KR101256106B1 - Klebsiella oxytoca M1 Capable of Producing 2,3-butanediol in High Yield - Google Patents

Abstract

The present invention relates to a Klebsiella oxytoca M1 strain having a high yield of 2,3-butanediol. In the strain of the present invention, 2,3-butanediol can be produced in a high yield within a short time, thereby enabling mass production of 2,3-butanediol. On the other hand, using the strain of the present invention can also produce acetoin, a precursor of 2,3-butanediol.

Description

Klebsiella oxytoca M1 Capable of Producing 2,3-butanediol in High Yield with excellent yield of 2,3-butanediol

The present invention is excellent Krebsciella 2,3-butanediol production yield Oxytoca M1 strain and a method for producing 2,3-butanediol using the same. In addition, the present invention is excellent Klebsiella acetoin production yield Oxytoca M1 strain and a method for producing acetoin using the same.

Recently, the production of renewable energy using biotechnology has attracted much attention for lack of fossil fuels, rising oil prices and environmental conservation. Among the renewable energy, 2,3-butanediol can be applied to various fields industrially. It is a chemical used in many fields such as high octane aviation fuels, print inks, lacquers, synthetic fragrances, synthetic rubbers, drugs, medicine, fumigants and emulsifiers. 2,3-butanediol can be produced by dehydration as a fuel additive, methyl-ethyl-ketone, and further dehydration can produce 1,3-butanediene, which is used as a raw material for synthetic rubber do.

A known microorganism by the current production of 2,3-butane diol to the erotic Pseudomonas dihydro-pillar (Aeromonas hydrophila), the Bacillus Subtilis ( Bacillus) subtilis ) , Brevibacillus Brevis S1 (Brevibacillus brevis S1), Corynebacterium Gulutamicom ( Corynebacterium glutamicum), enter into bakteo Aero jeneseu (Enterobacter aerogenes), keurep when Ella nurse California; also referred to as (Klebsiella pneumonia Aero bakteo Aero jeneseu (Aerobacter aerogenes)), when keurep Ella Oxy-City ( Klebsiella oxytica), Lactobacillus brevis (Lactobacillusbrevis), Lactobacillus Kasei (Lactobacilluscasei), Lactobacillus helveticus carcass (Lactobacillus helveticus), Fran tarum Lactobacillus (Lactobacillus plantarum), Lactobacillus Coco rapti's Curse (Lactococcuslactis), Fort Scott Clark Pocono teeth (Leuconostoclactis), current Kono Stock mesen teroyi Death (Leuconostoc mesenteroides), subspecies Crescent Morris (subsp. cremoris), Oe no coarse coco O Enigma (Oenococcusoeni), Phedi OKO kusu Three pens Tosa mouse (Pediococcus pentosaceus), Raul telra agent and Terry (Raoultella terrigena), Serratia sense Marseille (Serratia marcescens) (Caspi, 2008), Fannie Bacillus miksa poly (Paenibacillus polymyxa) (Nakashimada et al. 2000 ), keulrep when Ella agent and Terry (Klebsiella terrigena) (Blomqvist et al . 1993), Aero bakteo Indolozins ( Aerobacter indologenes) and Bacillus Kenny Li Po Miss (Bacillus licheniformis ), (Nilegaonkaretal. 1992) and the like are known.

There are many microorganisms that produce 2,3-butanediol, but there are few microorganisms that can be applied industrially, so the production efficiency of 2,3-butanediol is diversified for diversification of technology and mass production of 2,3-butanediol. This requires the discovery and development of high microorganisms.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a new strain having excellent 2,3-butanediol production capacity. As a result, the present inventors have separated the microorganisms from certain soils in Korea, and have completed the present invention by identifying that the microorganisms have the ability to produce 2,3-butanediol in high yield.

Accordingly, an object of the present invention is a high yield of 2,3-butanediol production Klebsiella Oxytoca To provide M1.

Another object of the present invention is to provide a method for preparing 2,3-butanediol.

Still another object of the present invention is a high yield of acetoin Klebsiella Oxytoca To provide the M1 (KCCM11177P) strain.

Another object of the present invention is to provide a method for producing acetoin.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention is excellent Klebsiella 2,3-butanediol production yield Oxytoca Provide the M1 (KCCM11177P) strain.

