A yeast site-directed mutant gene gex1 D , recombinant vector, recombinant bacterium and their applications

Abstract

The present invention provides a yeast site-directed mutant gene gex1<supgt;D< / supgt>, a recombinant vector, a recombinant bacterium and their applications, belonging to the technical field of bioengineering. The nucleotide sequence of the yeast site-directed mutant gene gex1<supgt;D< / supgt> of the present invention is shown in SEQ ID NO.1, and the amino acid sequence encoded by this sequence is shown in SEQ ID NO.2. This amino acid sequence is a mutation of leucine at position 613 of the amino acid encoded by the gex1 gene to serine. The present invention uses site-directed mutagenesis technology to mutate leucine at position 613 of the gex1 gene in the S288C strain to serine. The acid tolerance of the site-directed mutant strain DX-2 is significantly improved, and it can be used as a potential chassis microorganism for fermenting short-chain organic acids, especially suitable for the later transformation and production of succinic acid.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a yeast site-directed mutant gene gex1 D , a recombinant vector, a recombinant bacterium and their applications. Background Art

[0002] Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) is a unicellular organism with a short life cycle, fast reproduction speed, tenacious vitality, relatively easy to culture, and a relatively 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 research 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 selection and breeding of yeasts with excellent acid tolerance are 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, Saccharomyces cerevisiae is inevitably affected by various environmental stresses, mainly including high acid, high sugar, high osmotic pressure, high ethanol, etc. These stresses can damage the cell structure of yeast and cause oxidative denaturation of the structures of important biological macromolecules such as proteins, thereby seriously affecting its growth, metabolism, and physiological functions. In a slightly acidic environment, Saccharomyces cerevisiae has a certain stress and adaptation ability, but when in a highly acidic condition, its activity still faces severe challenges.

[0004] Under acid stress, Saccharomyces cerevisiae adjusts gene expression and metabolism through signal molecules and signal transduction pathways. Currently, related research mainly focuses on the barrier functions of cell walls and cell membranes, maintaining intracellular pH homeostasis through proton pumps such as Pmalp, mitochondrial retrograde pathways, endoplasmic reticulum stress responses, etc. Acid stress is the most common abiotic stress encountered by yeast cells during the fermentation of acidic products such as organic acids, seriously affecting cell growth and metabolism. In order to alleviate the inhibitory effect of acid stress on cell growth and product synthesis, a large amount of neutralizing agents often need to be added during the production process, which not only increases the cost, makes the downstream purification process more complex, but also easily causes environmental pollution. Obtaining acid-tolerant Saccharomyces cerevisiae by studying the acid tolerance mechanism can well solve this problem. However, there is no relevant report on the research of gex1 gene mutation in acid-tolerant fermentation of yeast. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a yeast site-directed mutant gene gex1 D , a recombinant vector, a recombinant bacterium and their applications. The Saccharomyces cerevisiae containing this mutant gene can ferment in an acidic environment and can also ferment to produce succinic acid relative to the standard Saccharomyces cerevisiae strain.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a yeast site-directed mutant gene gex1 D The nucleotide sequence of the yeast site-directed mutant gene gex1 D is shown in SEQ ID NO.1.

[0008] Preferably, the amino acid sequence of the yeast site-directed mutant gene gex1 D is shown in SEQ ID NO.2.

[0009] The present invention provides a recombinant vector, which contains the yeast site-directed mutant gene gex1 D .

[0010] The present invention provides a recombinant bacterium, which contains the yeast site-directed mutant gene gex1 D or the recombinant vector.

[0011] Preferably, the starting strain of the recombinant bacterium includes Saccharomyces cerevisiae S288C strain.

[0012] The present invention also provides the application of the yeast site-directed mutant gene gex1 D , the recombinant vector or the recombinant bacterium in fermentation in an acidic environment.

[0013] Preferably, the pH value of the acidic environment is 2.3 - 3.5.

[0014] Preferably, the acidic environment includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid.

[0015] The present invention provides the application of the yeast site-directed mutant gene gex1 D , the recombinant vector or the recombinant bacterium in the production of organic acids.

