CN114032187B - Kluyveromyces marxianus strain from Tibetan yoghurt and application thereof - Google Patents
Kluyveromyces marxianus strain from Tibetan yoghurt and application thereof Download PDFInfo
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- CN114032187B CN114032187B CN202111133340.8A CN202111133340A CN114032187B CN 114032187 B CN114032187 B CN 114032187B CN 202111133340 A CN202111133340 A CN 202111133340A CN 114032187 B CN114032187 B CN 114032187B
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- 235000018368 Saccharomyces fragilis Nutrition 0.000 title claims abstract description 52
- 229940031154 kluyveromyces marxianus Drugs 0.000 title claims abstract description 52
- 235000013618 yogurt Nutrition 0.000 title claims abstract description 24
- 241000235650 Kluyveromyces marxianus Species 0.000 title claims abstract 11
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims abstract description 74
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims abstract description 72
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 69
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 57
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 52
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 27
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 27
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- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 1
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- 241000511937 Curvibasidium rogersii Species 0.000 description 1
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- 229920001202 Inulin Polymers 0.000 description 1
- 244000062241 Kaempferia galanga Species 0.000 description 1
- 235000013421 Kaempferia galanga Nutrition 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
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- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
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- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
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- 239000001384 succinic acid Substances 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
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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
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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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- 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
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- 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
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Abstract
The invention provides a Kluyveromyces marxianus strain from Tibetan yoghurt and application thereof, wherein the preservation number of the strain Kluyveromyces marxianus JBA-MBY-JT140K is CGMCC No.21385, the strain is separated from a Tibetan yoghurt sample, and can be used for producing gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) by utilizing glucose and xylose, and in addition, the strain can secrete more GABA to a fermentation culture medium at high temperature and under the condition of adding ethanol or acetic acid with proper concentration. The accuracy is reliable and reliable through observation by a chromogenic method and measurement by an ultra-high performance liquid chromatography-quadrupole mass spectrometer, so that the capability of the strain for producing GABA is revealed, accumulation of a large amount of GABA is detected in cells of the strain, and the strain has good GABA production capability, 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) for producing GABA from Tibetan yoghurt and application thereof.
Background
GABA is a naturally occurring four-carbon non-protein amino acid that is widely found in plants, animals, and microorganisms. GABA is an inhibitory neurotransmitter in the human body, mainly regulating synaptic transmission, relaxing nerves, thus helping sleep and anti-depression, and also has antihypertensive, antidiabetic and anticancer effects (Ngo et al, molecular, 2019, 24:2678), so GABA is added to many sleep-aiding drugs and health products. GABA can also be used as the main ingredient of skin care products for skin cells, and has effect in smoothing wrinkles (Han et al, mycobiology,2017, 45:199-203). At present, the GABA production route mainly comprises microbial synthesis, plant extraction, chemical synthesis and the like. Chemical synthesis and plant extraction have limitations such as environmental pollution and low yield, so that the synthesis of GABA by a microbial fermentation method has attracted attention of researchers.
At present, microorganisms for producing GABA comprise lactobacillus, saccharomycete and the like, wherein the saccharomycete has the advantages of strong stress resistance, no risk of phage pollution, easiness in industrial amplification culture, capability of simultaneously synthesizing various active substances such as vitamins, polysaccharides and the like, good biological safety and the like, so that the production of GABA by utilizing the saccharomycete is paid attention to by researchers. Many wild yeast and industrial yeast cells respond to a variety of stress pressures in adapting to ecological environment evolution and application to industrial production, to which the cells will react, accumulating stress protective substances to protect the cells from injury, and GABA is a stress protective substance, and it is reported that GABA content in Candida and Saccharomyces cerevisiae cells is increased under conditions of acetic acid (Wang et al, graphics, 2013, 17:150-159), high osmotic pressure (Ji et al, J Gen Appl Microbiol,2017, 64:84-89), and oxidative stress (Coleman et al, J Biol Chem,2001, 276:244-250), etc.