The present inventors have tried to develop a new strain having excellent 2,3-butanediol production capacity. As a result, the present inventors have isolated microorganisms from certain soils in Korea, and found that these microorganisms have the ability to produce 2,3-butanediol in high yield.

Krebschiela of the present invention Oxytoca The M1 strain has a very good ability to produce 2,3-butanediol. 2,3-butanediol has the chemical formula C ₄ H ₁₀ O ₂ and is one of the structural isomers of butanediol, and the chemical structure is:

Formula 1

Krebschiela of the present invention The oxytoca M1 strain has a good yield of 2,3-butanediol. As used herein, the term “excellent yield” is used to refer to 2,3-butanediol, which means that the amount of 2,3-butanediol produced in comparison to the amount of sugar consumed while culturing the microorganism is 0.25 g / g to 0.3 g / g, preferably 0.3 g / g to 0.35 g / g, most preferably 0.35 g / g to 0.5 g / g .

According to another aspect of the present invention, the present invention provides a method for preparing 2,3-butanediol, comprising the following steps:

(a) Krebschiela of the present invention Oxytoca Culturing the M1 strain in a medium comprising a carbon source; And

(b) recovering 2,3-butanediol from the medium.

The carbon source used in the medium used in the method of the present invention includes conventional carbohydrates which can be added as a carbon source to the medium, preferably starch, glucose, acetic acid, paraffin, hydrocarbons, glucose, galactose, fructose , Xylose, sucrose, arabinose or lactose, more preferably glucose, galactose, fructose, xylose or sucrose, most preferably xylose or sucrose It is not limited to this.

The medium used in the method of the present invention preferably comprises a nitrogen source. The nitrogen source is preferably an organic nitrogen source, more preferably yeast extract, proteose peptone No. 3 and peptone, most preferably yeast extract.

According to a preferred embodiment of the present invention, when sugar is used as the carbon source, the concentration of sugar is 10 g / L to 120 g / L, more preferably 30 g / L to 110 g / L, more preferably 50 g / L. L to 100 g / L, even more preferably 60 g / L to 100 g / L. Even when 100 g / L or more sugar is used, the bacteria grow, but the degree is weak, and the growth of bacteria is inhibited at a sugar concentration of 150 g / L or more.

According to a preferred embodiment of the present invention, the culture temperature of the step of culturing in the medium is 25 ℃ to 35 ℃, more preferably 26 ℃ to 34 ℃, even more preferably 27 ℃ to 33 ℃ even more preferably Is 28 degreeC-32 degreeC Most preferably, it is 29 degreeC-31 degreeC.

According to a preferred embodiment of the invention, the culturing is carried out in aerobic conditions.

As used herein, the term “aerobic condition” refers to an environment in which an amount of oxygen exists to allow an aerobic strain to survive, and “aerobic condition” refers to an environment in which oxygen amount is low or no oxygen exists so that an aerobic strain cannot survive. Means.

According to another aspect of the present invention, the present invention provides a Krebsciella oxytoca M1 (Accession No. 000000) strain having a high yield of acetoin .

Acetoin is also called "3-hydroxybutanone" or "acetyl methyl carbinol" as a precursor to 2,3-butanediol. The chemical formula of acetoin is C ₄ H ₈ O ₂ and the chemical structure is:

(2)

According to another aspect of the invention there is provided a method for preparing acetoin comprising the following steps:

(a) Krebschiela of the present invention Oxytoca Culturing the M1 strain under aerobic conditions comprising a carbon source; And

(b) recovering acetoin from the medium.

According to a preferred embodiment of the present invention, when sugar is used as the carbon source, the concentration of sugar is 10 g / L to 120 g / L, more preferably 30 g / L to 110 g / L, more preferably 50 g / L. L to 100 g / L, even more preferably 60 g / L to 100 g / L.

According to another preferred embodiment of the present invention, the culture temperature of the step of culturing in the medium is 32 ℃ to 42 ℃, more preferably 33 ℃ to 41 ℃, even more preferably more than 34 ℃ to 40 ℃ Preferably it is 35 degreeC-39 degreeC Most preferably, it is 36 degreeC-38 degreeC.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a high yield of 2,3-butanediol Krebsciella Oxytoca Provided for the M1 (KCCM11177P) strain.

