KR101781294B1 - High growth Escherichia coli using glycerol as carbon source - Google Patents

Abstract

The present invention relates to a novel Escherichia coli which uses glycerol as a carbon source, which is a strain of Escherichia coli that has been improved to use glycerol as a carbon source more quickly and effectively than natural Escherichia coli, glycerol-containing microorganism preparation containing the Escherichia coli strain as an active ingredient And a method for the metabolism of glycerol using the Escherichia coli strain. The E. coli strain improved through the present invention grows faster than naturally occurring E. coli using glycerol as a unique carbon source, so that the non-productivity of the bioconversion process can be greatly increased if it is used. In addition, the E. coli strain may be usefully used as a platform strain of a biotransformation technology for converting glycerol to a useful product in the future.

Description

{High growth Escherichia coli using glycerol as carbon source}

The present invention relates to a novel Escherichia coli which uses glycerol as a carbon source, which is a strain of Escherichia coli that has been improved to use glycerol as a carbon source more quickly and effectively than natural Escherichia coli, glycerol-containing microorganism preparation containing the Escherichia coli strain as an active ingredient And a method for the metabolism of glycerol using the Escherichia coli strain.

As a result of global depletion of fossil fuels, environmental pollution caused by the use of fossil fuels, and surging oil prices, the development of clean and environmentally friendly alternative energy has been steadily developed. Biodiesel is attracting attention as one of such energy fuels. Biodiesel is produced mainly in Europe where diesel vehicles are widely used. In Korea, as the air pollution becomes serious, government efforts to disseminate biodiesel are being made, and production volume is increasing every year. In fact, biodiesel has been reported to have nearly tripled production in the US in 2012 compared to 2010, and this trend is expected to continue in the future.

Biodiesel is a single alkyl ester mixture with long fatty acid rings, which converts the original oil to the desired ester and uses lipester ester transesterification to remove free fatty acids. In this process, unlike non-aromatic vegetable oil, it has combustion characteristics very similar to light oil, so it can replace light oil in most of the currently used cases. In addition, unlike diesel, it is an oxygen-containing fuel containing about 10% (%) of oxygen. In the case of combustion, oxygen contained in biodiesel causes more complete combustion, and air pollution is 40 to 60% There is an advantage that it is less than ideal.

However, glycerol, which is produced as a by-product of lipid ester exchange reaction used in the production process of biodiesel, is pointed out as a major problem related to biodiesel. Purified glycerol can be used as a raw material for fine chemicals, pharmaceuticals production, and cosmetics, but unrefined glycerol, which is produced as a byproduct of the biodiesel production process, can be produced by a high concentration of unreacted fatty acid salt and methanol, Peroxides, and biodiesel components, and the purity is very low. Unpurified glycerol can be purified by methods such as distillation, adsorption, and extraction, but it has limitations in increasing the recovery rate by purification. In addition, it is very difficult to create added value through refining. Therefore, untreated glycerol is being culled or buried, and it is becoming a new source of environmental pollution. Therefore, it is required to develop a technology that can utilize the non-purified glycerol industrially generated in the biodiesel production process. Therefore, many researches have been made to utilize biotransformation technology as one of these utilization technologies.

Biotransformation technology is a technology field that has been researched early to replace it due to the environmental pollution problems due to the development of chemical processes in various industries. As a result, many chemical processes are being replaced by bioreactors. Biotransformation technology is widely used in the areas of biological correction, food processing, fine chemical manufacturing, and pharmaceutical production, which turn harmful substances into nontoxic substances. In recent years, there have been a lot of studies to produce industrially useful materials using microorganisms, yeast, fungi and the like which can grow nutrients of the non-purified glycerol produced in the biodiesel production process using these biotransformation technologies. For example, Escherichia coli, Kluyvera genus, Corynebacterium genus, or Clostridium genus bacteria that can utilize glycerol as a substrate using transformation or other methods are prepared from glycerol Research results for producing and using ethanol, butanol, 1, 2-propanediol, carotenoid, 3-hydroxypropionic acid, or amino acid have been reported (patent KR 10-1223816).

