CN111218409A

CN111218409A - High-salt-tolerance saccharomyces cerevisiae strain, and construction method and application thereof

Info

Publication number: CN111218409A
Application number: CN201911183990.6A
Authority: CN
Inventors: 贺斌; 刘华; 曾斌; 涂雅怡
Original assignee: Jiangxi Science and Technology Normal University
Current assignee: Jiangxi Science and Technology Normal University
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2020-06-02

Abstract

The invention provides a high-salt-tolerance saccharomyces cerevisiae strain, and a construction method and application thereof. In this technical scheme, an oleoyl synthetase encoding gene of aspergillus oryzae 3042 is extracted, transformed into a natural yeast strain by a vector, and the resulting recombinant bacterium individually expresses an oleoyl synthetase (Ole) derived from aspergillus oryzae 3042. According to the morphological characteristics and the search result of the 26S rRNA gene sequence in Genbank, the strain is identified as Saccharomyces cerevisiae (Saccharomyces cerevisiae). Experimental verification shows that the strain can grow and propagate in a high-salt environment of soy sauce brewing, and the unsaturated fatty acid content of the strain is higher than that of the conventional strain. The strain can ensure the smooth fermentation process under the condition of high salt and shorten the fermentation period to a certain extent, and the product has higher unsaturated fatty acid content, thereby being an excellent strain for brewing soy sauce.

Description

High-salt-tolerance saccharomyces cerevisiae strain, and construction method and application thereof

Technical Field

The invention relates to the technical field of industrial microorganisms, in particular to a high-salt-resistant saccharomyces cerevisiae strain, a construction method and application thereof.

Background

Soy sauce is one of the most main traditional seasonings in China, aspergillus and yeast are main fermentation microorganisms in soy sauce brewing, protease of the microorganisms can degrade proteins in raw materials into small molecules and polypeptides which are easy to be absorbed by human bodies, and metabolites of the microorganisms, such as small molecule alcohol, aldehyde, acid, ester, unsaturated fatty acid, phenols and the like, can also provide flavor for the soy sauce.

According to different fermentation processes of soy sauce brewing, the method can be divided into a solid salt-free state, a low-salt solid state and a high-salt dilute state. The solid state fermentation without salt and with low salt provides products which mainly comprise medium and low grade products, and the flavor of the products is not as good as that of the high salt dilute state fermentation process. Along with the continuous improvement of the living standard of people, the market demand for high-quality brewed seasonings is stronger and stronger, and salt-free and low-salt solid fermentation products can not meet the market demand. The soy sauce prepared by high-salt dilute fermentation (10% -15% NaCl) has good taste, rich unsaturated fatty acid content and is a high-grade export product. In the process of high-salt dilute state fermentation, salt-tolerant saccharomyces cerevisiae is generally required to be added in order to improve the flavor of the soy, so that the single flavor and the unsaturation caused by aspergillus oryzae fermentation in the soy brewing process are improved, and the flavor and the grade of the soy are comprehensively improved.

At present, the commonly used saccharomyces cerevisiae strain is severely inhibited from growing under the condition of 10% salt concentration, and the content of unsaturated fatty acid is low, especially the content of oleic acid is only about 7%. Oleic acid is an important monounsaturated fatty acid and plays an important role in stress tolerance of yeast, such as ethanol tolerance, salt stress tolerance and the like. The high-salt dilute environment can cause the water activity and cytoplasm composition in the cell of the saccharomyces cerevisiae to be obviously changed, and the enzyme in the cell membrane and the bacteria body is damaged, thereby inhibiting the growth and fermentation of the saccharomyces cerevisiae. Therefore, the breeding of the high-salt-resistant saccharomyces cerevisiae is very important for improving the flavor and the quality of the soy sauce.

Disclosure of Invention

The invention aims to provide a high-salt-resistant saccharomyces cerevisiae strain, a construction method and application thereof aiming at the technical defects of the prior art, so as to solve the technical problem that the salt tolerance of the conventional saccharomyces cerevisiae strain in the prior art needs to be improved.

Another technical problem to be solved by the present invention is that the conventional Saccharomyces cerevisiae strains have a low content of unsaturated fatty acids.