Kluyveromyces marxianus (Kluyveromyces marxianus) is a GRAS (Generally Recognized as Safe) grade microorganism approved by the United states FDA, and the national health and family planning Committee of the national republic of China approved the yeast as an edible fungus in 2013. Kluyveromyces marxianus has strong high temperature resistance, can grow at 52 ℃ and has a fast growth rate at 30-40 ℃. The yeast can utilize various carbon sources, such as glucose, xylose, inulin, lactose, galactose, arabinose, etc., as a single substrate for fermentation to produce products such as enzymes, amino acids, organic compounds, and ethanol (J.Biol. Wang Dongmei, 2020, 37:1-10). Several strains of native Kluyveromyces marxianus have been shown to produce GABA using glucose (Perpetulini et al, J Appl Microbiol 2020, 129:1609-1619), with a highest yield of 7.78mg/L detected.
Xylose is the most abundant monosaccharide except glucose in nature, for example, lignocellulose biomass can be hydrolyzed to generate a lot of xylose, and microorganisms which can utilize xylose can ferment by taking lignocellulose hydrolysate as a substrate, so that industrial production is performed to save cost, but the production of active substances by utilizing yeast which can naturally utilize xylose is more beneficial to fully utilizing lignocellulose resources for biorefinery.
Disclosure of Invention
Aiming at the defects in the prior art, the primary purpose of the invention is to provide a Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K which is a Tibetan yoghurt source. In particular to a yeast strain Kluyveromyces marxianus JBA-MBY-JT140K which can utilize glucose and xylose and produce GABA under the stress induction of high temperature, ethanol or acetic acid from a Tibetan yoghurt source, and the strain has the capacity of producing GABA, can respond to the stress induction of high temperature, ethanol or acetic acid and has great application potential.
It is a further object of the present invention to provide the use of the above strain for the production of GABA.
It is a third object of the present invention to provide the use of the above strain for the production of GABA from lignocellulosic biomass.
To achieve the above primary object, the solution of the present invention is:
kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K from Tibetan yoghurt is preserved in China general microbiological culture Collection center (CGMCC) on 12 months and 17 days in 2020, with a preservation address: the collection number of the microbiological institute of China academy of sciences is CGMCC No.21385. Classification name Kluyveromyces marxianus, survival was examined.
The isolation, purification and screening process of the JBA-MBY-JT140K strain is as follows: diluting a yoghurt sample by 10 times with normal saline, coating on a YPD solid culture medium flat plate, placing the flat plate in a constant temperature incubator at 30 ℃ for inversion culture for 1-2 days, taking out a bacterial colony on the flat plate, carrying out streak culture on the bacterial colony on a new YPD culture medium flat plate, taking out a single bacterial colony after single bacterial colony is obtained, streaking again, repeating for three times, finally taking out the single bacterial colony on the YPD liquid culture medium, placing the flat plate in a shaking table at 30 ℃ for culture for 1-2 days, and preserving the flat plate with glycerol with the final concentration of 30% (v/v) to obtain the yeast strain JBA-MBY-JT140K.
The single colony of Kluyveromyces marxianus JBA-MBY-JT140K is creamy to light yellow, has smooth edges, slightly reflective, raised colony and easy to pick up, cells under a microscope are oval and elliptical, and are identified as Kluyveromyces marxianus related strains by sequencing and analyzing through NCBI database blast based on a molecular marker of 26s rDNAD1/D2 sequence in a fungal genome and named Kluyveromyces marxianus JBA-MBY-JT140K through morphological feature observation. It is isolated from Tibetan yoghurt samples, can utilize glucose and xylose at 30 ℃ and can produce GABA under stress induction of high temperature, ethanol or acetic acid.
As one embodiment of the present invention, the strain has a sequence as shown in SEQ ID NO. 1.
To achieve the above secondary object, the solution of the present invention is:
the application of Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K derived from Tibetan yoghurt in GABA production is provided.
As one embodiment of the present invention, kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K, which is a source of Tibetan yoghurt, produces GABA using glucose and xylose as carbon sources.