On the other hand, in the case of a specific microorganism such as E. coli, there is an advantage that glycerol can be grown as a carbon source without the need of a transformation step, and the mechanism of its utilization is well known. Glycerol on the outside of E. coli cells enters the cell using GlpF, which is aquaglyceroporin, as a glycerol transporter without consumption of energy, and then glycerol kinase (GlpK), glycerol-3-phosphate di Glycerol-3-phosphate dehydrogenase, and triosephosphate isomerase (TpiA), and the metabolic process of glycerol is regulated in various forms. Escherichia coli can grow glycerol as a carbon source. However, when glycerol and glucose are present together, it is known that they exclusively perform diauxic growth using glucose only in post-glycerol. Therefore, when wild type Escherichia coli is cultured in a medium containing glycerol as the only carbon source, the growth rate is lower than that of Escherichia coli using glucose. Recently, Escherichia coli W introduced into the biotransformation process has a disadvantage in that the growth rate is low when glycerol is used as a substrate, despite the ability to use a wide carbon source. Therefore, it is necessary to develop an Escherichia coli strain which overcomes the above-mentioned disadvantages that can be usefully used for the bioconversion technology of glycerol as a useful product.

The present invention has been made to develop a novel E. coli which uses glycerol more rapidly and more effectively than Escherichia coli, which is a natural type, from E. coli which grow at a high growth rate, among living organisms which are used for bio- , Escherichia coli W was subcultured continuously in a medium containing glycerol as the only carbon source. As a result, it was confirmed that an Escherichia coli modified strain with improved non-growth rate was produced as compared with natural Escherichia coli.

Accordingly, it is an object of the present invention to provide a novel hypertrophic Escherichia coli strain using glycerol as a carbon source.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a novel hypertrophic Escherichia coli strain CY02 (Accession No. KCTC 12782BP) using glycerol as a carbon source.

In one embodiment of the present invention, the E. coli strain CY02 may be derived from Escherichia coli W.

In another embodiment of the present invention, the E. coli strain CY02 may be one produced by subculturing in a culture medium containing glycerol as a unique carbon source.

In another embodiment of the present invention, the Escherichia coli strain is selected from the group consisting of galactoside transport system permease, RS15750 (PEP-protein phosphotransferase) and RS18510 (global transcriptional regulator CRP). And glpK (glycerol kinase) may be inserted, deleted, or substituted by amino acid substitution.

The present invention also provides a glycerol / microbial preparation using the above E. coli strain CY02 (Accession No. KCTC 12782BP) as an active ingredient.

In addition, the present invention provides a method of glycerol metabolism comprising the step of treating said Escherichia coli strain CY02 (Accession No. KCTC 12782BP) to glycerol.

The novel E. coli strain improved through the present invention grows faster than natural E. coli using glycerol as a unique carbon source, so that the non-productivity of the bioconversion process can be greatly increased by using it. In addition, the E. coli strain may be usefully used as a platform strain of a biotransformation technology for converting glycerol to a useful product in the future.

Fig. 1 shows growth rates in a culture medium containing glycerol of CY02, natural E. coli W, and Escherichia coli W3110, which is a common laboratory strain, as the sole carbon source.
FIG. 2A shows the growth curves of a novel E. coli strain CY02, a wild-type E. coli W, and a general laboratory strain E. coli W3110 transformed with the pZA-GADΔC / E89Q vector expressing GADΔC / E89Q, a mutant of glutamate decarboxylase And FIG. 2B shows the concentration of gamma aminobutyric acid (GABA) produced in the culture medium of the E. coli.
FIG. 3 is a graph showing the number of genes mutated as a result of conducting a genome analysis on E. coli W, ALE population, and CY02 E. coli strains.
Fig. 4 shows the results of genomic analysis of Escherichia coli W, ALE population, and CY02 Escherichia coli strains, showing the degree of mutation, the type of mutation, and the gene function for each gene.

The present invention relates to a novel hypertrophic Escherichia coli strain which uses glycerol as a carbon source more quickly and effectively than natural Escherichia coli.

Accordingly, the present invention provides a novel hypertrophic Escherichia coli strain using glycerol as a unique carbon source.

The term " carbon source " used in the present invention means an organic molecule that supplies C (carbon), H (hydrogen), and O (oxygen) as elements necessary for microorganism growth. Organic molecules such as sucrose and glucose are used as carbon sources of microorganisms to supply energy and form electron transport and organic molecular skeletons. The novel E. coli strain derived from E. coli W of the present invention uses glycerol as a carbon source.

As used herein, the term " Escherichia coli " means gram-negative bacterium and refers to a bacterium commonly found in the intestines of warm-blooded animals including humans, and can be easily cultured in a laboratory environment. E. coli can survive through a variety of substrates, and E. coli and its associated bacteria have the ability to deliver DNA through bacterial conjugation, transduction, or modification.

The strain of the present invention is Escherichia coli , preferably Escherichia coli CY02 (Accession No. KCTC 12782BP), and may be derived from Escherichia coli W.

