Yeast site-directed mutagenesis gene gex1D, recombinant vector, recombinant bacterium and application of yeast site-directed mutagenesis gene gex1D

Abstract

The invention provides a yeast site-directed mutagenesis gene gex1D, a recombinant vector, recombinant bacteria and application of the yeast site-directed mutagenesis gene gex1D, and belongs to the technical field of bioengineering. The nucleotide sequence of the yeast site-directed mutagenesis gene gex1D is shown as SEQ ID NO.1, the amino acid sequence coded by the sequence is shown as SEQ ID NO.2, and the amino acid sequence is obtained by mutating the 613th leucine of the amino acid coded by the gex1 gene into serine. The 613-site leucine of the gex1 gene in the S288C strain is mutated into serine by adopting a site-specific mutagenesis technology, the acid resistance of the site-specific mutagenesis strain DX-2 is obviously improved, and the site-specific mutagenesis strain DX-2 can be used as a potential chassis microorganism for fermenting short-chain organic acids and is particularly suitable for later transformation and production of succinic acid.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology and specifically relates to a yeast site-directed mutation gene gex1. D , recombinant vectors, recombinant bacteria and their applications. Background Art

[0002] Saccharomyces cerevisiae Saccharomyces cerevisiae ) is a single-celled organism with a short life cycle, fast reproduction rate, strong vitality, relatively easy cultivation, and high genetic recombination rate. Therefore, it is widely used in industry and has high economic value. As a model strain for functional genomics research, Saccharomyces cerevisiae is widely used in the study of molecular genetic mechanisms, physiological and biochemical functions, metabolic pathways, and the identification of protein interactions in organisms, providing a good research object for analyzing the genetic structure of complex traits.

[0003] The breeding of yeast with excellent acid resistance is not only applicable to the traditional brewing field, but also crucial for the development of some metabolites with important industrial value, such as various organic acid products. During the fermentation process, brewer's yeast is inevitably affected by a variety of environmental stresses, mainly including high acid, high sugar, high osmotic pressure, high ethanol, etc. These stresses can destroy the cell structure of yeast and cause oxidative denaturation of the structure of important biomacromolecules such as proteins, thereby seriously affecting its growth, metabolism and physiological functions. In a slightly acidic environment, brewer's yeast has a certain stress and adaptability, but when it is in highly acidic conditions, its activity still faces severe challenges.

[0004] Under acid stress, Saccharomyces cerevisiae regulates gene expression and metabolism through signal molecules and signal transduction pathways. At present, relevant research mainly focuses on the barrier function of cell walls and cell membranes, maintaining intracellular pH homeostasis through proton pumps such as Pmalp, mitochondrial retrograde pathways, and endoplasmic reticulum stress responses. Acid stress is the most common abiotic stress encountered by yeast cells during the fermentation of acidic products such as organic acids, which seriously affects cell growth and metabolism. In order to alleviate the inhibitory effect of acid stress on cell growth and product synthesis, a large amount of neutralizers are often required during the production process, which not only increases costs and makes the downstream purification process more complicated, but also easily causes environmental pollution. This problem can be well solved by studying the acid resistance mechanism to obtain acid-resistant Saccharomyces cerevisiae. However, there are no reports on the effect of gex1 gene mutation on yeast acid fermentation. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a yeast site-directed mutation gene gex1 D , recombinant vectors, recombinant bacteria and their applications. The brewer's yeast containing the mutant gene can ferment in an acidic environment and can also ferment and produce succinic acid compared to standard brewer's yeast strains.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a yeast site-directed mutation gene gex1 D , the yeast site-directed mutation gene gex1 D The nucleotide sequence is shown in SEQ ID NO.1.

[0007] Preferably, the yeast site-directed mutation gene gex1 D The amino acid sequence is shown in SEQ ID NO.2.

[0008] The present invention provides a recombinant vector, wherein the recombinant vector contains the yeast site-directed mutation gene gex1 D .

[0009] The present invention provides a recombinant bacterium, wherein the recombinant bacterium contains the yeast site-directed mutation gene gex1 D or the recombinant vector.

[0010] Preferably, the starting strain of the recombinant bacteria includes Saccharomyces cerevisiae S288C strain.

[0011] The present invention also provides the yeast site-directed mutation gene gex1 D , and use of the recombinant vector or the recombinant bacteria in fermentation in an acidic environment.

[0012] Preferably, the pH value of the acidic environment is 2.3-3.5.

[0013] Preferably, the acidic environment includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid.

[0014] The present invention provides the yeast site-directed mutation gene gex1 D , and use of the recombinant vector or the recombinant bacteria in producing organic acids.

