CN107227323B - Construction and application of high-yield selenomethionine saccharomyces cerevisiae - Google Patents

Abstract

The invention discloses construction and application of high-yield selenomethionine selenium-rich saccharomyces cerevisiae. The target fragment containing the strong promoter is combined with the yeast genome by homologous recombinationMET6Exchanging promoter region of gene to obtainMET6An overexpression strain. The invention obtains the saccharomyces cerevisiae by screeningMET6The mutant strain SZ1 was overexpressed. Then the mutant strain is used for selenium enrichment operation, the mutant strain is cultured to a stable stage under the induction condition of galactose, and Na with the final concentration of 2.5mM is added₂SeO₃And incubating for 24h to obtain the selenium-enriched yeast with high yield of selenomethionine. The invention firstly uses the saccharomyces cerevisiaeMET6The gene modification is related to the selenium enrichment of saccharomyces cerevisiae. The content of the selenomethionine in the selenium-enriched cells of the strain is increased by at least two times compared with that of wild yeast cells.

Description

Construction and application of a high-yielding selenomethionine Saccharomyces cerevisiae

technical field

The invention belongs to the technical field of Saccharomyces cerevisiae engineering, in particular to a method for constructing a MET6 overexpression mutant strain of Saccharomyces cerevisiae, and also relates to the application of the MET6 overexpression mutant strain of Saccharomyces cerevisiae.

Background technique

Selenium is one of the essential elements of the human body, with functions such as anti-oxidation, enhancing immunity, detoxification, and promoting basal metabolism. Insufficient intake of selenium can lead to Keshan disease, cancer, cardiovascular and other diseases. Therefore, adequate intake of selenium plays an important role in maintaining health. The Chinese Nutrition Society has formulated the reference intake of dietary selenium for Chinese residents, and proposed that the average dietary selenium requirement for adults is 41 μg/d, and the recommended intake is 50 μg/d. Selenomethionine is a low toxicity organoselenide, which can be metabolized and retained in human and animal body, and is superior to other selenium compounds in nutrition, and is an ideal selenium supplement.

As the main strain in the fermentation industry, Saccharomyces cerevisiae also has strong selenium tolerance and enrichment ability. The selenium enrichment capacity is as high as 3000 μg/g, which can convert inorganic selenium into organic selenium, of which more than 90% of the selenium exists in the form of selenomethionine. Selenium-enriched yeast has become the preferred selenium supplement due to its strong food safety and high bioavailability.

Improving the selenium-enriching ability of yeast is an important topic, and traditional solutions generally start from the optimization of medium components and the optimization of culture conditions (temperature, rotation speed, ventilation, pH, etc.). The technology utilizes the powerful yeast genetics operating system to further regulate the synthesis of intracellular selenomethionine, and transform the cellular selenium metabolism, thereby improving the synthesis capacity of intracellular selenomethionine.

The methionine synthase encoded by the MET6 gene can catalyze the production of methionine from homocysteine, which is one of the main pathways of intracellular methionine synthesis in Saccharomyces cerevisiae. So far, there is no report on the modification of Saccharomyces cerevisiae MET6 gene to improve the synthesis ability of intracellular selenomethionine.

SUMMARY OF THE INVENTION

The purpose of the present invention is to construct a MET6 overexpression mutant strain of Saccharomyces cerevisiae, the mutant strain overexpresses the MET6 gene, and the ability to synthesize selenomethionine is improved when enriched with selenium.

Another object of the present invention is to provide a method for constructing a MET6 overexpression mutant strain of Saccharomyces cerevisiae. After constructing a MET6 overexpression mutant strain of Saccharomyces cerevisiae by using a microbial genetic method, the mutant strain is used for high selenium production when enriched with selenium. methionine.

In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

Application of a MET6 gene overexpression mutant of Saccharomyces cerevisiae in improving the synthesis of selenomethionine.

A method for constructing a MET6 gene overexpression mutant strain of Saccharomyces cerevisiae comprises the following steps: exchanging a target fragment containing a strong promoter with the promoter region of the MET6 gene on the yeast genome through homologous recombination to obtain a MET6 overexpression strain.

A construction method of Saccharomyces cerevisiae MET6 gene overexpression mutant strain SZ1, the steps are as follows:

Construction of Saccharomyces cerevisiae MET6 overexpression mutant SZ1: The principle of homologous recombination knockout of Saccharomyces cerevisiae gene, primer design, and the method of knockout fragment transformation were carried out according to the method described in the literature ( Yeast , 1998, 14: 953-962), the method is Current general methods for gene knockout in Saccharomyces cerevisiae;

1) Primer design: Design primers based on the MET6 gene sequence of Saccharomyces cerevisiae (GenBank: BK006943.1):

Upstream primer MET6-P1:

TTTTTGTTAAACTCTTCCTTTATCATAAAAAAGCAAGCATCTAAGAGCATAAAGGGAATAAGGGCGACAC;

Downstream primer MET6-P2:

TGATATGTACTTTGAAATTATATTGGTATTTTGTTTCTTAGAGTGTTGTGCGGTTTTTTCTCCTTGACGTT;