[0016] Preferably, the organic acids include one or more of L-malic acid, pyruvic acid, citric acid and succinic acid.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a yeast site-directed mutant gene gex1 D , a recombinant vector, a recombinant bacterium and their applications. Saccharomyces cerevisiae S288C was irradiated with ultraviolet light for mutagenesis, and a strain DX-1 with significantly improved acid resistance was obtained by flow sorting. Its genome was sequenced, and it was found that its gex1 DThe gene undergoes mutation, and leucine (L, Leu) at position 613 is replaced by serine (S, Ser). Using site-directed mutagenesis technology, leucine (L, Leu, TTA) at position 613 of the gex1 gene in the S288C strain is mutated to serine (S, Ser, TCA). The acid tolerance of the site-directed mutant strain DX-2 is significantly improved, and it can be used as a potential chassis microorganism for fermenting short-chain organic acids, especially suitable for the late modification and production of succinic acid. Brief Description of the Drawings

[0019] Figure 1 It is the gel electrophoresis map of gex1 gene amplification.

[0020] Figure 2 It is the map of PY26TEF-gex1 vector.

[0021] Figure 3 It is the verification map of positive clone sequencing.

[0022] Figure 4 It is the morphological map of S288C and DX-2 strains under the microscope (40×). Among them, A is the morphological map of S288C strain, and B is the morphological map of site-directed mutant DX-2 strain.

[0023] Figure 5 It is the OD 600 situation of S288C and DX-2 strains in YPD medium and YPD medium with different pH values.

[0024] Figure 6 It is the high-performance liquid chromatography detection and comparison map of the organic acid content in the 24-hour fermentation broth of S288C and DX-2 strains.

[0025] Figure 7 It is Figure 6 the enlarged map of the succinic acid peak in the high-performance liquid chromatography detection and comparison map. Detailed Embodiments

[0026] The present invention provides a yeast site-directed mutant gene gex1 D The nucleotide sequence of the yeast site-directed mutant gene gex1 D is shown in SEQ ID NO.1. The mutant gene gex1 of the present invention D mutates TTA at positions 1837-1839 of the gex1 gene to TCA. The amino acid sequence encoded by the nucleotide sequence of the present invention is shown in SEQ ID NO.2, and leucine (L, Leu, TTA) at position 613 of the amino acid encoded by the gex1 gene is mutated to serine (S, Ser, TCA).

[0027] The present invention also provides a recombinant vector, and the recombinant vector contains the yeast site-directed mutant gene gex1D The original vector used for the recombinant vector in the present invention is PY26TEF-GPD.

[0028] The present invention also provides a recombinant bacterium, which contains the yeast site-directed mutant gene gex1 D or the recombinant vector. The starting strain of the recombinant bacterium in the present invention includes but is not limited to the Saccharomyces cerevisiae S288C strain.

[0029] The present invention also provides the application of the yeast site-directed mutant gene gex1 D , the recombinant vector or the recombinant bacterium in fermentation in an acidic environment. The pH value of the acidic environment in the present invention is 2.1 - 3.5. The acidic environment in the present invention includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid. The yeast site-directed mutant gene gex1 D can improve the acid tolerance of Saccharomyces cerevisiae.

[0030] The present invention also provides the application of the yeast site-directed mutant gene gex1 D , the recombinant vector or the recombinant bacterium in the production of organic acids. The organic acids in the present invention include one or more of L-malic acid, pyruvic acid, citric acid and succinic acid, and the organic acid is preferably succinic acid.

[0031] In the present invention, unless otherwise specified, all components or reagents or culture media are commercially available products well-known to those skilled in the art.

[0032] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the examples in the present invention. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. All other examples obtained by those of ordinary skill in the art based on the examples in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0033] Example 1 Screening of Mutant Genes

[0034] Saccharomyces cerevisiae (Saccharomyces cerevisiae, S288C Baosai Biology) was cultured in YPD liquid medium (Qingdao Haibo Biology) to obtain a culture solution in the logarithmic phase; the culture solution in the logarithmic phase was centrifuged, the supernatant was discarded, and cells were obtained. The cells were serially diluted with physiological saline to obtain dilution solutions with different dilution multiples. 100 μL of each dilution solution was taken and spread on a YPD solid medium (Qingdao Haibo Biology) plate, and cultured upside down at 30 °C. The dilution gradient was determined based on the number of colonies being 100 - 300 for subsequent experiments, and the determined dilution gradient was 10 -4 .

[0035] Preheat the ultraviolet lamp in advance for 30 min. Take 3 mL of bacterial liquid with dilution gradients of 10 -4 and place it in a 60 mm petri dish for ultraviolet treatment for 0 s, 30 s, 60 s, 90 s, 120 s, and 150 s. Set 3 parallel experiments for each treatment time. Take 100 μL of the mutagenized bacterial liquid and evenly coat it on the YPD solid medium plate, and incubate it upside down at 30 °C for 24 h, then perform colony counting. The ultraviolet treatment time with a lethality rate of about 90% is used as the subsequent ultraviolet mutagenesis treatment time, and the determined ultraviolet mutagenesis time is 120 s.

