CN107227323A - A kind of structure of high yield selenomethionine saccharomyces cerevisiae and application - Google Patents

Abstract

The invention discloses the construction and application of a selenium-enriched Saccharomyces cerevisiae with high yield of selenomethionine. The target fragment containing a strong promoter was exchanged with the promoter region of the MET6 gene on the yeast genome by homologous recombination to obtain a MET6 overexpression strain. The present invention screens and obtains the MET6 overexpression mutant strain SZ1 of Saccharomyces cerevisiae. Then use the mutant strain for selenium-enrichment operation, culture the mutant strain under galactose-induced conditions to the stationary phase, add Na ₂ SeO ₃ at a final concentration of 2.5 mM, and incubate for 24 hours to obtain selenium-enriched yeast with high production of selenomethionine . The present invention for the first time correlates Saccharomyces cerevisiae MET6 gene modification with Saccharomyces cerevisiae selenium enrichment. After selenium enrichment, the intracellular selenomethionine content of the strain is at least twice that of the wild-type yeast cells.

Description

Construction and application of a high-yield selenomethionine Saccharomyces cerevisiae

technical field

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

Background technique

Selenium is one of the essential elements for the human body. It has the functions of anti-oxidation, enhancing immunity, detoxification, and promoting basic 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 human health. The Chinese Nutrition Society has formulated the reference intake of dietary selenium for Chinese residents, and proposed that the average adult dietary selenium requirement is 41 μg/d, and the recommended intake is 50 μg/d. Selenomethionine is an organic selenium compound with low toxicity, which can be metabolized and retained in human body and animal body. It 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 ability is as high as 3000 μg/g, which can convert inorganic selenium into organic selenium, and more than 90% of selenium exists in the form of selenomethionine. Selenium-enriched yeast has become the preferred selenium supplement additive due to its strong food safety and high bioavailability, and is widely used in animal and poultry breeding, as well as human selenium supplementation.

Improving the selenium-enriching ability of yeast is an important issue, and traditional solutions generally start from the optimization of medium components and optimization of culture conditions (temperature, rotation speed, ventilation, pH, etc.). This technology uses a powerful yeast genetics operating system to further regulate the synthesis of intracellular selenomethionine, and transform the selenium metabolism of cells, thereby improving the synthesis ability 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 improving the synthesis ability of intracellular selenomethionine by modifying the MET6 gene of Saccharomyces cerevisiae.

Contents of the invention

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

Another object of the present invention is to provide a method for constructing a MET6 overexpression mutant strain of Saccharomyces cerevisiae. After using microbial genetics to construct a MET6 overexpression mutant strain of Saccharomyces cerevisiae cells, the mutant strain is used for high selenium production during selenium enrichment substitute methionine.

In order to achieve the above object, the present invention adopts the following technical solutions:

The application of a MET6 gene overexpression mutant strain of Saccharomyces cerevisiae in improving the synthesis of selenomethionine.

A method for constructing a mutant strain of Saccharomyces cerevisiae MET6 gene overexpression, the steps of which are as follows: through homologous recombination, a target fragment containing a strong promoter is exchanged with the promoter region of the MET6 gene on the yeast genome to obtain a MET6 overexpression strain.

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

Construction of Saccharomyces cerevisiae MET6 overexpression mutant strain SZ1: Saccharomyces cerevisiae gene homologous recombination knockout principle, primer design, knockout fragment transformation method was 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 Saccharomyces cerevisiae MET6 gene sequence (GenBank: BK006943.1):

Upstream primer MET6-P1:

TTTTTGTTAAACTCTTCCTTTATTCATAAAAAAGCAAGCATCTAAGAGCATAAAGGGAATAAGGGCGACAC;

Downstream primer MET6-P2:

TGATATGTACTTTGAAATTATATTGGTATTTTGTTTCTTAGAGTGTTGTCGGTTTTTTTCTCTTGACGTT;

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

3) Overexpression of the MET6 gene: the knockout fragment amplified by PCR was introduced into cells of S. The MET6 overexpression mutant strain SZ1 ( Saccharomyces cerevisiae SZ1) of Saccharomyces cerevisiae with Ura synthesis gene was obtained by Ura marker screening. This strain has been preserved. Depository unit: China Type Culture Collection Center, address: China. Wuhan. Wuhan University, deposit Date: June 12, 2017, deposit number CCTCC No: M2017326, classification name: Saccharomyces cerevisiae SZ1.

