CN114032187A - Kluyveromyces marxianus strain derived from Tibetan yogurt and application thereof - Google Patents
Kluyveromyces marxianus strain derived from Tibetan yogurt and application thereof Download PDFInfo
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- CN114032187A CN114032187A CN202111133340.8A CN202111133340A CN114032187A CN 114032187 A CN114032187 A CN 114032187A CN 202111133340 A CN202111133340 A CN 202111133340A CN 114032187 A CN114032187 A CN 114032187A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention provides a Kluyveromyces marxianus strain derived from Tibetan yogurt and application thereof, wherein the preservation number of the Kluyveromyces marxianus JBA-MBY-JT140K is CGMCC No.21385, the strain is separated from a Tibetan yogurt sample, and can produce gamma-aminobutyric acid (GABA) by using glucose and xylose. The accuracy is reliable and credible through color development observation and ultra-high performance liquid chromatography-quadrupole mass spectrometer determination, so that the GABA production capacity of the strain is revealed, and the accumulation of a large amount of GABA is detected in the cell of the strain, thereby indicating that the strain has good GABA production capacity and has potential and development value for food production.
Description
Technical Field
The invention belongs to the technical field of microbial resources and application, and particularly relates to a Kluyveromyces marxianus strain (Kluyveromyces marxianus) capable of producing GABA and derived from Tibetan yogurt and application thereof.
Background
GABA is a naturally occurring four-carbon nonprotein amino acid widely found in plants, animals and microorganisms. GABA is an inhibitory neurotransmitter in human body, mainly regulates synaptic transmission, relaxes nerves to play a role in improving sleep and resisting depression, and also has antihypertensive, antidiabetic and anticancer effects (Ngo, etc., Molecules,2019, 24:2678), so GABA is added into many sleep-improving medicines and health-care products. GABA can also be used as main ingredient of skin care product and act on skin cells, and has wrinkle smoothing effect (Han et al, Mycobiology,2017,45: 199-203). At present, the GABA generation approaches mainly comprise microbial synthesis, plant extraction, chemical synthesis and the like. Chemical synthesis and plant extraction have the limitations of environmental pollution, low yield and the like, so that the synthesis of GABA by a microbial fermentation method attracts the attention of researchers.
At present, microorganisms for producing GABA comprise lactic acid bacteria, saccharomycetes and the like, wherein the saccharomycetes have the advantages of strong stress resistance, no risk of bacteriophage pollution, easiness in industrial amplification culture, capability of simultaneously synthesizing various active substances such as vitamins and polysaccharides, good biological safety and the like, so that the GABA production by utilizing the saccharomycetes is concerned by researchers. In response to a plurality of stress stresses in adaptation to ecological environment evolution and application to industrial production of a plurality of wild yeasts and industrial yeast cells, the cells respond to the stress and accumulate stress protective substances to protect the cells from damage, and GABA is a stress protective substance and is reported to be increased in the intracellular GABA content of candida and saccharomyces cerevisiae under the conditions of acetic acid (Wang et al, Omics,2013,17: 150-.
Kluyveromyces marxianus (Kluyveromyces marxianus) is a GRAS (generally Recognized as safe) grade microorganism certified by the U.S. FDA, and is approved as an edible strain by the national health and family planning committee of the people's republic of China in 2013. Kluyveromyces marxianus has strong high temperature resistance, some can grow at 52 ℃, and the growth rate is high at 30-40 ℃. The yeast can utilize various carbon sources, such as glucose, xylose, inulin, lactose, galactose, arabinose, etc., as single substrates for fermentation to produce products such as enzymes, amino acids, organic compounds, and ethanol (biological journal of wang dong mei, 2020,37: 1-10). Several natural Kluyveromyces marxianus strains have been shown to produce GABA from glucose (Perpetuini et al, J Appl Microbiol,2020,129:1609-1619) with the highest detectable yield of 7.78 mg/L.