The invention aims to solve the technical problem of how to improve the salt tolerance and the unsaturated fatty acid content of the conventional saccharomyces cerevisiae strain by a genetic engineering means.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a high-salt-resistant Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain with the preservation number of CGMCCNO: 14916.

preferably, the 26S rRNA nucleotide sequence of the Saccharomyces cerevisiae strain is shown in SEQ ID NO. 1.

On the basis of the technical scheme, the invention further provides a construction method of the saccharomyces cerevisiae strain, which comprises the following steps:

1) extracting total RNA of Aspergillus oryzae 3042, performing reverse transcription to synthesize a first cDNA chain, and performing PCR amplification by using the first cDNA chain as a template and fragments with sequences of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.4 as primers;

2) connecting the amplification product obtained in the step 1) to a T vector by using T4 ligase, digesting and cloning a vector pMD19 by using restriction enzyme double digestion, connecting overnight at 16 ℃ by using T4DNA ligase, transforming a host bacterium by using the connection product, culturing and amplifying the transformed bacterium, selecting positive clones, extracting plasmids, and selecting transformants with correct sequencing; digesting the positive clone and an expression vector PYES2 by using restriction enzyme double digestion, connecting the positive clone and the expression vector PYES2 by using T4DNA ligase at 16 ℃ overnight, and selecting a recombinant plasmid containing a target gene;

3) and (3) transforming the recombinant plasmid obtained in the step 2) into a competent yeast cell, wherein the obtained transformant is the high-salt-tolerance saccharomyces cerevisiae strain.

Preferably, the transformation in step 3) is performed by mixing a premix containing the recombinant plasmid with competent yeast cells and suspending the yeast cells in the premix; then water bath is carried out for 30min at the temperature of 30 ℃, and the mixture is uniformly mixed once every 10 min; and then hot shocking with 42 deg.C water bath for 30min, and mixing once every 10 min.

Preferably, the premix comprises the following components: PEG240 mu L, LiAc36 mu L of 1M, Carrier DNA10 mu L, the recombinant plasmid 5 mu L; sterile deionized water is added to make up to 360 mu L.

On the basis of the technical scheme, the invention further provides the application of the saccharomyces cerevisiae strain for brewing soy sauce.

On the basis of the technical scheme, the invention further provides application of the saccharomyces cerevisiae strain for executing fermentation.

Preferably, in this application, the following medium is used as the seed medium or fermentation medium for the saccharomyces cerevisiae strain: adding water into 10g of yeast extract, 20g of peptone and 20g of glucose to 1000 mL; sterilizing at 110 deg.C for 20min at pH 7.0.

Preferably, in this application, the seed culture conditions are as follows: selecting a single colony to be inoculated into a container, wherein the liquid loading amount of the seed culture medium in the container is 1/5 of the total volume; shaking and culturing at 30 deg.C and 200r/min for 24 h.

Preferably, in this application, the fermentation culture conditions are as follows: inoculating a culture solution obtained by seed culture at a ratio of 10% (v/v) into another container, wherein the liquid content of the fermentation medium in the container is 1/10 of the total volume; shaking and culturing at 30 deg.C and 200r/min for 48 h.

The high-salt-resistant Saccharomyces cerevisiae strain adopted by the invention is preserved in the China general microbiological culture collection management center, the address of the strain is No.3 of Xilu No.1 of Beijing Korean district, the preservation number of the strain is CGMCC NO: 14916.

The technical effects of the invention are concentrated in the following aspects:

1. according to the salt tolerance test of the embodiment example, the salt tolerance test of FIG. 3 and FIG. 4 shows that the Saccharomyces cerevisiae of the present invention grows well in the presence of 5% and 10% sodium chloride, which indicates that the Saccharomyces cerevisiae of the present invention has strong salt tolerance and is suitable for the industrial application in the production of high-salt dilute brewed soy sauce.

2. According to the third embodiment, after the saccharomyces cerevisiae is cultured in a YPD medium with 10% of sodium chloride at 30 ℃ for 2 days, the content of unsaturated fatty acid is obviously increased, and a foundation is provided for improving the quality of soy sauce.

3. The saccharomyces cerevisiae strain disclosed by the invention has high salt tolerance, can shorten the fermentation period in the high-salt dilute state brewing process, can produce soy sauce rich in unsaturated fatty acid, and provides an excellent strain for industrial production of the soy sauce.