[0015] Preferably, the organic acid includes one or more of L-malic acid, pyruvic acid, citric acid and succinic acid.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a yeast site-directed mutation gene gex1 D , recombinant vectors, recombinant bacteria and their applications, ultraviolet irradiation was used to induce mutagenesis of Saccharomyces cerevisiae S288C, and the strain DX-1 with significantly improved acid resistance was obtained by flow sorting, and its genome was sequenced, and it was found that gex1 DThe gene mutated, and the leucine (L, Leu) at position 613 was replaced by serine (S, Ser). The site-directed mutagenesis technique was used to mutate the leucine (L, Leu, TTA) at position 613 of the gex1 gene in the S288C strain to serine (S, Ser, TCA). The acid resistance of the site-directed mutant strain DX-2 was significantly improved, and it can be used as a potential chassis microorganism for the fermentation of short-chain organic acids, especially for the later transformation and production of succinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the gel electrophoresis diagram of gex1 gene amplification.

[0018] Figure 2 This is the map of the PY26TEF-gex1 vector.

[0019] Figure 3 This is a sequence verification diagram of the positive clones.

[0020] Figure 4 The morphological images of the S288C and DX-2 strains under a microscope (40×) are shown in Figure 1. A is the morphological image of the S288C strain, and B is the morphological image of the site-directed mutation DX-2 strain.

[0021] Figure 5 OD of S288C and DX-2 strains in YPD medium and YPD medium with different pH values 600 Condition.

[0022] Figure 6 This is a comparison chart of the HPLC detection of organic acid content in the 24h fermentation broth of S288C and DX-2 strains.

[0023] Figure 7 for Figure 6 Enlarged view of the succinic acid peak in the HPLC detection comparison diagram. DETAILED DESCRIPTION

[0024] The present invention provides a yeast site-directed mutation gene gex1 D , the yeast site-directed mutation gene gex1 D The nucleotide sequence of the mutant gene gex1 is shown in SEQ ID NO.1. D TTA mutates to TCA at positions 1837-1839 of the gene gex1. The amino acid sequence encoded by the nucleotide sequence of the present invention is shown in SEQ ID NO.2, and the amino acid sequence mutates to serine (S, Ser, TCA) at position 613 of the amino acid encoded by the gex1 gene from leucine (L, Leu, TTA).

[0025] The present invention also provides a recombinant vector, wherein the recombinant vector contains the yeast site-directed mutation gene gex1D The original vector used in the recombinant vector of the present invention is PY26TEF-GPD.

[0026] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium contains the yeast site-directed mutation gene gex1 D Or the recombinant vector. The starting strain of the recombinant bacteria of the present invention includes but is not limited to the Saccharomyces cerevisiae S288C strain.

[0027] The present invention also provides the yeast site-directed mutation gene gex1 D , the use of the recombinant vector or the recombinant bacteria in fermentation in an acidic environment. The pH value of the acidic environment of the present invention is 2.1-3.5. The acidic environment of the present invention includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid. The yeast site-directed mutation gene gex1 of the present invention D It can improve the acid resistance of brewer's yeast.

[0028] The present invention also provides the yeast site-directed mutation gene gex1 D , use of the recombinant vector or the recombinant bacteria in producing organic acids. The organic acid of the present invention includes one or more of L-malic acid, pyruvic acid, citric acid and succinic acid, and the organic acid is preferably succinic acid.

[0029] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0030] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1 Screening of mutant genes Saccharomyces cerevisiae (S288C, Baosai Bio) was cultured in YPD liquid medium (Qingdao Haibo Bio) to obtain a culture medium in the logarithmic phase; the culture medium in the logarithmic phase was centrifuged and the supernatant was discarded to obtain the bacteria. The bacteria were gradient diluted with physiological saline to obtain dilutions of different dilution multiples. 100 μL of the dilutions were spread on YPD solid medium (Qingdao Haibo Bio) plates and inverted at 30°C. The dilution gradient was determined when the number of colonies was 100-300 for subsequent experiments. The determined dilution gradient was 10 -4 .

[0032] Turn on the UV lamp to preheat for 30 minutes in advance, and take the dilution gradient of 10-4 3mL of bacterial solution was placed in a 60mm culture dish for UV treatment for 0s, 30s, 60s, 90s, 120s and 150s, and 3 parallel experiments were set for each treatment time. 100μL of the mutagenic bacterial solution was evenly spread on a YPD solid culture medium plate, inverted and cultured at 30℃ for 24h, and the colonies were counted. The UV treatment time with a lethality of about 90% was used as the subsequent UV mutagenesis treatment time, and the UV mutagenesis time was determined to be 120s.

