KR20200070799A - Microorganism strain having improved growth characteristics under acidic condition - Google Patents

Abstract

The present invention relates to an E. coli mutant strain having improved acid resistance under acidic condition, and a method for producing an organic acid by culturing the same.

Description

Microorganism strain having improved growth characteristics under acidic condition

The present invention relates to an E. coli mutant strain having improved acid resistance under acidic conditions, and a method for culturing the same to produce an organic acid.

The organic acid production process using microorganisms consists of a high concentration cell culture and an organic acid production fermentation process. Due to the by-products produced during cell culture, acetic acid or the final product organic acid, the environment in the fermenter is lowered to acidic (about pH 5.0), thereby inhibiting the growth of microorganisms and production of target substances. During the fermentation process, the base is continuously added to maintain the cell activity to maintain the neutral (about pH 7.0) constant. In this case, the excess base used is the cost of raw materials or the addition of desalting cost in the subsequent separation and purification process. Cause problems.

Therefore, for efficient organic acid production, it is required to develop an acid-resistant strain in which growth and target material production are maintained even under acidic conditions.

Japanese Patent Publication No. 2010-000025 (2010.01.07)

Against this background, the present invention provides a mutant strain that grows at a high concentration in a specific cell number or more and produces an organic acid in comparison with a non-mutated control group in an acidic medium environment.

Thus, as an example, it provides an E. coli mutant strain with improved acid resistance under acidic conditions.

In another example, a method of producing an organic acid, comprising culturing the E. coli mutant strain.

According to one aspect of the present invention, there is provided an E. coli mutant strain having improved acid resistance under acidic conditions. In one embodiment, the E. coli mutant strain has improved acid resistance under acidic conditions and can produce an organic acid.

According to another aspect of the present invention, a method for producing an organic acid is provided, comprising culturing the E. coli mutant.

Specifically, in the present invention, a random mutation was added to E. coli to select a species that grows in an acidic environment and compared with a non-mutated species, and compared to a non-mutated control group in an acidic (approximately pH 4.0) medium environment. The mutants growing at a high concentration were selected, and the strain was deposited on September 20, 2018 at KCTC (Korean Colletion for Type Cultures, Korea Research Institute of Bioscience and Biotechnology), and assigned accession number KCTC18714P.

As used herein, "mutant strain" or "mutant strain" refers to a strain having a change in DNA sequence compared to a parent strain that has not undergone mutation. Such variation may be a change in the DNA sequence by insertion, addition, substitution, deletion or inversion of the base in the DNA sequence. In addition, the mutation can be a missense, nonsense, or frameshift mutation.

As used herein, "acid resistance" refers to a cultivated property that shows sustained growth when cultured in an environment below pH 7.0.

As used herein, "organic acid" is a term encompassing an organic compound having an acidity, and for example, refers to an organic compound containing a carboxyl group or a sulfone group. These organic acids may be produced from organic substances including proteins, fats, and carbohydrates, and may be produced as intermediate products or major by-products in metabolism of various microorganisms, for example, wood-based or fiber-based biomass or waste biomass. It can be produced inexpensively from the back. The organic acid herein is not limited to a specific organic acid, 3-hydroxypropionic acid (3-hydroxypropionic acid, 3-HP), acetic acid, succinic acid, fumaric acid, malic acid, lactic acid, citric acid, butyric acid, oxalic acid, tartaric acid, palmitic acid , Benzoic acid, aspartic acid, glutamic acid, and the like, and preferably, 3-hydroxypropionic acid (3-HP).

The E. coli mutant of the present invention may have resistance to an acidic environment or an organic acid, and may have resistance to, for example, 3-hydroxypropionic acid or acetic acid, which is a by-product generated in the production process.

The E. coli mutant of the present invention can be advantageously used as a host for producing organic acids or mass-producing foreign recombinant proteins due to its improved acid resistance under acidic conditions. The acidic condition refers to an environment with a pH of less than 7.0, for example pH 4.0 to pH 5.0.

Thus, the E. coli mutant of the present invention may have the ability to synthesize organic acids, for example, the ability to synthesize 3-hydroxypropionic acid.

The E. coli strain of the present invention may itself be a strain genetically engineered to produce an organic acid, including a gene involved in organic acid production, to have an organic acid production capacity or to enhance the organic acid production capacity. The production of the organic acid includes those produced in a strain, those produced in a cell and secreted outside the cell, or a combination thereof.