Drawings

FIG. 1 is a graph showing the result of amplification of a target gene in an embodiment of the present invention; in the figure, M is DL10000 Marker, 1 and 2 are Ole1, and 3 and 4 are Ole 2.

FIG. 2 is a diagram showing the results of enzyme digestion verification and positive strains of Saccharomyces cerevisiae transformed with key enzyme genes by PCR screening of bacterial liquid in the embodiment of the present invention; in part a: in the figure, M is DL10000 Marker, 1-6 is the recombinant strain screening of PYES2+ Ole1, and 7-12 is the recombinant strain screening of PYES2+ Ole 2; in part B: in the figure, M is DL10000 Marker, 1-3 is double enzyme digestion verification of PYES2+ Ole1 recombinant plasmid, and 4-6 is double enzyme digestion verification of PYES2+ Ole2 recombinant plasmid.

FIG. 3 is a diagram showing the results of detecting the salt tolerance of Saccharomyces cerevisiae, a transgene enzyme gene, in accordance with an embodiment of the present invention; in the figure, WT is wild-type Saccharomyces cerevisiae Y0000, D9D1 is Ole1 transgenic Saccharomyces cerevisiae, and D9D2 is Ole2 transgenic Saccharomyces cerevisiae.

FIG. 4 is a diagram showing the results of fermentation verification of Saccharomyces cerevisiae, a transfer key enzyme gene in accordance with an embodiment of the present invention; in the figure, D9D1 is an Ole1 transgenic Saccharomyces cerevisiae, and D9D2 is an Ole2 transgenic Saccharomyces cerevisiae.

Detailed Description

Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Approximating language, as used herein in the following examples, may be applied to identify quantitative representations that could permissibly vary in number without resulting in a change in the basic function. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

EXAMPLE one (construction and identification of recombinant plasmid)

1. Amplification of target Gene

Culturing Aspergillus oryzae 3042 at 30 deg.C for 3 days, sampling, quickly freezing in liquid nitrogen, and storing in-70 deg.C refrigerator; total RNA was extracted using TRIzol Reagent (Invitrogen, USA) according to the kit instructions provided by the company. After the extraction, residual genomic DNA was removed from the total RNA by using DNase I (RNase-free) from Fermentas. The total RNA was then reverse transcribed to synthesize the first strand cDNA, according to the instructions of the TIANCcript cDNA first strand synthesis kit (Beijing Tiangen Biotechnology Co., Ltd.). Designing primer to amplify according to the target gene sequence, and recovering the specific fragment.

The primers are as follows (underlined sections are restriction sites):

Ole1-F(GAGCTCATGTCTTCCACAGCTATCCCCAAGC)；

Ole1-R(CTCGAGCTATTTGGAAAGCTTGGCCGGAGGA)；

Ole2-F(GAATTCATGGCCGAGTTGCACAACAGAAAAG)；

Ole2-R(AAGCTTTTATTTGCTATCATCAGCCCTAACA)。

2. construction and characterization of expression vectors

Connecting the recovered target fragment to a T vector by using T4 ligase, digesting and cloning the vector pMD19 by using restriction enzyme double digestion, connecting overnight at 16 ℃ by using T4DNA ligase, transforming a host bacterium by using a connection product, coating a transformed bacterium liquid on a flat plate, culturing overnight, selecting a positive clone, extracting a plasmid, sending to a marine engineering sequencing for verification, and selecting a transformant with correct sequencing. The positive clone and an expression vector PYES2 are digested by restriction enzyme double digestion, T4DNA ligase is used for overnight connection at 16 ℃, recombinant plasmids containing target genes are selected according to the method, and bacterial liquid PCR and double digestion verification are carried out.

Example two (obtaining of recombinant bacteria)

1. Transformed Saccharomyces cerevisiae

Yeast competent cells, which were stored at-80 ℃ were thawed on ice. Preparing a premixed solution: PEG240 mu L, 1M LiAc36 mu L, Carrier DNA10 mu L, target plasmid 5 mu L, adding sterile deionized water to make up to 360 mu L. Adding the premixed solution containing the target plasmid into the competent cells, and repeatedly blowing and sucking the sediment to thoroughly suspend the yeast cells in the premixed solution. Incubating in 30 deg.C water bath for 30min, and mixing once every 10 min; heating in 42 deg.C water bath for 30min, and mixing once every 10 min. Centrifuging at 12000rpm for 1min, removing supernatant, adding 100 μ L sterile deionized water into the precipitate for suspension, coating the bacterial solution on SD-Ura culture medium plate, and culturing at 30 deg.C for 2 days.