In another aspect of the present invention, the present invention provides glycerol versus a microorganism preparation containing the Escherichia coli strain CY02 (Accession No. KCTC 12782BP) as an active ingredient.

In another embodiment of the present invention, the present invention provides a method of glycerol metabolism comprising treating said Escherichia coli strain CY02 (Accession No. KCTC 12782BP) to glycerol.

The method for culturing the Escherichia coli of the present invention can be carried out according to a conventional method known in the art, that is, according to a conventional temperature, culture conditions. Such a culturing process can be easily adjusted according to the strain selected by those skilled in the art. Preferably, Escherichia coli W can be subcultured in a culture medium containing glycerol as a sole carbon source for about 40-50 days at intervals of 5-15 hours, more preferably for about 44 days at intervals of 8-9 hours So that the growth rate can be improved.

The culture medium contains glycerol as the only carbon source, and the pH of the culture can be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner during the culture. In addition, foaming can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture. In addition, oxygen or oxygen-containing gas may be injected into the culture to maintain the aerobic state of the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without injecting gas to maintain anaerobic and microorganism conditions. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C.

In one embodiment of the present invention, the natural type E. coli W was repeatedly subcultured in a medium containing glycerol as the only carbon source to obtain colonies. Among the selected improved strains CY02, K-12 It was confirmed that the growth rate was much higher than that of W3110 (see Example 1).

In another embodiment of the present invention, the Escherichia coli strain CY02, the wild-type Escherichia coli W, and the Escherichia coli W3110 are transfected with a vector expressing a glutamate decarboxylase mutant, transformed into a glycerol culture medium, cultured in a fresh medium As a result, it was confirmed that CY02 exhibits an excellent non-growth rate and high productivity of gamma-aminobutyric acid, thereby confirming that the high specific growth rate of the modified strain can be used for improving productivity of the useful product (see Example 2) .

In another embodiment of the present invention, a genomic analysis was performed to see which genes were mutated in common in the ALE population and the modified strain CY02 of the present invention, compared with the wild-type Escherichia coli W. As a result, galactoside transport system permease (mglC), RS15750 (PEP-protein phosphotransferase) and RS18510 (global transcriptional regulator CRP), which encode various enzyme proteins. And glpK (glycerol kinase) gene sequences, mutation, or mutation of the gene by single base mutation.

Thus, Escherichia coli CY02, a new Escherichia coli strain developed through the embodiment of the present invention, was deposited on April 1, 2015 (Accession No. KCTC 12782 BP) at the Microbiological Resource Center of the Korea Biotechnology Research Institute under the Budapest Treaty.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Improvement of Escherichia coli

Recently, E. coli W introduced into the biotransformation process has not improved the non-growth rate through laboratory evolution despite its ability to use a wide range of carbon sources. Thus, the present invention aims to improve the non-growth rate of Escherichia coli W through laboratory evolution.

Escherichia coli W was added to a medium containing glycerol as the sole carbon source (6.4 g / L Na ₂ HPO ₄揃 7H ₂ O, 3.0 g / L KH ₂ PO ₄ , 0.5 g / L NaCl, 1.0 g / L NH ₄ Cl, ₄ , 0.1 mM CaCl ₂ , 0.2% glycerol) and subcultured in fresh medium every 8 to 9 hours. The colonies obtained by repeating this for 44 days were plated on a solid medium to obtain 10 colonies, which were named CY01 to CY10, respectively. CY02, one of the 10 colonies, was selected and the absorbance at 600 nm was measured until 12 hours after culturing in order to measure the growth rate together with Escherichia coli W, a natural strain, and K-12 series Escherichia coli W3110, The growth rate of each Escherichia coli was compared with time.

As a result, as shown in Fig. 1, Escherichia coli strain CY02 showed much higher growth rate than Escherichia coli W and W3110. Also, Escherichia coli W showed a lower final cell concentration than W3110, whereas the modified strain CY02 showed a higher final cell concentration than W3110. From the above results, it was found that the Escherichia coli modified strain CY02 can utilize glycerol faster and more effectively than Escherichia coli W of the natural type.

Therefore, the strain was designated as Escherichia coli W CY02 (Escherichia coli W CY02), and the strain was deposited at the Korea Research Institute of Bioscience and Biotechnology (Accession No. KCTC 12782BP).