Xylose is a monosaccharide which is the most abundant in nature except glucose, for example, lignocellulose biomass can be hydrolyzed to produce a lot of xylose, and microorganisms capable of utilizing xylose can ferment lignocellulose hydrolysate serving as a substrate by using xylose hydrolysate, so that industrial production is carried out to save cost.
Disclosure of Invention
Aiming at the defects in the prior art, the primary object of the invention is to provide a Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K from Tibetan sour milk. Specifically provides a Tibetan yogurt source yeast strain Kluyveromyces marxianus JBA-MBY-JT140K capable of producing GABA under the stress induction of high temperature, ethanol or acetic acid by utilizing glucose and xylose, and the strain has the GABA producing capability and can respond to the stress induction of high temperature, ethanol or acetic acid, thereby having great application potential.
It is a secondary object of the present invention to provide the use of the above strain in the production of GABA.
The third purpose of the invention is to provide the application of the strain in producing GABA by utilizing lignocellulose biomass.
In order to achieve the above primary object, the solution of the present invention is:
kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K from Tibetan yogurt is preserved in China general microbiological culture Collection center (CGMCC) at 12-17 th of 2020, with the preservation address: the collection number of the microorganism research institute of Chinese academy of sciences, No.3 Xilu No.1 of Beijing, Chaoyang, and the institute of microorganisms is CGMCC No. 21385. The taxonomic name is Kluyveromyces marxianus, and survival was detected.
The separation, purification and screening processes of the JBA-MBY-JT140K strain are as follows: diluting a yoghourt sample by 10 times by using normal saline, coating the diluted yoghourt sample on a YPD solid culture medium plate, placing the plate in a constant temperature incubator at 30 ℃ for inverted culture for 1-2 days, picking out a bacterial colony on the plate until the bacterial colony grows out, carrying out streak culture on the bacterial colony on a new YPD culture medium plate, picking out a single bacterial colony after obtaining the single bacterial colony, carrying out streak culture again, repeating the three times, finally picking out the single bacterial colony to a YPD liquid culture medium, placing the YPD liquid culture medium in a shaking table at 30 ℃ for 1-2 days, and preserving the single bacterial colony with 30% (v/v) of glycerol to obtain a yeast strain JBA-MBY-JT 140K.
The single colony of the Kluyveromyces marxianus JBA-MBY-JT140K related by the invention is cream-colored to yellowish, has smooth edges, is slightly reflective, has raised colonies, is easy to pick up, and cells under a microscope are oval and elliptical, are observed by morphological characteristics of the cells, are sequenced based on a molecular marker of a 26s rDNNAD 1/D2 sequence in a fungal genome and are identified as a Kluyveromyces marxianus related strain through comparison analysis of NCBI database blast, and are named as Kluyveromyces marxianus JBA-MBY-JT 140K. It is isolated from Tibetan sour milk samples, can utilize glucose and xylose at 30 ℃, and can produce GABA under the stress induction of high temperature, ethanol or acetic acid.
As an embodiment of the invention, the strain has a sequence shown as SEQ ID NO. 1.
To achieve the above secondary object, the solution of the present invention is:
the Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K derived from Tibetan yogurt is applied to GABA production.
As an embodiment of the present invention, Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K derived from Tibetan yogurt produces GABA using glucose and xylose as carbon sources.
As an embodiment of the present invention, Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K derived from Tibetan yogurt produces GABA under the induction of high temperature, ethanol or acetic acid stress.
As an embodiment of the present invention, the elevated temperature is 30-37 deg.C, more preferably 37 deg.C, at which a large amount of GABA can accumulate.
As an embodiment of the present invention, the concentration of ethanol is 3-5% (v/v), more preferably 3% (v/v), at which a large amount of GABA is accumulated.
As an embodiment of the present invention, the concentration of acetic acid is 3 to 5g/L, more preferably 3g/L, at which a large amount of GABA is accumulated.