[0033] The above-determined ultraviolet mutagenesis time was used to induce mutagenesis of Saccharomyces cerevisiae (S288C) by ultraviolet irradiation to obtain a mutant strain. The obtained mutant strain was subjected to an acid resistance test: the original strain and the mutant strain were cultured in different pH mediums, and the strain DX-1 with significantly improved acid resistance was screened according to the OD value. The genome of strain DX-1 was sequenced, and it was found that its gex1 gene was mutated, and the 613th leucine (L, Leu, TTA) was replaced by serine (S, Ser, TCA).

[0034] Example 2 gex1 sequence amplification 1. DNA extraction: Saccharomyces cerevisiae (S288C, Baosai Biotechnology) was streaked and cultured on YPD solid medium (Qingdao Haibo Biotechnology). A single clone was picked and inoculated into YPD liquid medium. After culturing for 24 h, Saccharomyces cerevisiae DNA was extracted using the Yeast Genomic DNA Extraction Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0035] 2. Primer design: Based on the whole genome data of S288C strain in the National Center for Biotechnology Information (NCBI) database, primers gex1-F and gex1-R were designed to amplify the gex1 gene (and the restriction sites SacII and PacI were introduced at the same time). The primers for the gex1 gene are as follows: gex1-F: 5'-CCGCGGATGAGTTCTAGTGTTGTTG-3' (SEQ ID NO.3), gex1-R: 5'-TTAATTAATCAACATCCTAATTTCTTGTTACT-3' (SEQ ID NO.4).

[0036] 3. PCR amplification: Using the genomic DNA of the S288C strain extracted above as a template, the gex1 gene was amplified using the primers designed above. The PCR amplification reaction system is as follows: the total system is 50 μL, DNA template 2 μL, gex1-F and gex1-R primers 1 μL each, dNTP 4 μL, PCR Buffer (with Mg 2+ ) 5μL, Taq polymerase 0.25μL and ddH2O 36.75μL. The PCR amplification procedure was as follows: 94℃ pre-denaturation for 3min, 35 cycles (94℃ denaturation for 30s, 55℃ annealing for 2min and 72℃ extension for 2min), and then extension at 72℃ for 10min after the cycle, and stored at 4℃. The amplified sequence was verified by agarose gel electrophoresis. The gel band position was between 1500-2000bp and closer to 2000bp, which was basically consistent with the 1862bp of the gex1 gene ( Figure 1 ).

[0037] Example 3 Connection, transformation and identification of cloning vectors Example 2 After the PCR amplification is completed, 3 μL of dNTP and 0.5 μL of Taq enzyme are directly added to the high-fidelity enzyme PCR tube (system is 50 μL) and the reaction is continued at 72°C for 10-20 minutes, and then the amplified gene fragment is purified and recovered using a gel recovery kit (TaKaRa). The recovered product is connected to the pMD19-T cloning vector (TaKaRa), and the connection vector is transformed into Escherichia coli DH5α competent cells using the calcium chloride method. Positive transformation is screened using blue-white spots, and the monoclonal recombinant plasmid pMD19-T-gex1 is extracted using a plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.) and sequenced. The plasmid sequencing results show that the obtained gex1 sequence is correct and has 100% homology with the gene sequence in the gene library (NCBI Reference Sequence: NC 001135.5).

[0038] Example 4 Construction of the recombinant expression vector PY26TEF-gex1 The pMD19-T-gex1 and PY26TEF-GPD vectors constructed above (Beijing Zhuangmeng International Biogene Technology Co., Ltd.) were double-digested using the rapid endonucleases SacII and PacI of Beyotime Biotechnology, and the target digestion products were purified and recovered using a gel recovery kit (TaKaRa). The digestion products were ligated using 1μL of Beyotime Biotechnology T4 ligase. Transformed into Escherichia coli DH5α, inoculated on a resistance plate containing Amp for screening. After 12 hours, single clones were picked for colony PCR identification, and positive clones were picked in LB liquid culture medium containing Amp resistance, cultured at 37°C and 160r / min, and centrifuged to collect bacteria after 12 hours. The plasmid was extracted and sequenced using the primer sequences of gex1-F (SEQ ID NO.3) and gex1-R (SEQ ID NO.4) (Sangon Biotechnology Co., Ltd.). The results showed that the gex1 sequence was correct, and the constructed recombinant expression vector was named PY26TEF-gex1 ( Figure 2 ).