For example, when the mutant strain is a strain producing 3-HP, several pathways are known as a pathway for biosynthesizing 3-HP from a carbon substrate such as glycerol, but a representative example is as follows: First, glycerol dihydra Glycerol is converted to 3-hydroxypropionaldehyde (3-HPA), an intermediate chain through a dehydration reaction by a third agent, and 3-hydroxypropionaldehyde (aldehyde dehydrogenase (ALDH)). 3-HPA) is oxidized to 3-HP. Therefore, in order to further increase 3-HP production, the mutant strain may be a strain in which genes of enzymes involved in a pathway for biosynthesis of 3-HP are additionally introduced. For example, a gene encoding glycerol dehydratase and/or a gene encoding aldehyde dehydrogenase can be further introduced.

The term "glycerol dehydratase" or "diol dehydratase" is a polypeptide having an enzyme activity that catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA). Says Glycerol dihydratase or diol dihydratase can be coenzyme B12 dependent or non-dependent. The enzyme is, but is not limited to, Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium ), Klebsiella oxytoca, or Clostridium butyricum. The enzyme, regardless of its origin, is an enzyme having an enzyme activity that catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA) is included in the scope of the present application, and a gene encoding it is dhaB can do.

The term, “aldehyde dehydrogenase” is an enzyme that converts aldehydes to carboxylic acids, from 3-hydroxypropionaldehyde (3-HPA) to 3-hydroxypropionic acid (3-HP). Refers to a polypeptide having an enzyme activity that catalyzes the conversion of. Aldehyde dehydrogenase, regardless of its origin, is an enzyme that has enzymatic activity that catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 3-hydroxypropionic acid (3-HP). It is included, and examples of the gene encoding it include puuC and the like.

In addition, the mutant of the present application may further include a gene encoding glycerol dehydratase reactivase for reactivating glycerol dehydratase in order to further increase 3-HP production.

The term, "glycerol dehydratase reactivase" is an enzyme that acts to maintain catalytic activity by re-activating irreversibly inactivating glycerol dehydratase during action. Glycerol dihydratase Reactivase may be any of generally known, for example, Klebsiella sp., Citroacter sp., Lactobacillus sp. ), Salmonella sp. strain, etc., but is not limited thereto. Glycerol dihydratase reactivase may be DhaFG, GdrAB, DdrAB, etc., and genes encoding them may exemplify dhaFG, gdrAB, ddrAB, and the like.

Introduction to the strain means that the target gene is introduced into the host cell, such that the nucleic acid can be cloned as an extrachromosomal factor or by chromosomal integration. For example, vectors comprising genes related to organic acid synthesis can be introduced into host organisms. As another example, an integration vector containing an organic acid synthesis related gene can be introduced into a strain to be inserted into the genome of the strain. The tissue-specificity of the insertion can be controlled, for example, by the vector route of administration. As another example, a non-integrated vector comprising an organic acid synthesis related gene can be introduced into a host organism by being introduced into the host organism.

As used herein, a vector refers to a gene construct comprising essential regulatory elements operably linked to express a gene insert encoding a target protein in a cell of an individual, and includes various forms such as plasmid, virus vector, bacteriophage vector, cosmid vector, etc. Vectors of can be used.

It is preferred that the gene inserted into the vector and transferred is irreversibly fused into the genome of the host cell so that gene expression in the cell is stably maintained for a long period of time. Such vectors can include transcriptional and translational expression control sequences that enable the gene to be expressed in a selected host. Expression control sequences may include promoters for conducting transcription, any operator sequences for regulating such transcription, and/or sequences that regulate termination of transcription and translation. The initiation codon and termination codon are generally regarded as part of the nucleic acid sequence encoding the protein of interest, and must exhibit action in the individual when the gene construct is administered and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible. In addition, in the case of a replicable expression vector, the origin of replication may be included. In addition, an enhancer, a non-translated region at the 3'end of the desired gene, a selection marker (eg, antibiotic resistance marker), or a replicable unit may be appropriately included. The vector can be self-replicating or integrated into the host genomic DNA.

Each component in the vector must be operably linked to each other, and the linking of these component sequences can be performed by ligation (linking) at a convenient restriction enzyme site, and in the absence of such a site, the conventional method is used. According to the synthetic oligonucleotide adapter (oligonucleotide adapter) or linker can be performed using.