In order to achieve the third object, the solution of the invention is:
the Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K derived from Tibetan yogurt is applied to GABA production by using lignocellulose biomass.
Due to the adoption of the scheme, the invention has the beneficial effects that:
firstly, the Kluyveromyces marxianus strain producing GABA is separated and screened from Tibetan sour milk, the accuracy is reliable and credible through chromogenic observation and ultra performance liquid chromatography-quadrupole mass spectrometer (Waters, USA) measurement, so that the GABA producing capability of the Kluyveromyces marxianus strain is revealed, and the accumulation of a large amount of GABA is detected in cells of the Kluyveromyces marxianus strain, so that the potential and development value of GABA produced by the Kluyveromyces marxianus strain for food production are shown.
Secondly, the Kluyveromyces marxianus strain is activated by liquid, and a large amount of GABA can be accumulated when the Kluyveromyces marxianus strain is fermented for 2 days in a fermentation medium taking glucose and xylose as carbon sources at 37 ℃.
Third, the Kluyveromyces marxianus strain of the present invention produces GABA under the induction of high temperature of 37 ℃, 3% (v/v) ethanol or 3g/L acetic acid stress.
Drawings
FIG. 1 is a schematic diagram showing the cell morphology of the strain under 400-fold microscope in example 1 of the present invention.
FIG. 2 shows the growth of the strain in example 3 of the present invention fermented for 48h with different concentrations of glucose and xylose as carbon sources.
FIG. 3 is a graph showing the GABA production yield of the strain in example 3 of the present invention by fermentation for 48 hours using glucose and xylose at different concentrations as carbon sources.
FIG. 4 shows the growth of the fermentation broth of the strain of example 4 of the present invention after fermentation for 48h induced by high temperature, ethanol or acetic acid.
FIG. 5 shows the GABA production yield of the strain broth obtained in example 4 of this invention after fermentation for 48h induced by high temperature, ethanol or acetic acid.
FIG. 6 shows the amount of GABA accumulated in cells of the strain in example 5 of the present invention after fermentation induced by xylose as a carbon source or ethanol for 48 hours.
The Kluyveromyces marxianus strain JBA-MBY-JT140K has been preserved in China general microbiological culture Collection center (CGMCC) at 12-17 th of 2020, with the preservation address: the collection number of the microorganism research institute of Chinese academy of sciences, No.3 Xilu No.1 of Beijing, Chaoyang, and the institute of microorganisms is CGMCC No. 21385.
Detailed Description
The invention provides a Kluyveromyces marxianus strain derived from Tibetan yogurt and application thereof.
The Kluyveromyces marxianus JBA-MBY-JT140K is separated, purified and identified from a yogurt sample collected from the Tibet region, can produce GABA by utilizing xylose and glucose, and improves extracellular GABA secretion under various environmental conditions such as high temperature, ethanol or acetic acid (namely the Kluyveromyces marxianus utilizes xylose and glucose, and high temperature, ethanol or acetic acid are exogenously added to induce GABA to be released to the extracellular space), thereby showing good development and application potentials of the Kluyveromyces marxianus strain.
The present invention will be further described with reference to the following examples.
Example 1 screening and biological identification of Yeast strains
1) Screening of Yeast strains
Diluting a yoghourt sample by 10 times with normal saline, coating the diluted yoghourt sample on a YPD solid culture medium plate, placing the YPD solid culture medium plate in a constant temperature incubator at 30 ℃ for inverted culture for 1-2 days, picking out a bacterial colony on the plate until the bacterial colony grows out, carrying out streak culture on the bacterial colony on a new YPD culture medium plate, picking out a single bacterial colony after obtaining the single bacterial colony, carrying out streak culture again, repeating for three times, finally picking out the single bacterial colony to a YPD liquid culture medium, placing the YPD liquid culture medium in a shaking table at 30 ℃ for 1-2 days, and preserving the single bacterial colony with 30% (v/v) of final concentration to obtain a yeast strain.