Example 2. Verification of bioconversion reaction using an improved E. coli strain

To verify that the high specific growth rate of the CY02 E. coli strain improved by the method of Example 1 can be utilized to improve the productivity of the useful product, CY02 can be used as a functional food, a functional cosmetic raw material, Production of Gamma-Amino Butyric Acid (GABA) was confirmed.

Gamma aminobutyric acid is synthesized through the decarboxylation of the amino acid glutamate. It is known that glutamate decarboxylase acts at this time. When glutamate decarboxylase mutant (GADΔC / E89Q), which acts at neutral pH, is used, gamma aminobutyric acid can be produced along with cell growth. GADΔC / E89Q, a mutant of glutamate decarboxylase, was cloned into the expression vector pZAmcs (Expressys, USA), and the plasmid pZA-GADΔC / E89Q was introduced into each of Escherichia coli CY02, W and W3110 and transformed. Each of the E. coli transformed to express the GAD? C / E89Q enzyme was taken as a single colony in a solid medium, and 5 ml of glycerol medium (6.4 g / L Na ₂ HPO ₄ .7H ₂ O, 3.0 g / L KH ₂ PO ₄ , 0.5 g / L NaCl, 1.0 g / L NH ₄ Cl, 2 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.2% glycerol, 5 mg / L chloramphenicol) and seeded for 16 hours. Growth rate and gamma aminobutyric acid production were investigated by inoculation in 50 ml of fresh medium of the same composition.

As a result, as shown in Fig. 2, it was confirmed that CY02 exhibited an excellent non-growth rate as compared with E. coli W and W3110, and exhibited remarkably high gamma aminobutyric acid productivity. From the above results, it was confirmed that the Escherichia coli strains improved by the present invention can be usefully used in biotransformation technology for producing useful products using glycerol as a sole carbon source.

Example 3. Genetic mutation verification of the improved E. coli strain

The inventors of the present invention sought to verify whether the novel CY02 Escherichia coli strain of the present invention was improved into a hyperproliferative strain having glycerol as the only carbon source by mutating any of the genes in comparison with Escherichia coli W of the natural type. To do this, mutated genes were examined by genomic analysis of wild type Escherichia coli W (ATCC 9637), ALE population, and improved CY02 E. coli strains.

The ALE population represents a group of evolved Escherichia coli that reached the last stage of laboratory evolution of E. coli W, and CY02 is a single strain selected in this population.

As shown in FIG. 3, 68, 93, and 100 gene mutations were analyzed in Escherichia coli W, ALE population, and CY02 E. coli strains, and the same genes without mutation of the strains existed, There were 5, 9, and 18 genes with the same mutation in each strain.

Furthermore, as shown in FIG. 4, the major mutations in the ALE population and the CY02 E. coli strain were found to be mutations in the cluster 5 region of mglC, RS15750, RS18510, and glpK, respectively, as compared to E. coli W . More specifically, mglC is a gene having the function of permease of galactoside transport system. In the CY02 Escherichia coli strain, the deletion of the nucleic acid in the gene region resulted in deletion of function. RS15750 gene has PEP-protein phosphotransferase function and CY02 Escherichia coli It was shown that the function of the strain was lost due to the insertion of the nucleic acid in the gene site. In addition, RS18510 and GLPK locus have single nucleotide variation (SNV). Global transcriptional regulator RS18510, which has CRP function, was substituted with histidine (Arg210His) in arginine at position 210 and glycerol kinase GlpK, the amino acid at position 236 was replaced by threonine with methionine (Thr236Met). Therefore, it can be understood that Escherichia coli W was modified to CY02 Escherichia coli strain having high viability by the four major gene mutations.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Korea Biotechnology Research Institute KCTC12782BP 20150401

Claims (6)

Escherichia coli strain CY02 (Accession No. KCTC 12782BP), which utilizes glycerol as the only carbon source.
The method according to claim 1,
The Escherichia coli strain CY02 (Accession No. KCTC 12782BP) is characterized in that it is derived from Escherichia coli W.
The method according to claim 1,
The Escherichia coli strain CY02 (Accession No. KCTC 12782BP) is produced by subculturing in a culture medium containing glycerol as a unique carbon source.
The method according to claim 1,
The Escherichia coli strains are selected from the group consisting of galactoside transport system permease, PEP-protein phosphotransferase (PEP-protein phosphotransferase), global transcriptional regulator CRP (global transcription regulator CRP), and glycerol kinase E. coli strain CY02 (Accession No. KCTC 12782BP), characterized in that a mutation has occurred through insertion, deletion, or amino acid substitution in the amino acid sequence of SEQ ID NO: delete delete

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