As one embodiment of the present invention, kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K, which is a source of Tibetan yoghurt, produces GABA under the induction of high temperature, ethanol or acetic acid stress.
As one embodiment of the present invention, the elevated temperature is 30-37℃, more preferably 37℃, at which a substantial amount of GABA can be accumulated.
As one 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 can be accumulated.
As one embodiment of the present invention, the concentration of acetic acid is 3-5g/L, more preferably 3g/L, at which a large amount of GABA can be accumulated.
To achieve the third object, the solution of the present invention is:
the application of the Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K derived from Tibetan yoghurt in GABA production by utilizing lignocellulose biomass.
By adopting the scheme, the invention has the beneficial effects that:
firstly, the invention separates and screens a strain Kluyveromyces marxianus producing GABA from Tibetan yoghurt, and the accuracy is reliable and reliable through observation by a chromogenic method and measurement by an ultra-high performance liquid chromatography-quadrupole mass spectrometer (Waters, USA), so that the capability of producing GABA is revealed, and accumulation of a large amount of GABA is detected in cells of the strain, so that the potential and development value of the GABA produced by the strain in food production are shown.
The Kluyveromyces marxianus strain of the invention is activated by liquid, and a large amount of GABA can be accumulated in 2 days of fermentation by using glucose and xylose as a fermentation medium of a carbon source at 37 ℃.
Third, the Kluyveromyces marxianus strain of the invention produces GABA under stress induction of 3% (v/v) ethanol or 3g/L acetic acid at 37 ℃.
Drawings
FIG. 1 is a schematic diagram showing cell morphology of the strain of example 1 of the present invention under a 400-fold microscope.
FIG. 2 shows the growth of the strain of example 3 of the present invention fermented for 48 hours with different concentrations of glucose and xylose as carbon sources.
FIG. 3 shows GABA yields obtained by fermenting the strain of example 3 of the present invention with different concentrations of glucose and xylose as carbon sources for 48 hours.
FIG. 4 shows the growth of the strain broth of example 4 of the present invention after high temperature, ethanol or acetic acid induced fermentation for 48 hours.
FIG. 5 shows GABA production from the strain fermentation broth of example 4 of the present invention by high temperature, ethanol or acetic acid-induced fermentation for 48 hours.
FIG. 6 shows GABA content of 48h intracellular accumulation of the strain of example 5 of the present invention by using xylose as a carbon source or ethanol.
The Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 12 months 17 of 2020, and has a preservation address: the collection number of the microbiological institute of China academy of sciences is CGMCC No.21385.
Detailed Description
The invention provides a Kluyveromyces marxianus strain from Tibetan yoghurt and application thereof.
According to the invention, a Kluyveromyces marxianus strain Kluyveromyces marxianus JBA-MBY-JT140K is separated, purified and identified from a yoghurt sample collected in a Tibetan region, and can be used for producing GABA by utilizing xylose and glucose, and the extracellular GABA secretion is improved under various environmental conditions such as high temperature, ethanol or acetic acid (namely Kluyveromyces marxianus is used for utilizing xylose and glucose, and the extracellular GABA is induced to be released by exogenous addition of high temperature, ethanol or acetic acid), so that the strain has good development and application potential.
The invention is further illustrated by the following examples.
Example 1 screening and biological identification of Yeast strains
1) Screening of Yeast strains
Diluting a yoghurt sample by 10 times with normal saline, coating on a YPD solid culture medium flat plate, placing the flat plate in a constant temperature incubator at 30 ℃ for inversion culture for 1-2 days, taking out a bacterial colony on the flat plate, carrying out streak culture on the bacterial colony on a new YPD culture medium flat plate, taking out a single bacterial colony after single bacterial colony is obtained, streaking again, repeating for three times, finally taking out the single bacterial colony on the YPD liquid culture medium, placing the YPD liquid culture medium in a shaking table at 30 ℃ for 1-2 days, and preserving the YPD liquid culture medium by using glycerol with the final concentration of 30% (v/v) to obtain the yeast strain.