2) Identification of Kluyveromyces marxianus strains
The formulation of the medium of this example is as follows (sterilization conditions are both 115 ℃ for 20min),
GABA producing strains solid medium (1L): 10g of yeast powder, 20g of peptone, 20g of glucose, 20g of agar, 0.001g of bromocresol green, 0.02g of glyoxylic acid and 0.02g of succinic acid.
YPD liquid medium (1L): 10g of yeast powder, 20g of peptone and 20g of glucose. And (5) autoclaving.
YPD solid Medium (1L): 10g of yeast powder, 20g of peptone, 20g of glucose and 20g of agar. And (5) autoclaving.
YPD fermentation medium (1L): 10g of yeast powder, 20g of peptone and 20/40g of glucose. And (5) autoclaving.
YPX fermentation medium (1L): 10g of yeast powder, 20g of peptone and 20/40g of xylose. And (5) autoclaving.
The cell status of the yeast strain was observed under a 400X microscope after culturing the strain in YPD plates for about 2 days; the cells are oval and elliptical, and the cell size is (3.1-4.2X 1.3-2.3) μm. The vegetative propagation mode is unilateral budding. A micrograph of the strain at 400 Xmicroscope under YPD is shown in FIG. 1.
The strain is subjected to genome extraction, and the taxonomic identification of 26s rDNA D1/D2 sequence sequencing is carried out. Genome-templated PCR amplification was performed by fungal 26s rDNA D1/D2 primers (forward 26 s-F: 5'-GCATATCGGTAAGCGGAGGAAAAG-3' SEQ ID NO. 2; reverse 26 s-R: 5'-GGTCCGTGTTTCAAGACGG-3' SEQ ID NO. 3). Blast (http:// blast-t.ncbi.nlm.nih.gov/blast. cgi) homology alignment was performed with the finally determined 26s rDNA D1/D2 sequence, which was most similar to Kluyveromyces marxianus CBS712 strain with a maximum similarity of 100%, thus identifying this strain as Kluyveromyces marxianus.
Wherein the JBA-MBY-JT140K 26s rDNA D1/D2 sequence (SEQ ID NO.1) is as follows:
GCTCAAATTTGAAATCTGGCGTCTTCGACGTCCGAGTTGTAATTTGAAGAAGGCGA CTTTGTAGCTGGTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTGAGAATCC CGTGTGGCGAGGATCCCAGTTATTTGTAAAGTGCTTTCGACGAGTCGAGTTGTTTGGGA ATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATA GCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGT ACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGCGTTTGCTTCGGCTTTC GCTGGGCCAGCATCAGTTTTAGCGGTTGGATAAATCCTCGGGAATGTGGCTCTGCTTCGG TAGAGTGTTATAGCCCGTGGGAATACAGCCAGCTGGGACTGAGGATTGCGACTTTTGTC AAGGATGCTGGCGT。
the morphological characteristics and culture of the strain JBA-MBY-JT140K of the present example: after culturing for 2d on an YPD plate at 30 ℃, a single colony is cream-colored to faint yellow, has smooth edge, is slightly reflective, and is easy to pick up. Microscopic characteristics: after 2 days of YPD plate culture, the cells were oval and elliptical in shape and the cell size was (3.1-4.2X 1.3-2.3) μm. The vegetative propagation mode is unilateral budding. Kluyveromyces marxianus JBA-MBY-JT140K was able to grow on xylose and increased GABA production (7.78mg/L) 35-fold over the previously reported Kluyveromyces marxianus strain on glucose production (Perpetuini et al, J Appl Microbiol,2020,129: 1609-.
Example 2, JBA determination of GABA production in MBY-JT140K fermentation broth
This example measured the GABA content in the fermentation broth of JBA-MBY-JT 140K.