2. Identification and preservation of Positive clones

And (3) carrying out PCR identification on the bacteria liquid by using a specific primer on the positive clone, mixing the bacteria liquid with a positive detection result with 30% of glycerol, placing the mixture in a glycerol tube, and storing the mixture in an ultra-low temperature refrigerator at the temperature of-70 ℃ for later use.

EXAMPLE III (26S rRNA identification experiment of Saccharomyces cerevisiae strain of the invention)

Extraction and purification of yeast genomic DNA was carried out by the method of Sambrook et al (1989). PCR amplification of 26S rRNA gene sequences is carried out according to the method of Solieri et al (2007) by taking extracted and qualified genome DNA as a template and selecting a primer pair NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3'), and gene sequence sequencing is completed by Shanghai worker-sequencing company.

Example four (the intracellular fatty acid content of Saccharomyces cerevisiae of the present invention)

1. Preparation of samples

Taking 120mg of a sample, precisely weighing, placing the sample in a10 mL test tube with a plug, adding 2mL of 0.5mol/L potassium hydroxide methanol solution, charging nitrogen, placing the sample in a water bath at 60 ℃ for 20min, cooling the sample after oil droplets are dissolved, adding 2mL of 15% boron trifluoride methanol solution, charging nitrogen, placing the sample in a water bath at 60 ℃ for 6min, cooling the sample, precisely adding 2mL of n-hexane, shaking the sample, adding 2mL of saturated sodium chloride solution, standing the mixture, and taking an upper solution;

2. chromatographic separation column

Supelco SP-2340(30mm×0.25mm×0.2μm)；

Temperature: the column temperature is 165 ℃, the vaporization chamber is 215 ℃, and the detector is 215 ℃;

carrier gas flow rate (column front pressure): 150kpa of high purity nitrogen; 70kpa of hydrogen; air 50 kpa.

Under the chromatographic condition, a sample is subjected to pretreatment and then is injected with 2 mu L of sample, and the fatty acid composition of the saccharomyces cerevisiae is calculated by using retention time for qualitative determination, peak area for quantitative determination and a normalization method. The main fatty acid components of the saccharomyces cerevisiae strain are shown in table 1, and mainly exist in the forms of oleic acid, palmitic acid, palmitoleic acid, arachidic acid and stearic acid (table 1). Compared with the prior saccharomyces cerevisiae cultured on a high-salt (10% NaCl) culture medium, the saccharomyces cerevisiae strain has the advantages that the unsaturated fatty acid content is improved by 20%, and the oleic acid content is improved by more than 10%.

TABLE 1 major fatty acid composition of Saccharomyces cerevisiae of the present invention

Example V (salt tolerance experiment of Saccharomyces cerevisiae of the invention)

YPD medium was prepared. NaCl is added, the concentration is set to be 0%, 5%, 10% and 15%, the wild type Saccharomyces cerevisiae strain Y0000 and the strain of the invention are diluted according to the proportion of 1:1, 1:10, 1:100, 1:1000 and 1:10000, and 5 mu L of the diluted strain is respectively inoculated on YPD solid culture media with different salt concentrations. The results are shown in FIG. 3, where the Saccharomyces cerevisiae strains according to the invention grew well in the presence of 5% and 10% sodium chloride, whereas the prior Saccharomyces cerevisiae strains did not grow substantially in the presence of 10% sodium chloride.

Preparing YPD liquid culture medium, adding NaCl in 0%, 5%, 10% and 15%, inoculating wild Saccharomyces cerevisiae strain Y0000 and the strain of the present invention in the same OD value, shaking culture at 30 deg.c and 200r/m for 2 days, measuring the light absorption value at 600nm wavelength, diluting the concentrated bacterial liquid properly, and multiplying the diluted value by the dilution factor to obtain the practical value of the culture liquid. The results are shown in FIG. 4, the Saccharomyces cerevisiae strain of the present invention has good fermentation conditions in the presence of 5% and 10% sodium chloride, while the existing Saccharomyces cerevisiae has basically stopped fermentation in the presence of 10% sodium chloride.