[0039] Example 5 Construction of site-directed mutagenesis strains 1. Design of mutation primers: Using PY26TEF-gex1 as the starting vector, according to the principle of reverse overlap extension PCR, a pair of mutation primers gex1-F' and gex1-R' were designed by one-step method to introduce mutation sites, where the underlined letters are the mutation sites, corresponding to amino acid 613. The primers are as follows: gex1-F': 5'-CATAATCGAAGTAACAAGAAATCAGGATGTTGA-3' (SEQ ID NO.5), gex1-R': 5'-TCAACATCCTGATTTCTTGTTACTTCGATTATG-3' (SEQ ID NO.6).

[0040] 2. PCR amplification: Using the PY26TEF-gex1 vector as a template, the mutant primers constructed above were used for PCR amplification. The reaction system for PCR amplification was: 50 μL total system, 2 μL template, 2 μL gex1D-F' and 2 μL gex1D-R', 25 μL 2×PhantaMaster Mix, 19 μL ddH2O. The PCR amplification program was: 94°C pre-denaturation for 5 min, 30 cycles (94°C denaturation for 30 s, 55°C annealing for 30 s and 72°C extension for 5 min), and then extended at 72°C for 5 min after the cycle.

[0041] 3. Amplification product processing and transformed strains: Treat the above PCR product with DpnⅠ to eliminate the original vector template. The DpnⅠ reaction system is as follows: The total system is 50 μL, including 1 μL of DpnⅠ, 5 μL of Quick cut Buffer, and 44 μL of PCR product. The DpnⅠ reaction procedure is: 37°C for 2 h, 75°C for 15 min, and 4°C ∞. Purify the product PY26TEF-gex1 D Transform DH5α, inoculate it on a resistance plate containing Amp for screening, and select positive clones for sequencing verification to check if mutations have occurred. After Figure 3 sequencing, the results show that the leucine (L, Leu, TTA) at position 613 of the gex1 gene in the amplification product of the mutant primer has mutated to serine (S, Ser, TCA).

[0042] Use the quick Saccharomyces cerevisiae competent cell preparation kit of Zhuangmeng Biotechnology Co., Ltd. to transform the verified correct PY26TEF-gex1 D recombinant vector into Saccharomyces cerevisiae S288C. Use SD-URA yeast defective medium (Beijing Coolaber Technology Co., Ltd.) to screen the transformants and obtain the site-directed mutant strain DX-2.

[0043] Example 6 Strain acid tolerance verification experiment Activate Saccharomyces cerevisiae S288C and the site-directed mutant strain DX-2 on YPD plates, then pick single colonies and inoculate them into 10 mL YPD shaking tubes (liquid volume 3 mL) respectively. Observe the cell morphology under a microscope (40×) ( Figure 4 ). Culture the cells until the OD 600 reaches the range of 1 - 1.5, and then inoculate them into 250 mL flasks containing YPD medium (pH = 6.5) and YPD medium with different pH values (pH = 5.3, pH = 4.1, pH = 3.5, pH = 3, pH = 2.8, pH = 2.5, and pH = 2.3) at an inoculation amount of 1 v / v% (liquid volume 50 mL). Culture at 30°C and 200 r / min, observe the growth of different cells, and take samples to detect the OD value of the cells when the cells become significantly turbid (Table 1). Among them, the above YPD media with different pH values are adjusted with L-malic acid, As can be seen from Table 1, Saccharomyces cerevisiae S288C and the site-directed mutant strain DX-2 grow well in YPD medium and YPD medium with pH = 5.3, pH = 4.1, and pH = 3.5 within 20 h, with no significant difference; when 2.3 < pH < 3.5, the growth of the site-directed mutant strain DX-2 is significantly better than that of the S288C strain. From Figure 4 the microscope photos, it can be seen that the site-directed mutant strain DX-2 grows in clusters, while the S288C strain grows as single colonies, showing significant differences in morphology.

[0044] Table 1 Growth of S288C and DX-2 strains in medium with different pH values

[0045] Example 7 Experimental verification of the acid resistance of strains to different organic acids Activate Saccharomyces cerevisiae S288C and site-directed mutant strain DX-2 on YPD plates, then pick single colonies and inoculate them into 10mL shake tubes (3mL) filled with YPD medium. Detect OD values ​​within the range of 1-1.5, and inoculate them into 250mL YPD medium and pH=3 YPD medium shake flasks (50mL) at an inoculum volume of 1v / v%, respectively. Cultivate at 30℃, 200r / min for 36h, take samples, and detect OD. The test results are as follows: Figure 5 The pH of the YPD medium at 3 was adjusted using L-malic acid, citric acid, lactic acid, succinic acid, and pyruvic acid.