Specifically, the method comprises inoculating JBA-MBY-JT140K strain stored in refrigerator at-80 deg.C 20 μ L into 1mL YPD liquid culture medium, culturing at 30 deg.C in 200rpm shaking table for 24 hr, transferring into 100mL YPD seed culture medium (i.e. YPD liquid culture medium), culturing at 30 deg.C in shaking table for 18 hr, and culturing according to initial OD600Inoculating into YPD fermentation medium at 0.1, sealing with gas-permeable sealing film, fermenting at 30 deg.C and 200rpm for 48 hr, sampling, centrifuging at 8000rpm for 3min, collecting supernatant, diluting by 50 times,mixing well, filtering with 0.22 μm filter membrane, and determining GABA content with ultra high performance liquid chromatography-quadrupole mass spectrometer, wherein the peak emergence time of GABA is 1.010 min.
Example 3, JBA-MBY-JT140K production of GABA Using glucose and xylose
This example measured the ability of JBA-MBY-JT140K to utilize glucose and xylose to produce GABA.
Specifically, the method comprises inoculating JBA-MBY-JT140K strain stored in refrigerator at-80 deg.C 20 μ L into 1mL YPD liquid culture medium, culturing at 30 deg.C in 200rpm shaking table for 24 hr, transferring into 100mL YPD seed culture medium (i.e. YPD liquid culture medium), culturing at 30 deg.C in shaking table for 18 hr, and culturing according to initial OD600Inoculating to YPD (glucose 20g/L and glucose 40g/L) and YPX (xylose 20g/L and xylose 40g/L) fermentation medium (as shown in figure 2) for 0.1, sealing with gas-permeable sealing membrane, fermenting at 30 deg.C and 200rpm for 48 hr, sampling, centrifuging at 8000rpm for 3min, diluting the supernatant by 50 times, mixing with 0.22 μm filter membrane, measuring content with ultra performance liquid chromatography-quadrupole mass spectrometer, measuring light absorption value at 600nm with full wavelength GABA analyzer, and measuring OD in the strain fermentation liquid600As shown in FIG. 2, the GABA content is shown in FIG. 3.
Example 4, JBA-MBY-JT140K production of GABA in response to stress induction by high temperature, ethanol or acetic acid
This example measured the GABA content of JBA-MBY-JT140K at high temperature, with ethanol or acetic acid added for fermentation.
Specifically, the method comprises inoculating JBA-MBY-JT140K strain stored in refrigerator at-80 deg.C 20 μ L into 1mL YPD liquid culture medium, culturing at 30 deg.C in 200rpm shaking table for 24 hr, transferring into 100mL YPD seed culture medium (i.e. YPD liquid culture medium), culturing at 30 deg.C in shaking table for 18 hr, and culturing according to initial OD600Inoculating to YPD (glucose 20g/L) fermentation medium (shown in figure 4) for 0.1, adding into fermentation broth according to concentration of ethanol 3% (v/v) and ethanol 5% (v/v), respectively, acetic acid 3g/L and acetic acid 5g/L, and control group without any addition of any condition, sealing with gas-permeable sealing film, fermenting at 30 deg.C and 200rpm for 48 hr, sampling, standing at high temperature at 37 deg.C and 200rpm for 48 hr, sampling, and cutting at 8000rpmCentrifuging for 3min, collecting supernatant, diluting 50 times, mixing with 0.22 μm filter membrane, measuring GABA content with ultra high performance liquid chromatography-quadrupole mass spectrometer, measuring light absorption value at 600nm with full wavelength microplate reader, and measuring OD in the strain fermentation broth600As shown in FIG. 4, the GABA content is shown in FIG. 5.
Example 5, JBA-MBY-JT140K intracellular GABA content under xylose fermentation or ethanol stress conditions
This example measured the intracellular GABA content of JBA-MBY-JT140K under xylose fermentation or ethanol stress conditions.