EXAMPLE six

1. A high-salt-resistant Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain is preserved in China general microbiological culture Collection center (CGMCC) in 11 months and 16 days in 2017, and the preservation number is CGMCC NO: 14916.

2. the nucleotide sequence of 26S rRNA of the high-salt-resistance saccharomyces cerevisiae is shown as SEQ ID NO.1 in a sequence table.

3. YPD seed/fermentation medium: adding water into 10g of yeast extract, 20g of peptone and 20g of glucose to 1000mL, sterilizing at the temperature of 110 ℃ for 20min, and adding water into the mixture to obtain a mixture with the pH value of 7.0; agar powder 20g, pH7.0 was added to the slant medium.

4. The culture conditions are as follows:

seed culture: single colonies were picked from YPD plates and inoculated into a flask for culture at a liquid loading of 50mL/250mL at 30 ℃ at 200r/min for 24 h.

And (3) shake flask fermentation culture: inoculating the cultured seed culture solution into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% (v/v) for culturing, measuring the light absorption value under the wavelength of 600nm after culturing for 48h at the fermentation temperature of 30 ℃ at 200r/min, properly diluting the bacterium solution with high concentration, and multiplying the value obtained after dilution by the dilution multiple to obtain the actual value of the culture solution.

5. Determination of fatty acid content (gas chromatography-Mass Spectrometry, GC-MS)

1) Preparation of samples: taking 120mg of a sample, precisely weighing, placing the sample in a10 mL test tube with a plug, adding 2mL of 0.5mol/L potassium hydroxide methanol solution, charging nitrogen, placing the sample in a water bath at 60 ℃ for 20min, cooling the sample after oil droplets are dissolved, adding 2mL of 15% boron trifluoride methanol solution, charging nitrogen, placing the sample in a water bath at 60 ℃ for 6min, cooling the sample, precisely adding 2mL of n-hexane, shaking the sample, adding 2mL of saturated sodium chloride solution, standing the mixture, and taking an upper solution;

2) a chromatographic separation column: supelco SP-2340(30 mm. times.0.25 mm. times.0.2 μm);

3) temperature: the column temperature is 165 ℃, the vaporization chamber is 215 ℃, and the detector is 215 ℃;

The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-salt-resistant saccharomyces cerevisiae strain has a preservation number of CGMCC NO: 14916.

2. the Saccharomyces cerevisiae strain according to claim 1, wherein the 26S rRNA of the Saccharomyces cerevisiae strain has a nucleotide sequence shown in SEQ ID No. 1.

3. The method of constructing a s.cerevisiae strain according to claim 1, comprising the steps of:

4. The method according to claim 3, wherein the transformation in step 3) comprises mixing a premix containing the recombinant plasmid with competent yeast cells to suspend the yeast cells in the premix; then water bath is carried out for 30min at the temperature of 30 ℃, and the mixture is uniformly mixed once every 10 min; and then hot shocking with 42 deg.C water bath for 30min, and mixing once every 10 min.

5. The method of construction according to claim 4, characterized in that the premix comprises the following ingredients: PEG240 mu L, LiAc36 mu L of 1M, Carrier DNA10 mu L, the recombinant plasmid 5 mu L; sterile deionized water is added to make up to 360 mu L.

6. Use of a strain of saccharomyces cerevisiae according to claim 1 for brewing soy sauce.

7. Use of a strain of saccharomyces cerevisiae according to claim 1 for performing a fermentation.

8. Use according to claim 7, characterized in that in the use, the following medium is used as seed medium or fermentation medium for the strain of Saccharomyces cerevisiae: adding water into 10g of yeast extract, 20g of peptone and 20g of glucose to 1000 mL; sterilizing at 110 deg.C for 20min at pH 7.0.

9. Use according to claim 8, wherein the seed culture conditions are as follows: selecting a single colony to be inoculated into a container, wherein the liquid loading amount of the seed culture medium in the container is 1/5 of the total volume; shaking and culturing at 30 deg.C and 200r/min for 24 h.

10. Use according to claim 9, wherein the fermentation conditions are as follows: inoculating a culture solution obtained by seed culture at a ratio of 10% (v/v) into another container, wherein the liquid content of the fermentation medium in the container is 1/10 of the total volume; shaking and culturing at 30 deg.C and 200r/min for 48 h.