An application of the MET6 gene overexpression mutant strain SZ1 of Saccharomyces cerevisiae to increase the synthesis of selenomethionine, the steps are:

1) Selenium enrichment process: inoculate MET6 overexpression mutant strains SZ1 and BY4742 from the plate in 2 mL of YPGal medium (yeast extract: 1%, peptone: 2%, galactose: 2%), 200 rpm /min, shake culture at 30°C for 24 h; add Na ₂ SeO ₃ with a final concentration of 2.5 mM to the medium, continue 200 rpm/min, shake culture at 30°C for 24 h, and complete selenium enrichment;

2) Collection of intracellular selenomethionine: Selenized yeast cells (0.5 g, wet weight) were collected, washed with PBS solution and aliquoted into cryopreservation tubes filled with acid-washed glass beads (Sigma). Break the cells with a Mini-Beadbeater-16 glass bead cell breaker, shake for 1 min, and incubate on ice for 1 min; collect the supernatant after centrifugation, add 200 mg of Trypsin and 10×TCS Buffer (1 M Tris-Cl pH 7.5, 100 mM CaCl ₂ , 5% SDS) and incubated at 37 °C for 18 h; after centrifugation, the supernatant was filtered with a 0.22 μm filter membrane, 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 After membrane filtration, use size exclusion chromatography to remove incompletely digested proteins (the chromatographic column is Superdex200 HR 10/30 Column; the instrument is Shimadzu Chromatographic Separation System), and the 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 wavelength is set at 220 nm and 280 nm, and samples with retention times of 80-89 min and 101-104.5 min are collected ;

3) Continue to inject the collected samples into high-performance liquid chromatography-inductively coupled plasma-mass spectrometer (HPLC-ICP-MS) for further confirmation, and its mobile phase composition is formic acid/ammonium formate (25mM), 0.1% (v /v) TFA, pH 3.1, 3% (v/v) methanol; the standard selenomethionine was purchased from Sigma; The contents of selenomethionine were 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 S. cerevisiae MET6 gene with S. cerevisiae selenium enrichment for the first time. Using the powerful yeast genetics operating system, the MET6 overexpression mutant strain SZ1 was constructed, which overexpressed the MET6 gene, further regulated the synthesis of selenomethionine in the cell, and modified the selenium metabolism of the cell, thereby improving the cell growth rate. Synthetic capacity of endoselenomethionine. After the selenium enrichment of the strain, the intracellular selenomethionine content is increased by at least 2 times compared with the wild-type yeast cells.

Description of drawings

Fig. 1 is a schematic diagram of electrophoresis for identification of MET6 gene recombinant strain SZ1 by PCR.

PCR was performed with primers for MET6 gene promoter-specific homologous recombination verification, and it was found that a specific amplified band with a size of 2200 bp appeared in the swimming lane of the SZ1 template.

detailed description

Taking the construction and application of a high-yield selenomethionine-enriched Saccharomyces cerevisiae as an example, the present invention will be further described with reference to the accompanying drawings.

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

[Example 1] A method for constructing Saccharomyces cerevisiae MET6 overexpression mutant strain SZ1

Construction of Saccharomyces cerevisiae MET6 overexpression mutant strain SZ1: Saccharomyces cerevisiae gene homologous recombination knockout principle, primer design, knockout fragment transformation method was 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 Saccharomyces cerevisiae MET6 gene sequence (GenBank: BK006943.1):

Upstream primer MET6-P1:

TTTTTGTTAAACTCTTCCTTTATCATAAAAAAGCAAGCATCTAAGAGCATAAAGGGAATAAGGGCGACAC;

Downstream primer MET6-P2:

TGATATGTACTTTGAAATTATATTGGTATTTTGTTTCTTAGAGTGTTGTCGGTTTTTCTCCTTGACGTT;

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

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

[Example 2] An application of the MET6 overexpression mutant strain of Saccharomyces cerevisiae in improving the synthesis of selenomethionine