(Ii) Using the strains of the present invention, 2,3-butanediol can be produced at increased production rates and yields.

1 is a diagram showing the results of a systematic analysis using 16S rDNA nucleotide sequence of bacteria having high similarity to the nucleotide sequence of Krebsiella oxytoca M1 in Mega program.
Figure 2a is 30 ℃, Figure 2b is a graph showing the results of quantifying the product using gas chromatography while culturing the strain of the present invention in a 37 ℃ incubator, respectively. The values in the graph represent the average of the results of two experiments.
Figure 3a is aerobic environmental medium, Figure 3b is a graph showing the result of quantifying the product using gas chromatography while culturing the strain of the present invention in an anaerobic environmental medium. The values in the graph represent the average of the results of two experiments.

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 embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Production strain selection, 2,3-butanediol production measurement and opening analysis

 Example 1 Sampling and Screening of 2,3-Butanediol Production Strains

Samples were taken from the Manlypo tidal flat soil of Mohang-ri, Mohang-ri, Sowon-myeon, Taean-gun, Chungcheongnam-do, Korea. Since Klebsiella oxytoca is an anaerobic bacterium, 10 g of Manalipo soil is selected during the enrichment process (K ₂ HPO ₄ 0.5 g / L, KH ₂ PO ₄ 0.5 g / L, (NH ₄ ) ₂ SO ₄ 3 g / L, MgSO ₄ H ₂ O 0.2 g / L, CaCl ₂ H ₂ O 0.02 g / L, FeSO ₄ H ₂ O 0.00092 g / L, SL7 1 mL, 1 g / L, MES 19.52 g / L, glycerol 30 g / L, pH titrated to 6 using 5N KOH, pantothenic acid capsules 0.0108 g / L, nicotinic acid 0.0108 g / L , 0.2688 g / L myoinositol, 0.0108 g / L thiamine, 0.0108 g / L pyridoxine, 0.0022 g / L para-aminobenzoic acid, 0.0003 g / L d-biotin) into a 500 mL flask containing 200 mL anaerobic and Aerobic culture was carried out, respectively. After two days of cross-culture, 1 mL of the culture solution was added to a tube containing 9 mL of 0.85% NaCl, followed by serial dilution, and 2,3-butanediol solid selective medium (agar in the same component as the liquid selection medium). 14 g / L added), please add the components and their amounts). After incubation in a 30 ℃ incubator and inoculated into a liquid selection medium for each colony shape, the culture solution was quantified by gas chromatography (Agilent technology 6890N Network GC system) equipped with a flame ionization detector (FID) for two days. For the column, HP-INNOWAX (30 mx 250 μm × 0.25 μm, Agilent technology) was used.

Example  2: 16 S rDNA  Gene sequencing

16S rDNA gene was amplified by PCR (Polymerase Chain Reaction) technique using genome DNA as a template in the strain producing 2,3-butanediol. Primers used for PCR amplification were 27 forward primers (5'-AGAGTTTGATCTGCTCAG-3 ') and 1492 reverse primers (5'-AAGGAGGTGATCCAGCCGCA-3'). PCR was carried out by 10 cycles of 94 ° C., 1 cycle at 94 ° C., 1 minute at 55 ° C., and 1 minute 30 seconds at 72 ° C. for 30 minutes and reaction at 72 ° C. for 10 minutes. The amplified 16S rDNA gene result was commissioned by Bionics for sequencing. Sequence analysis results are the same as in the first sequence of the sequence listing. The Eztxon blast program using the above nucleotide sequence showed 99.78% concordance with the Klebsiella oxytoca JCM 1665 strain. Phylogenetic analysis was performed in the Mega program using 16S rDNA nucleotide sequences of bacteria having high similarity to the above nucleotide sequence (see FIG. 1). As a result of the same analysis, it was found that the similarity with Krepciella oxytoca JCM 1665 was high. Granted. The isolated strain was stored at -70 ℃ in 25% glycerol.

Example  3: Various On carbon  According to 2,3- Butanediol  Production aspect

Krebsiela In the aspect in which the oxytoca M1 strain produces 2,3-butanediol, the availability of various carbon sources and the pattern of 2,3-butanediol production were observed. As the carbon source used in the experiments, glucose, galactose, fructose, and xantose, which were pentose sugars, were used as the carbon source, and sucrose, a disaccharide, was also used in the experiment.