2) Identification of Kluyveromyces marxianus Strain
The formulation of the medium of this example is as follows (sterilization conditions are 115℃for 20 min),
GABA producing strain screening solid Medium (1L): 10g of yeast powder, 20g of peptone, 20g of glucose, 20g of agar, 0.001g of bromocresol green, 0.02g of glyoxylate and 0.02g of succinic acid.
YPD liquid medium (1L): yeast powder 10g, peptone 20g and glucose 20g. And (5) autoclaving.
YPD solid medium (1L): yeast powder 10g, peptone 20g, glucose 20g and agar 20g. And (5) autoclaving.
YPD fermentation medium (1L): yeast powder 10g, peptone 20g, glucose 20/40g. And (5) autoclaving.
YPX fermentation medium (1L): yeast powder 10g, peptone 20g, xylose 20/40g. And (5) autoclaving.
After culturing the yeast strain in YPD plates for about 2 days, the cell state was observed under a 400x microscope; the cells are oval or elliptical, and the cell size is (3.1-4.2X1.3-2.3) μm. The asexual propagation mode is unilateral bud reproduction. Microscopic pictures of the strain under 400-fold microscope of YPD are shown in fig. 1.
The strain is subjected to genome extraction and classification identification of 26s rDNA D1/D2 sequence sequencing. PCR amplification was performed with genome as template by fungal 26s rDNA D1/D2 primer (forward 26s-F:5'-GCATATCGGTAAGCGGAGGAAAAG-3' SEQ ID NO.2; reverse 26s-R:5'-GGTCCGTGTTTCAAGACGG-3' SEQ ID NO. 3). Blast (http:// blast-t.ncbi.lm.nih.gov/blast.cgi) homology alignment was performed with the final 26s rDNA D1/D2 sequence, which was most similar to strain Kluyveromyces marxianus CBS, 100% for maximum similarity, thus identifying this strain as Kluyveromyces marxianus.
Wherein, the sequence of JBA-MBY-JT140K 26s rDNA D1/D2 (SEQ ID NO. 1) is as follows:
GCTCAAATTTGAAATCTGGCGTCTTCGACGTCCGAGTTGTAATTTGAAGAAGGCGA CTTTGTAGCTGGTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTGAGAATCCCGTGTGGCGAGGATCCCAGTTATTTGTAAAGTGCTTTCGACGAGTCGAGTTGTTTGGGA ATGCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATA GCGAACAAGTACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGGAAGGGCATTTGATCAGACATGGCGTTTGCTTCGGCTTTC GCTGGGCCAGCATCAGTTTTAGCGGTTGGATAAATCCTCGGGAATGTGGCTCTGCTTCGGTAGAGTGTTATAGCCCGTGGGAATACAGCCAGCTGGGACTGAGGATTGCGACTTTTGTC AAGGATGCTGGCGT。
morphological characteristics and culture of the strain JBA-MBY-JT140K of this example: after 2d incubation on YPD plates at 30deg.C, single colonies were creamy to pale yellow, smooth in edge, slightly reflective, raised and easily picked up. Microscopic features: after 2d incubation in YPD plates, the cells were oval or elliptical and the cell size was (3.1-4.2X1.3-2.3) μm. The asexual propagation mode is unilateral bud reproduction. Kluyveromyces marxianus JBA-MBY-JT140K is capable of xylose-utilizing growth and is 35-fold higher in GABA production (7.78 mg/L) than the Kluyveromyces marxianus strain reported in the literature using glucose (Perpetulini et al, J Appl Microbiol 2020, 129:1609-1619).
Example 2 determination of GABA yield in JBA-MBY-JT140K fermentation broth
The GABA content of the fermentation broth of JBA-MBY-JT140K was measured in this example.
