (Bio-Rad). Then, the chloramphenicol (5㎍ / ㎖) plated on a large amount of the solid medium is added to the LB medium were selected transformants, this B. subtilis (pHT43-ALAS) and named it to the Korea Microorganism Conservation Center (KCCM) in Yurim Building, Hongje 1-dong, Seodaemun-gu, Seoul, Korea The deposit was made on November 18, 2011 under the accession number KCCM11226P.

ALAS  With unintroduced strain ALA  Production comparison

In order to compare the ALA production between the ALA synthase-expressing Bacillus subtilis prepared in Example 1 and the ALA synthase-free Bacillus subtilis strains, the experiment was performed as follows.

That is, to measure the amount of ALA produced in the ALA synthase-expressing Bacillus subtilis and the ALA synthase-free Bacillus subtilis strains, 1M sodium acetate buffer (pH 4.7) 100 was added to 200 µl of the culture filtrate. Μl, 50 μl of acetyl acetone were added, followed by heating for 10 minutes, and cooling at room temperature to 2-methyl-3-acetyl-5-propionate pyrrole formed by MER (modified Ehrlich's reagent: -dimethylaminobenzaldehyde 0.2 g, glacial acetic acid 8 ml, 1 ml of 70% perchloric acid, 1 ml of DW) 350 µl was added, and the mixture was left at room temperature for 15 minutes.

The amount of the colored compound was measured at 555 nm using a Microplate Spectrophotometer (Biotech, USA) and then quantified by converting ALA production amount from the ALA standard curve.

As a result, as shown in Figure 2 ALA synthase expression Bacillus subtilis according to the present invention was confirmed to show a 1.4-fold increase in ALA production compared to the control ALA synthase unintroduced Bacillus subtilis strains. .

In addition, the following experiment was carried out to determine the ALA optimum yield production conditions of the recombinant strain according to the present invention.

ALA  Determination of optimum conditions of production and measurement of yield

3-1: ALA  Determination of temperature conditions for production optimization

B. subtilis produced in Example 1 In order to determine the optimum ALA production yield of the (pHT43-ALAS) strain was tested as follows. That is, the prepared B. subtilis (pHT43-ALAS) strain was cultured in LB medium containing 5 µg / ml of chloramphenicol. The culture temperature of the medium was incubated at 25 ° C., 30 ° C. and 37 ° C., respectively, and the yield of 5-aminolevulinic acid was measured.

As a result, as shown in Figure 3, ALA synthase expression of Bacillus subtilis (B. subtilis) strain according to the invention was found to exhibit the most ALA increased yield at about 30 ℃.

3-2: ALA  Determination of Concentration Conditions of Precursors for Production Optimization

B. subtilis produced in Example 1 In order to determine the optimum ALA production yield of the (pHT43-ALAS) strain was tested as follows. That is, the transformed B. subtilis -ALAS containing the ALAS gene prepared in Example 1 is used to produce ALA in a conventional chloramphenicol-containing LB medium for succinic acid, glycine and various minerals. Was incubated. In order to determine the optimum ALA production conditions, the succinic acid 2, 4, 6, 8, 10 and 12 g / L was added to the culture medium, the ALA production yield was measured, glycine 2, 4, 6, 8, After addition by concentration of 10 and 12 g / L, the yield of ALA was measured.

As a result, as shown in Figure 4, in the recombinant strain according to the invention succinic acid showed a high ALA production compared to the succinic acid-free medium at 8 to 12 g / L, in particular the highest yield at about 9 g / L . In addition, as shown in Figure 5, in the case of the recombinant strain according to the present invention showed a high ALA production in general compared to the succinic acid-free medium in the range of 2 to 12 g / L, 4 to 12 g / L The yield was better at, and the yield was better at 8-10 g / L, especially at about 8 g / L.

On the other hand, when cultured as described above in a medium containing both succinic acid and glycine, as shown in Figure 6, when added 1 g / L glycine and 9 g / L succinic acid to the culture medium, 1.5 times more than no addition ALA production was confirmed.

As a result, the recombinant strain according to the present invention was confirmed to show a high ALA production yield in a specific combination of glycine and succinic acid.

3-3: Optimized ALA  Under production conditions ALA  Production quantity measurement

Meanwhile, to measure the amount of ALA produced under the conditions of Example 3-2, 100 μl of 1M sodium acetate buffer (pH 4.7) and 50 μl of acetyl acetone were added to 200 μl of the culture filtrate, followed by heating for 10 minutes, followed by room temperature. 350 μl of MER (modified Ehrlich's reagent: -dimethylaminobenzaldehyde, 8 ml of glacial acetic acid, 1 ml of 70% perchloric acid, DW 1 ml) was prepared in 2-methyl-3-acetyl-5-propionate pyrrole formed by cooling in It was added and left at room temperature for 15 minutes.

The amount of the colored compound was measured at 555 nm using a Microplate Spectrophotometer (Biotech, USA) and then quantified by converting ALA production amount from the ALA standard curve.

As a result of measuring the production of ALA according to the incubation time under the optimized conditions of ALA, it was confirmed that the ALA production pattern as shown in Figure 7, and, as shown in Figure 8, when the recombinant microorganism according to the present invention is cultured in the optimum conditions It was confirmed that ALA production was about 2.1 times higher than that of the Bacillus subtilis strain which did not introduce ALAS derived from Zymomonas mobilis.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Korea Microorganism Conservation Center (overseas) KCCM11226P 20111128

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Claims (6)

Recombinant microorganism with improved ability to produce 5-aminolevulinic acid, wherein a Bacillus subtilis has introduced a recombinant vector containing a 5-aminolevulinic acid synthase gene of Zymonas mobilis. .
A recombinant microorganism having improved 5-aminolevulinic acid production capacity, in which a 5-aminolevulinic acid synthase gene of Zymonas mobilis is inserted into a chromosome of Bacillus subtilis.
The recombinant microorganism according to claim 1 or 2, wherein the 5-aminolevulinic acid synthase gene of Zymonas mobilis is a nucleic acid sequence represented by SEQ ID NO: 2.
The recombinant microorganism of claim 1, wherein the recombinant microorganism is derived from a Bacillus subtilis strain deposited with accession number KCCM11226P.
Culturing the recombinant microorganism of claim 1 or 2 in a culture medium to which 8 to 12 g / L succinic acid or 8 to 10 g / L glycine is added; And recovering 5-aminolevulinic acid from the culture medium.
Culturing the recombinant microorganism of claim 1 or 2 in a culture medium to which 1 to 2 g / L glycine and 3 to 9 g / L succinic acid are added; And recovering 5-aminolevulinic acid from the culture medium.

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