Specifically, the method comprises inoculating JBA-MBY-JT140K strain stored in refrigerator at-80 deg.C 20 μ L into 1mL YPD liquid culture medium, culturing in shaker at 30 deg.C and 200rpm for 24 hr, transferring into 100mL YPD seed culture medium (YPD liquid culture medium), culturing in shaker at 30 deg.C and 200rpm for 18 hr, and culturing according to initial OD6000.1 of the cells were inoculated into YPD (glucose 20g/L) and YPX (glucose 20g/L) fermentation media to which 3g/L of ethanol and YPX (xylose 40g/L) were added, respectively, sealed with a gas-permeable sealing film, fermented at 30 ℃ and 200rpm for 48 hours, then sampled at 50mL, centrifuged at 4 ℃ and 30000 rpm for 5 minutes, the cells were collected, washed twice with sterile deionized water, added with 4mL of 0.1mol/L hydrochloric acid (GR) solution, the cells were suspended, and then dispensed into five 1.5mL centrifuge tubes at 800. mu.L/tube. Wherein one tube of cell sap is centrifuged at 10000rpm for 2min, the supernatant is removed, the cell sap is placed in a 50 ℃ oven until the dry weight of the cells is constant, the dry weight of the cells is measured by a precision analytical balance, 500 mu L of glass beads with the diameter of 0.5mm are respectively added into the remaining four tubes, the tubes are placed in a metal module precooled at the temperature of minus 20 ℃, disrupting the cells with a cell disruptor, followed by centrifugation at 12000rpm at 4 ℃ for 5min to collect all supernatants in a 5mL centrifuge tube, combining the supernatants into one tube, then adding a proper volume of precooled sulfosalicylic acid (16.7 percent, w/v) until the final concentration is 4.5 percent (w/v), shaking and uniformly mixing, standing for 1h at 4 ℃, the supernatant was collected by centrifugation at 15000rpm for 30min at 4 ℃ and adjusted to pH 1.9 with 1mol/L NaOH, and after passing through a 0.22 μm filter, the GABA content was measured by an amino acid analyzer, the results are shown in FIG. 6.
As can be seen from FIG. 6, the Kluyveromyces marxianus JBA-MBY-JT140K identified in this example also contains a large amount of GABA resources in the cells, and the highest yield is 4816.14mg/g dry weight, which indicates the potential and application value of the Kluyveromyces marxianus JBA-MBY-JT140K in producing GABA as a microbial cell factory.
In conclusion, the invention provides a GABA-producing yeast strain separated from a yoghourt sample collected from the Tibet region of China, and the GABA-producing yeast strain is classified as Kluyveromyces marxianus through molecular identification. The strain is preserved in China (Beijing) general microorganism culture collection center in 12-17 th of 2020, and the original name is JBA-MBY-JT140K, and the preservation number is CGMCC No. 21385. Through preliminary screening, the strain is found to be capable of producing GABA, and the capacity of producing GABA of the strain is determined by using an ultra-high performance liquid chromatography-quadrupole mass spectrometer. The strain has strong high temperature resistance and good growth state at 37 ℃. The strain can produce GABA by using xylose and respond to the induction of various stress conditions such as high temperature, ethanol or acetic acid, most preferably, glucose is used as a carbon source at 37 ℃ and 3% (v/v) ethanol is added, or xylose is used as the carbon source at 37 ℃, so that the GABA production can be induced more obviously, and the highest yield reaches 275.09mg/L in a shake flask. In addition, the strain accumulates a large amount of GABA in cells, the highest yield is 4816.14mg/g dry weight, and the potential and the application value of the strain in producing GABA as a microbial cell factory are shown.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of additional inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Sequence listing