The preparation method comprises inoculating 20 μL of JBA-MBY-JT140K strain preserved at-80deg.C in 1mL YPD liquid medium, culturing in shaking table at 30deg.C and 200rpm for 24 hr, transferring to 100mL YPD seed medium (i.e. YPD liquid medium), culturing in shaking table at 30deg.C for 18 hr, and adjusting OD 600 Inoculating 0.1 into YPD fermentation medium, sealing with air-permeable sealing film, fermenting at 30deg.C and 200rpm for 48 hr, sampling, centrifuging at 8000rpm for 3min, diluting the supernatant 50 times, mixing, filtering with 0.22 μm filter membrane, and measuring GABA content with ultra-high performance liquid chromatography-quadrupole mass spectrometer with GABA peak time of 1.010min.
Example 3, JBA-MBY-JT140K GABA production Using glucose and xylose
This example measures the ability of JBA-MBY-JT140K to produce GABA using glucose and xylose.
The preparation method comprises inoculating 20 μL of JBA-MBY-JT140K strain preserved at-80deg.C in 1mL YPD liquid medium, culturing in shaking table at 30deg.C and 200rpm for 24 hr, transferring to 100mL YPD seed medium (i.e. YPD liquid medium), culturing in shaking table at 30deg.C for 18 hr, and adjusting OD 600 Inoculating to YPD (glucose 20g/L and glucose 40 g/L) and YPD X (xylose 20g/L and xylose 40 g/L) fermentation medium (shown in figure 2) 0.1, sealing with air-permeable sealing film, fermenting at 30deg.C and 200rpm for 48 hr, sampling, centrifuging at 8000rpm for 3min, collecting supernatantDiluting the solution by 50 times, mixing, filtering with 0.22 μm filter membrane, measuring GABA content by ultra-high performance liquid chromatography-quadrupole mass spectrometer, measuring light absorption value at 600nm by full wavelength enzyme-labeling instrument, and measuring OD in the strain fermentation broth 600 As shown in FIG. 2, the GABA content is shown in FIG. 3.
Example 4, JBA-MBY-JT140K Induction of GABA production in response to stress at high temperature, ethanol or acetic acid
The present example measures GABA content of JBA-MBY-JT140K under fermentation conditions of ethanol or acetic acid addition at high temperature.
The preparation method comprises inoculating 20 μL of JBA-MBY-JT140K strain preserved at-80deg.C in 1mL YPD liquid medium, culturing in shaking table at 30deg.C and 200rpm for 24 hr, transferring to 100mL YPD seed medium (i.e. YPD liquid medium), culturing in shaking table at 30deg.C for 18 hr, and adjusting OD 600 Inoculating to YPD (glucose 20 g/L) fermentation medium (shown in figure 4) 0.1, adding ethanol 3% (v/v) and ethanol 5% (v/v) respectively, adding acetic acid 3g/L and acetic acid 5g/L into fermentation broth, sealing with air-permeable sealing membrane without adding any condition, fermenting at 30deg.C and 200rpm for 48 hr, sampling, fermenting at 37deg.C and 200rpm for 48 hr, centrifuging at 8000rpm for 3min, diluting supernatant 50 times, filtering 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 enzyme-labeling instrument, and measuring OD in the strain fermentation broth 600 As 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 measures the intracellular GABA content of JBA-MBY-JT140K under xylose fermentation or ethanol stress conditions.