[0036] Using the determined ultraviolet mutagenesis time above, Saccharomyces cerevisiae (S288C) was mutagenized by ultraviolet irradiation to obtain mutant strains. Conduct an acid tolerance test on the obtained mutant strains: Place the original strain and the mutant strain in media with different pH values for cultivation, and screen the strain DX-1 with significantly improved acid tolerance according to the OD value. Genome sequencing was performed on strain DX-1, and it was found that its gex1 gene mutated, and leucine (L, Leu, TTA) at the 613th position was replaced by serine (S, Ser, TCA).

[0037] Example 2 Amplification of gex1 sequence

[0038] 1. DNA extraction: Streak-culture Saccharomyces cerevisiae (Saccharomyces cerevisiae, S288C, Baosai Biology) on YPD solid medium (Qingdao Haibo Biology), pick a single colony, inoculate it into YPD liquid medium, and after culturing for 24 h, use the yeast genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the DNA of Saccharomyces cerevisiae.

[0039] 2. Primer design: According to the whole-genome data of the S288C strain in the National Center for Biotechnology Information (NCBI) database, design primers gex1-F and gex1-R to amplify the gex1 gene (introducing restriction enzyme sites SacII and PacI 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).

[0040] 3. PCR Amplification: Using the genomic DNA of the S288C strain extracted above as a template, the gex1 gene was amplified with the primers designed above. The reaction system for PCR amplification is as follows: The total system is 50 μL, including 2 μL of DNA template, 1 μL each of gex1-F and gex1-R primers, 4 μL of dNTP, 5 μL of PCR Buffer (with Mg 2+ )), 0.25 μL of Taq polymerase, and 36.75 μL of ddH2O. The PCR amplification program is as follows: Pre-denaturation at 94°C for 3 min, 35 cycles (denaturation at 94°C for 30 s, annealing at 55°C for 2 min, and extension at 72°C for 2 min). After the cycles are completed, extend at 72°C for 10 min and store at 4°C. The amplified sequence was verified by agarose gel electrophoresis. The position of the gel band is between 1500 - 2000 bp and is closer to 2000 bp, which is basically consistent with the 1862 bp of the gex1 gene ( Figure 1 ).

[0041] Example 3 Ligation, Transformation, and Identification of Cloning Vectors

[0042] In the high-fidelity enzyme PCR tube (system is 50 μL) after the PCR amplification in Example 2, 3 μL of dNTP and 0.5 μL of Taq enzyme were directly added and the reaction continued at 72°C for 10 - 20 min. Then, the amplified gene fragment was purified and recovered using a gel extraction kit (TaKaRa). The recovered product was ligated with the pMD19-T cloning vector (TaKaRa), and the ligated vector was transformed into Escherichia coli DH5α competent cells using the calcium chloride method. Positive transformants were screened using blue-white screening, and the monoclonal recombinant plasmid pMD19-T-gex1 was extracted using a plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.) and sequenced. The plasmid sequencing results showed that the obtained gex1 sequence was correct and had 100% homology with the gene sequence in the gene bank (NCBI Reference Sequence: NC 001135.5).

[0043] Example 4 Construction of Recombinant Expression Vector PY26TEF-gex1

[0044] The above-constructed pMD19-T-gex1 and PY26TEF-GPD vectors (Beijing Zhuangmeng International Biotechnology Co., Ltd.) were double digested with the rapid restriction enzyme SacII and PacI from Beyotime Biotechnology, and the target digested products were purified and recovered using a gel extraction kit (TaKaRa). 1 μL of T4 ligase from Beyotime Biotechnology was used to ligate the digested products. The ligation products were transformed into Escherichia coli DH5α and inoculated onto a resistance plate containing Amp for screening. After 12 h, monoclonal colonies were picked for colony PCR identification. The positive clones were picked and cultured in LB liquid medium containing Amp resistance at 37 °C and 160 r / min. After 12 h, the bacteria were collected by centrifugation, and the plasmids were extracted. Sequencing verification was performed using the primer sequences gex1-F (SEQ ID NO.3) and gex1-R (SEQ ID NO.4) (Sangon Biotech Co., Ltd.). The results showed that the gex1 sequence was correct, and the constructed recombinant expression vector was named PY26TEF-gex1 ( Figure 2 ).