2) PCR amplification: PCR amplification was performed on plasmid pSP72-ura3-pGAL1 (available through Addgene) using upstream primer MET6-P1 and downstream primer MET6-P2 to obtain GAL1 containing the upstream and downstream sequences of the promoter of Saccharomyces cerevisiae MET6 gene Strong promoter fragments;

3) Overexpression of MET6 gene: The knockout fragment amplified by PCR was introduced into Saccharomyces cerevisiae BY4742 strain cells by LiAc/PEG transformation method (BY4742 strain was obtained from the China Type Culture Collection, which can be purchased), and passed The M ET6 overexpression mutant SZ1 ( Saccharomyces cerevisiae SZ1) of Saccharomyces cerevisiae with Ura synthetic gene was obtained by Ura marker screening. This strain has been deposited. Date: June 12, 2017, deposit number CCTCC No: M2017326, taxonomic name: Saccharomyces cerevisiae SZ1.

A kind of application of utilizing the MET6 gene overexpression mutant SZ1 of Saccharomyces cerevisiae in improving the synthesis of selenomethionine, the steps are:

1) Selenium enrichment process: MET6 overexpression mutant strain SZ1 and BY4742 were inoculated from the plate in 2 mL YPGal medium (yeast extract: 1%, peptone: 2%, galactose: 2%), 200 rpm/ min, shaking at 30°C for 24 hours; adding Na ₂ SeO ₃ with a final concentration of 2.5 mM to the medium, continuing at 200 rpm/min, shaking at 30° C for 24 hours, and completing the enrichment of selenium;

2) Collection of intracellular selenomethionine: Collect the selenized yeast cells (0.5 g, wet weight), wash with PBS solution, and distribute them into cryovials filled with acid-washed glass beads (Sigma). The cells were disrupted with a Mini-Beadbeater-16 glass bead cell disrupter, shaken for 1 min, and incubated on ice for 1 min; after centrifugation, the supernatant was collected, and 200 mg of Trypsin and 10×TCS Buffer (1 M Tris-Cl pH 7.5, 100 mM CaCl ₂ , 5% SDS) was incubated at 37 °C for 18 h; after centrifugation, the supernatant was filtered with a 0.22 μm filter, and 40 mg of protease 14 was added and incubated at 37 °C for 24 h; after centrifugation, the supernatant was filtered with a 0.22 μm filter. After membrane filtration, the incompletely digested proteins were removed by size exclusion chromatography (the chromatographic column was Superdex 200 HR 10/30 Column; the instrument was Shimadzu Corporation chromatographic separation system), and the mobile phase composition was 0.05M NaH ₂ PO ₄ - Na ₂ HPO ₄ (pH 7.2), mobile phase flow rate of 0.5 mL/min, column temperature of 30°C, UV detection wavelengths set to 220 nm and 280 nm, and collection retention times of 80-89 min and 101-104.5 min. sample;

3) Continue to inject the collected samples into high performance liquid chromatography-inductively coupled plasma-mass spectrometer (HPLC-ICP-MS) for further confirmation, and the mobile phase composition is formic acid/ammonium formate (25mM), 0.1% (v /v) TFA, pH 3.1, 3% (v/v) methanol; selenomethionine standard was purchased from Sigma; wild-type Saccharomyces cerevisiae cells BY4742 and MET6 overexpression mutant SZ1 were obtained after selenium enrichment. The content of selenomethionine was 7.806 μg/g SeMet and 21.15 μg/g SeMet, respectively.

Compared with the prior art, the present invention has the following advantages and effects:

The present invention associates Saccharomyces cerevisiae MET6 gene with Saccharomyces cerevisiae enrichment for the first time. Using the powerful yeast genetics operating system, a MET6 overexpression mutant SZ1 was constructed, overexpressing the MET6 gene, further regulating the synthesis of intracellular selenomethionine, and modifying the cellular selenium metabolism, thereby improving the cellularity of selenium metabolism. Synthetic capacity of endoselenomethionine. Compared with wild-type yeast cells, the intracellular selenomethionine content of the selenium-enriched strain increased by at least 2 times.

Description of drawings

Figure 1 is a schematic diagram of electrophoresis of a PCR identification of the MET6 gene recombinant strain SZ1.

PCR was performed with primers specific for homologous recombination verification of the MET6 gene promoter. It was found that a specific amplification band with a size of 2200 bp appeared in the lane of the SZ1 template.

Detailed ways

The present invention is further described below with reference to the accompanying drawings by taking the construction and application of a high-yielding selenomethionine selenium-enriched Saccharomyces cerevisiae as an example.

Saccharomyces cerevisiae BY4742 strain and plasmid pSP72-ura3-pGAL1 were obtained from China Type Culture Collection.