Microorganisms frozen in 50% of glycerol were seeded for overnight in 50 mL paper medium and paper medium containing a concentration of 50 g / L per 100 mL Erlenmeyer flask (K ₂ HPO _4). 13.7 g / L, KH ₂ PO ₄ 2 g / L, (NH ₄ ) ₂ HPO ₄ 3.3 g / L, (NH ₄ ) ₂ SO ₄ 6.6 g / L, MgSO ₄ 7H ₂ O 0.25 g / L, ZnSO ₇ H ₂ O 0.001 g / L, FeSO ₄ 7H ₂ O 0.05 g / L, MnSO ₄ H ₂ O 0.001 g / L, CaCl ₂ 2H ₂ O 0.01 g Seed cultured in 20 mL of / L and EDTA 0.05 g / L) were uniformly inoculated into each flask by 2-5% (v / v). During aerobic incubation at 150 rpm in a 30 ° C. incubator, microbial cultures were taken at regular intervals of 1 mL and the product was quantified using FID-equipped gas chromatography (Agilent technology 6890N Network GC system), and the column was HP-INNOWAX ( 30 mx 250 μm × 0.25 μm, Agilent technology) was used. The experiment was repeated twice.

Carbon source Glucose Galactose Fructose Xylose Sucrose 2,3-butanediol production concentration (g / L) 12.9 12.8 14.2 15.0 15.2

      Table 1 shows 2,3-butanediol production for various carbon sources.

Incubation for 48 hours resulted in the production of 2,3-butanediol as the main product from all the carbon sources used in the experiments as shown above, as well as several other substances. The final concentration of 2,3-butanediol in glucose is 12.9 g / L, 12.8 g / L in galactose, 14.2 g / L in fructose, 15.0 g / L in xylose and 15.2 g / L in sucrose It was found that 2,3-butanediol was produced using all carbon sources, and the produced concentration was not significantly different.

Example  4: 2,3- depending on the culture temperature Butanediol Birth  Aspect

20 mL of the paper medium with 50 g / L glucose was added to a sterile 100 mL flask with a carbon source of sucrose. Klebsiella cultured in the same medium the day before Oxytoca M1 Strains were inoculated 2-5% (v / v) in each flask. 1 mL each was incubated at 30 rpm and 37 ° C. incubator at 150 rpm and quantitated using gas chromatography (Agilent technology 6890N Network GC system) .The column was HP-INNOWAX (30 mx 250 μm x 0.25 μm, Agilent technology). The above experiment was repeated twice.

15.2 g / L of 2,3-butanediol was produced at 30 ° C and 11.6 g / L at 37 ° C. Thus, 30 ° C. produced 2,3-butanediol more efficiently than 37 ° C. Other byproducts, ethanol, succinic acid, lactate and formylate, were produced at similar concentrations. Acetoin is a precursor of 2,3-butanediol. Reversible reactions may occur with each other in the production of 2,3-butanediol. In other words, the amount of acetoin increases as the amount of 2,3-butanediol decreases in the second half of the production of 2,3-butanediol. In the case of acetoin when produced at 37 ℃ was produced faster than 30 ℃ (see Figure 2a and 2b).

Example  5: Anaerobic  And 2,3- in aerobic culture Butanediol  Production aspect

Krebsiela Oxytoka M1 is a permeable anaerobic bacterium that can grow in both anaerobic and oxygen aerobic environments. Therefore, experiments were conducted to compare the growth of bacteria and the observation of products in anaerobic and aerobic environments. Aerobic media was prepared by adding 20 mL of paper medium with 50 g / L sucrose into a sterile 100 mL Erlenmeyer flask. The anaerobic medium is placed in a sterile 125 mL serum battle with 20 mL of sucrose 50 g / L in a sterile 125 mL serum battle and blown with argon gas through an antibacterial filter for 30 min. The serum battle was sealed with a dog and an aluminum cap and then sterilized. The prepared medium was inoculated with 200 μl of the bacteria cultured in the same medium the day before. 1 mL of each was regularly taken while incubating at 150 rpm at 30 ° C. and analyzed using gas chromatography. Be using sucrose during keurep in the aerobic environment, Ella The production of 2,3-butanediol was 15.15 g / L when oxytoca M1 was cultured, and 9.31 g / L was produced when cultured in anaerobic environment, yielding 2,3-butanediol in higher yield than in aerobic environment. It can be seen that it can be obtained. The anaerobic culture did not produce acetoin (see FIGS. 3A and 3B).