[0045] Example 5 Construction of Site-Directed Mutant Strains

[0046] 1. Design of mutant primers: Using PY26TEF-gex1 as the starting vector, according to the principle of inverse overlapping extension PCR, a pair of mutant primers gex1-F’ and gex1-R’ were designed by a one-step method to introduce mutant sites. The underlined letters represent the mutant sites, corresponding to the 613th amino acid. The primers are as follows: gex1-F’: 5’-CATAATCGAAGTAACAAGAAATCAGGATGTTGA-3’ (SEQ ID NO.5), gex1-R’: 5’-TCAACATCCTGATTTCTTGTTACTTCGATTATG-3’ (SEQ ID NO.6).

[0047] 2. PCR amplification: Using the PY26TEF-gex1 vector as the template, PCR amplification was performed with the above-constructed mutant primers. The reaction system for PCR amplification was as follows: The total volume was 50 μL, including 2 μL of template, 2 μL each of gex1D-F’ and gex1D-R’, 25 μL of 2×Phanta Master Mix, and 19 μL of ddH2O. The PCR amplification program was as follows: Pre-denaturation at 94 °C for 5 min, 30 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 5 min), and then extension at 72 °C for 5 min after the cycle ended.

[0048] 3. Treatment of amplified products and transformation of strains: The above PCR products were treated with DpnⅠ to eliminate the original vector template. The DpnⅠ reaction system was as follows: The total volume was 50 μL, including 1 μL of DpnⅠ, 5 μL of Quick cut Buffer, and 44 μL of PCR products. The DpnⅠ reaction procedure was: 37°C for 2 h, 75°C for 15 min, and 4°C ∞. The purified product PY26TEF-gex1 D was transformed into DH5α and inoculated onto a resistance plate containing Amp for screening. Positive clones were selected for sequencing verification to determine whether mutations occurred. After Figure 3 sequencing, the results showed that the leucine (L, Leu, TTA) at position 613 of the gex1 gene in the amplified product using the mutant primer was mutated to serine (S, Ser, TCA).

[0049] The verified correct PY26TEF-gex1 D recombinant vector was transformed into Saccharomyces cerevisiae S288C using the Quick Yeast Competent Cell Preparation Kit of Zhuangmeng Biotechnology Co., Ltd. Transformants were screened using SD-URA yeast defective medium (Beijing Coolaber Technology Co., Ltd.) to obtain the site-directed mutant strain DX-2.

[0050] Example 6 Verification experiment on the acid tolerance of strains

[0051] Saccharomyces cerevisiae S288C and the site-directed mutant strain DX-2 were activated on YPD plates, and then single colonies were respectively picked and inoculated into 10 mL YPD shaking tubes (liquid loading volume 3 mL). The cell morphology was observed under a microscope (40×) ( Figure 4 ). The cells were cultured until the OD 600 reached a range of 1 - 1.5, and then inoculated 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 loading volume 50 mL). The cultures were carried out at 30°C and 200 r / min, and the growth of different cells was observed. Samples were taken to detect the OD value of the cells when the cells became significantly turbid (Table 1). Among them, the above YPD medium with different pH values was adjusted using L-malic acid,

[0052] As can be seen from Table 1, Saccharomyces cerevisiae S288C and the site-directed mutant strain DX-2 grew well within 20 h in YPD medium and YPD medium with pH = 5.3, pH = 4.1, and pH = 3.5, showing no significant difference. When 2.3 < pH < 3.5, the growth of the site-directed mutant strain DX-2 was significantly better than that of the S288C strain. From Figure 4Microscopic photographs showed that the site-directed mutant strain DX-2 grew in clusters, while the S288C strain grew as single colonies, with significant differences in morphology.

[0053] Table 1 Growth of S288C and DX-2 strains in media with different pH values

[0054]

[0055] Example 7 Experiment for verifying the acid tolerance of strains to different organic acids

[0056] Saccharomyces cerevisiae S288C and the site-directed mutant strain DX-2 were activated on YPD plates, and then single colonies were picked and inoculated into 10 mL shaking flasks containing YPD medium (liquid volume: 3 mL). The OD value was measured until it reached the range of 1 - 1.5, and then they were inoculated into 250 mL YPD medium and YPD medium with pH = 3 (liquid volume: 50 mL) in shaking flasks at an inoculation amount of 1 v / v%. Samples were taken after culturing at 30 °C and 200 r / min for 36 h, and the OD was measured. The test results are shown as follows. Figure 5 Among them, the YPD medium with pH = 3 was adjusted with L-malic acid, citric acid, lactic acid, succinic acid, and pyruvic acid respectively.