Examples of useful expression control sequences include early and late promoters of adenovirus, monkey virus 40 (SV40), mouse breast tumor virus (MMTV) promoter, long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalo Virus (CMV) promoter, Epstein virus (EBV) promoter, Loews Sacoma virus (RSV) promoter, RNA polymerase II promoter, T3 and T7 promoters, major operator and promoter regions of phage lambda, and prokaryotic or eukaryotic cells Or other sequences of construction and derivation known to control the expression of genes in their viruses and various combinations thereof.

Introducing the gene into a strain is suitable standard techniques as known in the art, for example, electroporation, electroinjection, microinjection, calcium phosphate co-precipitation (calcium phosphate) co-precipitation, calcium chloride/rubidium chloride method, retroviral infection, DEAE-dextran, cationic liposome method, polyethylene glycol-mediated uptake, Gene guns, etc. may be used, but are not limited thereto. At this time, the circular vector can be cut with an appropriate restriction enzyme and introduced into a linear vector form.

The present invention also provides a method for producing an organic acid, comprising culturing the E. coli strain as defined above.

The medium used for the culture of the strain must adequately satisfy the requirements of the specific strain. The medium may contain various carbon sources, nitrogen sources, personnel and trace element components. As a specific medium, LB (Luria Bertani) broth, SD (Sabouraud Dextrose) broth, or YM (Yeast medium) broth may be used, but is not limited thereto.

As a carbon source in the medium, glycerol may be included. In addition, monosaccharides, oligosaccharides, polysaccharides, monocarbon substrates, or mixtures thereof can be exemplified. Monosaccharides such as, for example, glucose and fructose; Oligosaccharides such as sucrose, maltose or lactose; Polysaccharides such as starch or cellulose; A single carbon substrate such as methanol, formaldehyde or formate can be exemplified. In addition, 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, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid may be exemplified, but are not limited thereto. These materials can be used individually or as a mixture.

Examples of nitrogen sources in the medium include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. It is not limited thereto. The nitrogen source can also be used individually or as a mixture.

Personnel in the medium may include, but are not limited to, potassium dihydrogen phosphate or dipotassium phosphate or the corresponding sodium-containing salt. In addition, the culture medium may include a metal salt such as magnesium sulfate or iron sulfate required for growth, or may include essential growth materials such as amino acids and vitamins, but is not limited thereto. The above-mentioned raw materials may be added batchwise or continuously in an appropriate manner to the culture during the culture process.

In addition, if necessary, the pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, and ammonia or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner. In addition, anti-foaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles.

The culture (fermentation) can be arranged in batch, fed-batch, or continuous mode depending on the recombinant microorganism. Fermentation conditions can be cultured in aerobic, micro-aerobic, and anaerobic conditions, and preferably can be carried out in a batch manner in micro-anaerobic conditions. The culture temperature and the culture time can be performed by appropriately selecting the characteristics of the host cell to be used. For example, oxygen or oxygen-containing gas (eg, air) may be injected into the culture to maintain aerobic conditions, and the temperature of the culture is usually 20°C to 45°C, preferably 25°C to 40°C Can be. Cultivation can be continued until the desired amount of organic acid is obtained.

Methods for recovering organic acids from fermentation media are known in the art. For example, organic acids can be obtained from cell media by performing centrifugation, chromatography, extraction, filtration, precipitation, or combinations thereof. Production of organic acids by microorganisms can be confirmed and quantified by suitable techniques well known in the art. As an example, HPLC techniques can be used to measure the amount of organic acid produced by a selected clone. The HPLC technique also helps to determine the purity of organic acids produced by microorganisms.

The acid resistance is improved under acidic conditions, and by using an E. coli mutant that produces organic acid, desired organic acid can be fermented and produced at a high concentration.

Figure 1 shows the results of evaluating the acid resistance by the inoculation concentration of E. coli mutant strains according to an embodiment of the present application.