MET6 overexpression mutant strains SZ1 and BY4742 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 200 rpm/min, and shake at 30°C for 24 h to complete selenium enrichment. The selenized yeast cells (0.5 g, wet weight) were collected, washed with PBS solution and divided into cryopreservation tubes filled with acid-washed glass beads (Sigma), and broken with a Mini-Beadbeater-16 glass bead cell breaker Cells were shaken for 1 min and incubated on ice for 1 min; after centrifugation, the supernatant was collected, added 200 mg of Trypsin and 10×TCS Buffer (1 M Tris-Cl pH 7.5, 100mM CaCl ₂ , 5% SDS) and incubated at 37 °C 18 h. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane, and 40 mg of protease 14 was added to incubate at 37°C for 24 h. After centrifugation, the supernatant was filtered with a 0.22 μm filter membrane, and the incompletely digested protein was removed by size exclusion chromatography (the chromatographic column was Superdex 200 HR 10/30 Column; the instrument was Shimadzu Chromatographic Separation System), and the mobile phase composition 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 wavelength is set at 220 nm and 280 nm, and the collection retention time is 80- 89 min and 101-104.5 min samples.

Continue to inject the collected samples into a high-performance liquid chromatography-inductively coupled plasma-mass spectrometer (HPLC-ICP-MS) for further confirmation, and its 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. The intracellular selenomethionine contents of BY4742 wild-type Saccharomyces cerevisiae cells and MET6 overexpression mutant SZ1 after selenium enrichment were 7.806 μg/g SeMet and 21.15 μg/gSeMet, 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 violating the spirit of the present invention.

SEQUENCE LISTING

<110> Wuhan University

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

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 70

<212>DNA

<213> Artificial sequence

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tttttgttaa actcttcctt tatcataaaa aagcaagcat ctaagagcat aaagggaata 60

agggcgacac 70

<210> 2

<211> 70

<212>DNA

<213> Artificial sequence

<400> 2

tgatatgtac tttgaaatta tattggtatt ttgtttctta gagtgttgtc ggttttttct 60

ccttgacgtt 70

Claims (4)

1. a kind of saccharomyces cerevisiaeMET6Gene overexpression mutant strain is improving the application of selenomethionine synthesis.

2. saccharomyces cerevisiae described in claim 1MET6The construction method of gene overexpression mutant strain, it is characterised in that include Following steps：According to the methods of homologous recombination described by document（Yeast, 1998, 14: 953-962）, it is strong by that will contain In the purpose fragment and Yeast genome of promoterMET6The promoter region of gene is exchanged, and is obtainedMET6Gene overexpression bacterium Strain.

3. saccharomyces cerevisiae according to claim 2MET6The construction method of gene overexpression mutant strain, it is characterised in that Comprise the following steps：

1）Design of primers：According to saccharomyces cerevisiaeMET6Gene order（GenBank: BK006943.1）Design primer：

Sense primer MET6-P1：

TTTTTGTTAAACTCTTCCTTTATCATAAAAAAGCAAGCATCTAAGAGCATAAAGGGAATAAGGGCGACAC；

Anti-sense primer MET6-P2：

TGATATGTACTTTGAAATTATATTGGTATTTTGTTTCTTAGAGTGTTGTCGGTTTTTTCTCCTTGACGTT；

2）PCR is expanded：Plasmid pSP72-ura3-pGAL1 is carried out using sense primer MET6-P1 and anti-sense primer MET6-P2 PCR is expanded, and is obtained and is contained saccharomyces cerevisiaeMET6The GAL1 strong promoter fragments of the promoter upstream and downstream sequence of gene；

3）MET6The overexpression of gene：PCR is expanded by obtained knockout fragment by LiAc/PEG method for transformation and imports wine brewing Yeast BY4742 strain cells, screening obtains the saccharomyces cerevisiae with Ura synthetic genesMET6Gene overexpression mutant strain SZ1。

4. the saccharomyces cerevisiae that the construction method of claim 3 is obtainedMET6Gene overexpression mutant strain, it is characterised in that it is classified It is named as Saccharomyces Cerevisiae in S Z1（Saccharomyces cerevisiaeSZ1）, China typical culture collection center is preserved in, Deposit number is CCTCC No: M2017326.