The preparation method comprises inoculating 20 μL of JBA-MBY-JT140K strain preserved at-80deg.C in 1mL YPD liquid medium, culturing in shaking table at 30deg.C and 200rpm for 24 hr, transferring to 100mL YPD seed medium (i.e. YPD liquid medium), culturing in shaking table at 30deg.C and 200rpm for 18 hr, and adjusting OD 600 0.1 is inoculated to Y respectivelyPD (glucose 20 g/L), YPD (glucose 20 g/L) were added to 3g/L ethanol and YPD (xylose 40 g/L) fermentation medium, sealed with a gas-permeable sealing film, fermented at 30℃for 48 hours at 200rpm, sampled for 50mL, centrifuged at 4℃for 5min at 30000 rpm, the cells were collected, washed twice with sterile deionized water, 4mL of 0.1mol/L hydrochloric acid (GR) solution was added to suspend the cells, and then split-packed into five 1.5mL centrifuge tubes at 800. Mu.L/tube. After centrifugation of one tube of the cell liquid at 10000rpm for 2min, the supernatant was removed, the cells were placed in a 50℃oven until the dry weight of the cells became constant, the dry weight of the cells was measured by a precision analytical balance, the remaining four tubes were each filled with 500. Mu.L of glass beads having a diameter of 0.5mm, placed in a pre-chilled metal module at-20℃and the cells were broken by a cell breaker, followed by centrifugation at 12000rpm for 5min at 4℃to collect all the supernatant in a 5mL centrifuge tube, the two tube supernatants were combined into one tube, then a suitable volume of pre-chilled sulfosalicylic acid (16.7%, w/v) was added to a final concentration of 4.5% (w/v), shaking and mixing was performed, standing at 4℃for 1h, centrifugation was performed for 30min at 15000rpm to collect the supernatant, pH was adjusted to 1.9 by 1mol/L NaOH, and after membrane filtration by a filter membrane filtration, the GABA content was detected by an amino acid analyzer, as shown in FIG. 6.
As can be seen from FIG. 6, the cell of Kluyveromyces marxianus JBA-MBY-JT140K strain identified in this example also contains a large amount of GABA resources, and the highest yield is 4816.14mg/g dry weight, which indicates the potential and application value in GABA production as a microbial cell factory.
In summary, the invention provides a GABA-producing yeast strain separated from a yogurt sample collected from a Tibetan region of China, which is classified as Kluyveromyces marxianus by molecular identification. The strain is preserved in China general microbiological culture Collection center (Beijing) in 12 months and 17 days 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 capability of producing GABA of the strain is determined by utilizing 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, responds to induction of various stress conditions of high temperature, ethanol or acetic acid, most preferably uses glucose as a carbon source and adds 3% (v/v) ethanol at 37 ℃, or uses xylose as a carbon source at 37 ℃ to obviously induce the generation of GABA, and the highest yield in shake flasks reaches 275.09mg/L. In addition, a large amount of GABA is accumulated in cells of the strain, and the highest yield is 4816.14mg/g dry weight, so that the potential and application value of the strain in GABA production as a microbial cell factory are shown.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein may be applied to other embodiments without the use of the additional inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art will appreciate that, in light of the principles of the present invention, improvements and modifications can be made without departing from the scope of the invention.
Sequence listing
<110> Galangal (group) Co., ltd
<120> Kluyveromyces marxianus strain derived from Tibetan yoghurt 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 (7)
1. A kluyveromyces marxianus (Kluyveromyces marxianus) strain JBA-MBY-JT140K from Tibetan yoghurt, which is characterized in that: the China general microbiological culture Collection center CGMCC (China general microbiological culture Collection center) is preserved in the year 1 and 22 of 2021, and the preservation address is: the collection number of the microbiological institute of China academy of sciences is CGMCC No.21385.
2. Use of a kluyveromyces marxianus (Kluyveromyces marxianus) strain JBA-MBY-JT140K from Tibetan yoghurt as claimed in claim 1 for the production of GABA.
3. The use according to claim 2, characterized in that: kluyveromyces marxianus (Kluyveromyces marxianus) strain JBA-MBY-JT140K from Tibetan yoghurt utilizes glucose and xylose as carbon sources to produce GABA.
4. The use according to claim 2, characterized in that: kluyveromyces marxianus (Kluyveromyces marxianus) strain JBA-MBY-JT140K from Tibetan yoghurt can produce GABA under the induction of high temperature, ethanol or acetic acid stress.
5. The use according to claim 4, characterized in that: the high temperature is 30-37 ℃.
6. The use according to claim 4, characterized in that: the concentration of the ethanol is 3-5% (v/v).
7. The use according to claim 4, characterized in that: the concentration of the acetic acid is 3-5g/L.
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| 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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