Example  6: sugar concentration On the gradient  According to 2,3- Butanediol  Production aspect

The production of 2,3-butanediol was observed using glucose with a carbon source concentration gradient. Glucose concentrations were 5 g / L, 10 g / L, 20 g / L, 40 g / L, 60 g / L, 80 g / L and 100 g / L, respectively. The mL was placed in a 100 mL Erlenmeyer flask. The previous day cultured in the same medium was inoculated 2-5% (v / v) each. 1 ml each was incubated at 30 ° C. at 150 rpm and quantitated using gas chromatography (Agilent technology 6890N Network GC system) .The column was HP-INNOWAX (30 mx 250 μm x 0.25 μm, Agilent technology). It was.

Krebsiela 2,3-butanediol production yield of oxytoca M1 (after 48 hours) Early
Glucose Concentration (g / L) real
Glucose concentration
(g / L) Concentration of Glucose
(g / L) 2,3-butanediol production concentration
(g / L) yield
(g / g) 5 5.0 5.0 0.5 0.09 10 9.3 9.3 2.1 0.22 20 13.4 13.4 5.6 0.41 40 42.3 42.3 11.8 0.28 60 69.3 69.3 26.2 0.38 80 90.0 76.6 26.8 0.35 100 104.0 77.6 27.7 0.36

As a result of measuring the production concentration of 2,3-butanediol for 48 hours after inoculation of the strain, the concentration of 2,3-butanediol showed a concentration of 25 g / L or more when the glucose concentration was 60 g / L or more. The maximum 2,3-butane concentration was 0.5 g / L at a glucose concentration of 5 g / L, 2.1 g / L at 10 g / L, 5.6 g / L at 20 g / L, and 11.8 g at 40 g / L. / L, 26.2 g / L at 60 g / L, 26.8 g / L at 80 g / L, 27.7 g / L at 100 g / L. This suggests that up to 100 g / L of sugar is not a major obstacle in the production of 2,3-butanediol, and 2,3-BD is produced and disappeared at low concentrations of 5, 10 g / L of carbon source. It was found that this is due to the use of 2,3-butanediol as a carbon source when the carbon source is insufficient in microbial growth and growth.

Example  7: Krebscheiler Oxytoca M1  and Krebscheiler Oxytoca DSM1686  2,3- of strains Butanediol  Production comparison

The 2,3-butanediol production of the two strains was compared at the concentration of 100 g / L using glucose as the carbon source. Keurep Ciel La oxy Cytokines KCTC1686 was purchased from Korea BRC and used as a control strain for comparison with the isolates we isolated as type strain. 20 mL of the paper medium with 100 g / L of glucose was placed in a 100 mL sterile flask, and 2-5% (v / v) of the inoculated bacteria in the same medium the day before. After culturing for 132 hours. The experiment was repeated twice. The maximum yield of 2,3-butanediol of the M1 strain was 53.4 g / L for 72 hours and the maximum yield of the KCTC strain was 45.3 g / L at the same time.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Microorganism Conservation Center (overseas) KCCM11177P 20110221

Claims (7)

Krebschiela with excellent yield of 2,3-butanediol Oxy cytokine M1 (KCCM11177P) strain.
Process for preparing 2,3-butanediol, comprising the following steps:
(a) Krebsciela of claim 1 Culturing the oxytoca M1 strain in a medium comprising a carbon source; And
(b) recovering 2,3-butanediol from the medium.
The method of claim 2, wherein the culture temperature of the step of culturing in the medium is characterized in that 25 ℃ to 35 ℃.
The method of claim 2, wherein said culturing is carried out under aerobic conditions.
Klebsiella with excellent yield of acetoin Oxy cytokine M1 (KCCM11177P) strain.
Method for preparing acetoin comprising the following steps:
(A) culturing the Klebsiella oxytoca M1 strain of claim 5 in aerobic conditions in a medium containing a carbon source; And
(b) recovering acetoin from the medium.
The method of claim 6, wherein the culture temperature of the step of culturing in the medium is characterized in that 34 ℃ to 42 ℃.

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