<110> Kalan (group) corporation
Kluyveromyces marxianus strain derived from Tibetan yogurt and application thereof
<141> 2021-09-22
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 484
<212> DNA
<213> Yeast (Curvibasidium rogersii)
<400> 1
gctcaaattt gaaatctggc gtcttcgacg tccgagttgt aatttgaaga aggcgacttt 60
gtagctggtc cttgtctatg ttccttggaa caggacgtca tagagggtga gaatcccgtg 120
tggcgaggat cccagttatt tgtaaagtgc tttcgacgag tcgagttgtt tgggaatgca 180
gctctaagtg ggtggtaaat tccatctaaa gctaaatatt ggcgagagac cgatagcgaa 240
caagtacagt gatggaaaga tgaaaagaac tttgaaaaga gagtgaaaaa gtacgtgaaa 300
ttgttgaaag ggaagggcat ttgatcagac atggcgtttg cttcggcttt cgctgggcca 360
gcatcagttt tagcggttgg ataaatcctc gggaatgtgg ctctgcttcg gtagagtgtt 420
atagcccgtg ggaatacagc cagctgggac tgaggattgc gacttttgtc aaggatgctg 480
gcgt 484
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artficial Sequence)
<400> 2
gcatatcggt aagcggagga aaag 24
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artficial Sequence)
<400> 3
ggtccgtgtt tcaagacgg 19
Claims (9)
1. Kluyveromyces marxianus strain Kluyveromyces marxianus from Tibetan yogurt is characterized in that: the culture is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 17 days in 2020, the preservation address is as follows: the collection number of the microorganism research institute of Chinese academy of sciences, No.3 Xilu No.1 of Beijing, Chaoyang, and the institute of microorganisms is CGMCC No. 21385.
2. Kluyveromyces marxianus strain Kluyveromyces marxianus as claimed in claim 1, wherein: the strain has a sequence shown as SEQ ID NO. 1.
3. Use of a Kluyveromyces marxianus strain Kluyveromyces marxianus derived from Tibetan yogurt as claimed in claim 1 in the production of GABA.
4. Use according to claim 3, characterized in that: kluyveromyces marxianus strain Kluyveromyces marxianus from Tibetan yogurt utilizes glucose and xylose as carbon sources to produce GABA.
5. Use according to claim 3, characterized in that: the Kluyveromyces marxianus strain Kluyveromyces marxianus from Tibetan yogurt produces GABA under the induction of high temperature, ethanol or acetic acid stress.
6. Use according to claim 5, characterized in that: the high temperature is 30-37 ℃.
7. Use according to claim 5, characterized in that: the concentration of the ethanol is 3-5% (v/v).
8. Use according to claim 5, characterized in that: the concentration of the acetic acid is 3-5 g/L.
9. Use of Kluyveromyces marxianus strain Kluyveromyces marxianus derived from Tibetan yogurt as claimed in claim 1 in production of GABA from lignocellulosic biomass.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733078A (en) * | 2020-07-24 | 2020-10-02 | 塔里木大学 | Method for producing gamma-aminobutyric acid by fermenting indigenous strains in sailima raw material |
| CN112251311A (en) * | 2020-09-17 | 2021-01-22 | 复旦大学 | Method for brewing cider fruits by using Kluyveromyces marxianus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733078A (en) * | 2020-07-24 | 2020-10-02 | 塔里木大学 | Method for producing gamma-aminobutyric acid by fermenting indigenous strains in sailima raw material |
| CN112251311A (en) * | 2020-09-17 | 2021-01-22 | 复旦大学 | Method for brewing cider fruits by using Kluyveromyces marxianus |
Non-Patent Citations (1)
| Title |
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| G PERPETUINI等: "γ-aminobutyric acid production by Kluyveromyces marxianus strains", 《J APPL MICROBIOL》, vol. 129, no. 6, pages 1609 - 1619 * |
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| CN115287204A (en) * | 2022-09-22 | 2022-11-04 | 山东锦鲤生物工程有限公司 | Kluyveromyces marxianus strain and application thereof |
| CN115287204B (en) * | 2022-09-22 | 2023-08-18 | 山东锦鲤生物工程有限公司 | Kluyveromyces marxianus strain and application thereof |
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