[0057] It can be seen from Figure 5 that the site-directed mutant strain DX-2 is far superior to Saccharomyces cerevisiae S288C in terms of acid tolerance, and shows different tolerances and preferences for different organic acids, and grows well in the environments of L-malic acid, succinic acid, and pyruvic acid.

[0058] Example 8 Comparative experiment on organic acid production by strains

[0059] The site-directed mutant strain DX-2 and the S288C strain obtained in Example 5 were respectively subjected to 250 mL shaking flask fermentation culture, and the types and contents of organic acids in the supernatant of the fermentation broth were detected by high performance liquid chromatography (HPLC). The required culture medium, culture conditions, and detection methods are as follows:

[0060] Preparation of seed liquid: The S288C strain and the site-directed mutant strain DX-2 were respectively prepared into glycerol tube strains for preservation. The preserved strains were respectively inoculated into YPD solid medium for activation, and single colonies of the activated strains were inoculated into 15 mL shaking flasks containing YPD liquid medium (liquid volume: 3 mL), and cultured at 30 °C and 220 r / min for 10 - 20 h until the logarithmic growth phase to obtain the seed liquid.

[0061] Flask fermentation culture: Take 1.5 mL of the seed liquid and inoculate it into a 250 mL baffled flask containing fermentation medium (the liquid loading is 50 mL) at an inoculation amount of 3% by volume. Ferment at 30 °C and 220 r / min. During the fermentation process, sample and detect the residual sugar at regular intervals, and add 80% glucose mother liquor to control the glucose content at 20 g / L - 30 g / L according to the sugar consumption. The fermentation broth obtained after 24 h of fermentation is centrifuged at 8000 r / min for 2 min, and the supernatant of the fermentation broth is filtered through a 0.22 μm water-based membrane to obtain the fermentation filtrate. The formula of the fermentation medium used is: 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.

[0062] Conditions for detecting organic acids in the fermentation filtrate by high performance liquid chromatography: Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 (5 μL, 4.6 nm × 250 nm); Mobile phase: 0.1 mol / L KH2PO4 solution, and the pH of the KH2PO4 solution is adjusted to 2.8 with phosphoric acid; Flow rate 0.5 mL / min; Detection wavelength 210 nm; Injection volume 20 μL; Column temperature 20 °C.

[0063] Liquid phase detection results Figure 6 and Figure 7 as shown (the purple line is the fermentation broth of DX-2 strain, and the blue line is the fermentation broth of S288C strain). It can be seen that L-malic acid, pyruvic acid and citric acid can be detected in the 24 h fermentation broth of S288C strain and DX-2 strain. Among them, succinic acid can be additionally detected in the fermentation broth of DX-2 strain, and the concentration of succinic acid in the fermentation broth > 1 g / L. It can be seen that DX-2 strain can be used as a potential chassis microorganism for fermenting short-chain organic acids, especially suitable for the later transformation and production of succinic acid.

[0064] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A yeast site-directed mutagenesis gene gex1 D , use of a recombinant vector or a recombinant bacterium in fermentation in an acidic environment; the pH value of the acidic environment is 2.3-3.5; The yeast site-directed mutagenesis gene gex1 D The nucleotide sequence is shown in SEQ ID NO.1; The yeast site-directed mutagenesis gene gex1 D The amino acid sequence is shown in SEQ ID NO.2; The recombinant vector contains the yeast site-directed mutation gene gex1 D ; The recombinant bacteria contains the yeast site-directed mutation gene gex1 D or the recombinant vector; The starting strain of the recombinant bacteria includes the Saccharomyces cerevisiae S288C strain.

2. The use according to claim 1, characterized in that The acidic environment includes one or more of L-malic acid, citric acid, lactic acid, succinic acid and pyruvic acid.

3. A yeast site-directed mutagenesis gene gex1 D , the use of recombinant vectors or recombinant bacteria in producing organic acids; The organic acid is one or more of L-malic acid, pyruvic acid, citric acid and succinic acid; The yeast site-directed mutagenesis gene gex1 D The nucleotide sequence is shown in SEQ ID NO.1; The yeast site-directed mutagenesis gene gex1 D The amino acid sequence is shown in SEQ ID NO.2; The recombinant vector contains the yeast site-directed mutation gene gex1 D ; The recombinant bacteria contains the yeast site-directed mutation gene gex1 D or the recombinant vector; The starting strain of the recombinant bacteria includes the Saccharomyces cerevisiae S288C strain.

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