Hereinafter, the present invention will be described in more detail by the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Selection of E. coli strain

To develop an acid-resistant strain, E. coli W3110 (ATCC 2325) was treated with a random mutant derivative, EMS (Ethyl methanesulfonate), to induce mutations, and then mutants grown in an acidic (pH 4.0) medium were selected. Specifically, the bacteria were inoculated into LB medium at pH 7.0 (Luria Bertani broth in distilled water (DW) at a concentration of 25 g/L and sterilized in an autoclave for 15 minutes at 121 degrees) and cultured overnight, and the cultured bacteria were It was re-inoculated in 5 ml LB medium having the same composition as and grown to OD ₆₀₀ 0.3. After centrifugation, the supernatant was removed, and 1 ml of 1X PBS buffer was added and resuspended. This process was repeated twice. After adding 35 ul of EMS to a resuspended state in 2 ml of 1X PBS buffer, the cells were cultured at 37°C for 45 minutes. After centrifugation, the supernatant was removed, and 1 ml of 1X PBS buffer was added and resuspended. This process was repeated twice. LB medium at pH 7.0 After the addition, the cells were incubated at 37°C for 45 minutes. The solution of the mixture of Luria Bertani broth in distilled water (DW) at a concentration of 25 g/L was lowered to pH with 5N HCl using a pH meter, and then sterilized in an autoclave for 15 minutes at 121 degrees, and 20 g of agar was used for plate solidification. /L to prepare the LB plate medium. After spreading the bacteria on the LB plate medium whose pH was adjusted to pH 7.0 or pH 4.0, the cells were cultured at 37°C for 3 days. Acid-resistant candidate groups #1, #4, #12, #13, #14, #27, and #31 growing in pH 4.0 LB plate medium were screened and reconfirmed.

Example 2. Confirmation of acid resistance of Escherichia coli mutant strain according to inoculation concentration

Since the initial inoculation concentration affects the pH 4.0 acidic medium when the strain exhibits acid resistance, the following experiment was conducted to confirm the variation according to the inoculation concentration. Specifically, control and acid-resistant strain candidate groups #1, #4, #12, #13, #14, #27, and #31 were cultured overnight in LB liquid medium at pH 7.0. After 1 ml of the culture solution was centrifuged, the supernatant was removed and resuspended by adding 1 ml of 1X PBS buffer. This process was repeated twice. Resuspended in 1 ml of 1X PBS buffer is assumed to be 1X, and the pH 4.0 solution is lowered to 5N HCl with a pH meter measurement of a solution mixed with 25 g/L concentration of distilled water in LB medium (Luria Bertani broth at pH 4.0) at 121 degrees. Sterilized in an autoclave for 15 minutes) Inoculated in 3 ml (1X: 30 ul / 5X: 150 ul / 7.5X: 225 ul, and the pH change due to the increase of the inoculation amount is minimal). Cultured overnight at 37° C. shaker to observe the culture status of the bacteria and measure the absorbance of OD ₆₀₀ .

As a result, as shown in FIG. 1, when the initial 5X inoculation was observed, the variation in OD measurement in the #14, #27, and #31 strains was more pronounced than in the 1X inoculation, thereby securing acid resistance. Therefore, among these, #14 strains were deposited with KCTC (Korean Colletion for Type Cultures, Korea Research Institute of Bioscience and Biotechnology) on September 20, 2018, and were given accession number KCTC18714P.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.

Korea Research Institute of Bioscience and Biotechnology KCTC18714P 20180920

Claims (7)

Improved acid resistance under acidic conditions,
E. coli strain KCTC18714P with acid resistance. According to claim 1, wherein the E. coli strain is characterized in that the acid resistance is improved under acidic conditions and produces an organic acid, E. coli strain KCTC18714P. According to claim 2, wherein the E. coli mutant strain is characterized in that the acid resistance is improved for the acid or the final product of the organic acid produced as a by-product during cell culture, E. coli mutant strain KCTC18714P. According to claim 3, E. coli mutant strain KCTC18714P, the acidic substance produced as a by-product in cell culture is acetic acid. According to claim 1, wherein the acidic condition is less than pH 7.0, or pH 4.0 to pH 5.0, E. coli strain KCTC18714P. The method of claim 2, wherein the organic acid is 3-hydroxypropionic acid (3-hydroxypropionic acid, 3-HP), acetic acid, succinic acid, fumaric acid, malic acid, lactic acid, citric acid, butyric acid, oxalic acid, tartaric acid, palmitic acid, benzoic acid, aspartic acid E. coli strain KCTC18714P, which is an acid or glutamic acid. A method of producing an organic acid, comprising culturing the E. coli strain of claim 1.

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