[Example 1] A construction method of S. cerevisiae MET6 overexpression mutant SZ1

Construction of Saccharomyces cerevisiae MET6 overexpression mutant SZ1: The principle of homologous recombination knockout of Saccharomyces cerevisiae gene, primer design, and the method of knockout fragment transformation were carried out according to the method described in the literature ( Yeast , 1998, 14: 953-962), the method is Current general methods for gene knockout in Saccharomyces cerevisiae;

1) Primer design: Design primers based on the MET6 gene sequence of Saccharomyces cerevisiae (GenBank: BK006943.1):

Upstream primer MET6-P1:

TTTTTGTTAAACTCTTCCTTTATCATAAAAAAGCAAGCATCTAAGAGCATAAAGGGAATAAGGGCGACAC;

Downstream primer MET6-P2:

TGATATGTACTTTGAAATTATATTGGTATTTTGTTTCTTAGAGTGTTGTGCGGTTTTTTCTCCTTGACGTT;

2) PCR amplification: PCR amplification was performed on plasmid pSP72-ura3-pGAL1 (available through Addgene) using upstream primer MET6-P1 and downstream primer MET6-P2 to obtain GAL1 containing the upstream and downstream sequences of the promoter of Saccharomyces cerevisiae MET6 gene Strong promoter fragments;

3) Overexpression of MET6 gene: The knockout fragment obtained in step 2) PCR amplification was introduced into Saccharomyces cerevisiae BY4742 strain cells by LiAc/PEG transformation method (BY4742 strain is from the China Type Culture Collection, which can be purchased) , and the M ET6 overexpression mutant SZ1 ( Saccharomyces cerevisiae SZ1) of Saccharomyces cerevisiae with Ura synthetic gene was obtained by Ura marker screening, which was deposited in the China Center for Type Culture Collection with the deposit number CCTCCNo: M2017326.

[Example 2] Application of a M ET6 overexpression mutant of Saccharomyces cerevisiae in improving the synthesis of selenomethionine

The MET6 overexpression mutant SZ1 and BY4742 strains were inoculated from the plate in 2 mL of YPGal medium (yeast extract: 1%, peptone: 2%, galactose: 2%), 200 rpm/min, 30°C Shake culture for 24 h; add Na ₂ SeO ₃ with a final concentration of 2.5 mM to the medium, continue to 200 rpm/min, shake at 30 °C for 24 h, and complete the selenium enrichment. The selenized yeast cells (0.5 g, wet weight) were collected, washed with PBS solution, and then aliquoted into cryovials filled with acid-washed glass beads (Sigma), and disrupted with a Mini-Beadbeater-16 glass bead cell disrupter The cells were shaken for 1 min and incubated on ice for 1 min; after centrifugation, the supernatant was collected, and 200 mg of Trypsin and 10×TCS Buffer (1 M Tris-Cl pH 7.5, 100 mM CaCl ₂ , 5% SDS) were added at 37 °C Incubate for 18 h. After centrifugation, the supernatant was filtered through a 0.22 μm filter, and then 40 mg of protease 14 was added and incubated at 37 °C for 24 h. After centrifugation, the supernatant was filtered with a 0.22 μm filter membrane, and the incompletely digested proteins were removed by size exclusion chromatography (the chromatographic column was Superdex 200 HR 10/30 Column; the instrument was Shimadzu Corporation’s chromatographic separation system), and its mobile phase composition is 0.05 M NaH ₂ PO ₄ -Na ₂ HPO ₄ (pH 7.2), the mobile phase flow rate is 0.5 mL/min, the column temperature is 30°C, the UV detection wavelengths are set to 220 nm and 280 nm, and the collection retention time is 80- 89 min and 101-104.5 min samples.

The collected samples were injected into high performance liquid chromatography-inductively coupled plasma-mass spectrometer (HPLC-ICP-MS) for further confirmation, and the mobile phase composition was formic acid/ammonium formate (25mM), 0.1% (v/v) ) TFA, pH 3.1, 3% (v/v) methanol. Selenomethionine standard was purchased from Sigma Company. The content of selenomethionine in wild-type Saccharomyces cerevisiae cells BY4742 and MET6 overexpression mutant SZ1 after Se enrichment was 7.806 μg/g SeMet and 21.15 μg/g SeMet, respectively.

The above description is an explanation of the present invention, not a limitation of the present invention. For the limited scope of the present invention, refer to the claims. The present invention can be modified in any form without departing from the spirit of the present invention.

SEQUENCE LISTING

<110> Wuhan University

<120> Construction and application of a high-yielding selenomethionine Saccharomyces cerevisiae

<160> 2

<170> PatentIn version 3.3

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ttttttgttaa actcttcctt tatcataaaa aagcaagcat ctaagagcat aaagggaata 60

agggcgacac 70

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<212> DNA

<213> Artificial sequences

<400> 2

tgatatgtac tttgaaatta tattggtatt ttgtttctta gagtgttgtc ggttttttct 60

ccttgacgtt 70

Claims (2)

1. A Saccharomyces cerevisiae MET6 gene overexpression mutant, characterized in that, its classification is named Saccharomyces cerevisiae SZ1 ( Saccharomyces cerevisiae SZ1), and the deposit number is CCTCC No: M2017326. 2. The application of the MET6 gene overexpression mutant of Saccharomyces cerevisiae according to claim 1 in improving the synthesis of yeast selenomethionine.

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