[0046] Depend on Figure 5 It can be seen that the site-directed mutant strain DX-2 is far superior to Saccharomyces cerevisiae S288C in terms of acid resistance, and shows different tolerance and preferences for different organic acids, and grows better in the environments of L-malic acid, succinic acid and pyruvic acid.

[0047] Example 8 Comparative experiment on production of organic acids by strains The site-directed mutant strain DX-2 and the S288C strain obtained in Example 5 were fermented in 250 mL shake flasks, and the types and contents of organic acids in the fermentation supernatants were detected by high performance liquid chromatography (HPLC). The required culture medium, culture conditions and detection methods are as follows: Preparation of seed solution: The S288C strain and the site-directed mutant strain DX-2 were prepared into glycerol tube strains for preservation. The preserved strains were inoculated into YPD solid medium for activation. After activation, a single colony was selected and inoculated into a 15mL YPD liquid medium shaking tube (liquid volume was 3mL), and cultured at 30°C and 220r / min for 10-20h until the logarithmic growth phase to obtain seed solution.

[0048] Shake flask fermentation culture: 1.5 mL of seed liquid was inoculated into a 250 mL baffle shake flask (50 mL of liquid volume) containing fermentation medium at a volume ratio of 3%, and fermentation culture was carried out at 30 ° C and 220 r / min. During the fermentation process, samples were taken at intervals to detect residual sugar, and 80% glucose mother solution was added according to the sugar consumption to control the glucose content to 20 g / L-30 g / L. The fermentation broth obtained after 24 hours of fermentation culture was centrifuged at 8000 r / min for 2 minutes, and the supernatant of the fermentation broth was passed through a 0.22 μm water membrane to obtain the fermentation filtrate. The fermentation medium used was formulated as follows: glucose 50 g / L, yeast powder 15 g / L, (NH4)2SO4 12 g / L, K2HPO4 5 g / L, CaCl2·H2O 0.1 g / L, ZnSO4·7H2O 0.5 g / L, MgSO4·7H2O 6 g / L.

[0049] The conditions for detecting organic acids in the fermentation filtrate by high performance liquid chromatography were as follows: chromatographic column Agilent ZORBAXEclipse XDB-C18 (5 μL, 4.6 nm × 250 nm); mobile phase: 0.1 mol / L KH2PO4 solution, the pH of the KH2PO4 solution was adjusted to 2.8 using phosphoric acid; flow rate 0.5 mL / min; detection wavelength 210 nm; injection volume 20 μL; column temperature 20 °C.

[0050] Liquid phase test results Figure 6 and Figure 7 As shown (the purple line is the fermentation broth of the DX-2 strain, and the blue line is the fermentation broth of the S288C strain). It can be seen that L-malic acid, pyruvic acid, and citric acid can be detected in the 24h fermentation broth of the S288C strain and the DX-2 strain. Among them, succinic acid can be additionally detected in the fermentation broth of the DX-2 strain, and the concentration of succinic acid in the fermentation broth is>1g / L. It can be seen that the DX-2 strain can be used as a potential chassis microorganism for the fermentation of short-chain organic acids, especially suitable for the later transformation and production of succinic acid.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A yeast site-directed mutagenesis gene gex1 D , characterized in that, The yeast site-directed mutagenesis gene gex1 D The nucleotide sequence is shown in SEQ ID NO.

1.

2. The yeast site-directed mutation gene gex1 as claimed in claim 1 D , characterized in that, The yeast site-directed mutagenesis gene gex1 D The amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector contains the yeast site-directed mutation gene gex1 according to claim 1 D .

4. A recombinant bacterium, characterized in that: The recombinant bacteria contains the yeast site-directed mutation gene gex1 according to claim 1 D Or the recombinant vector according to claim 3.

5. The recombinant bacterium according to claim 4, characterized in that The starting strain of the recombinant bacteria includes the Saccharomyces cerevisiae S288C strain.

6. The yeast site-directed mutation gene gex1 according to claim 1 or 2 D , Use of the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 or 5 in fermentation in an acidic environment; The pH value of the acidic environment is 2.3-3.

5.

7. The use according to claim 6, characterized in that The acidic environment includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid.

8. The yeast site-directed mutation gene gex1 according to claim 1 or 2 D , Use of the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 or 5 in producing organic acids.

9. The use according to claim 8, characterized in that The organic acid includes one or more of L-malic acid, pyruvic acid, citric acid and succinic acid.

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