A recombinant cerevisiae strain overexpressing Cna1 gene and its construction method and application

Abstract

The present invention discloses a recombinant saccharomyces cerevisiae strain overexpressing a Cna1 gene and a construction method thereof; the recombinant saccharomyces cerevisiae strain overexpresses the Cna1 gene. The nucleotide sequence of the Cna1 gene is SEQ ID No. 1, and the saccharomyces cerevisiae strain is Saccharomyces cerevisiae S288c. The present invention promotes the formation of a biofilm of the recombinant strain by overexpressing the Cna1 gene. At the same time, the present invention utilizes fungal signaling pathway genes for the first time in the production of ethanol based on biofilm immobilization fermentation, thereby increasing the amount of biofilm of the recombinant strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and specifically relates to a recombinant brewer's yeast overexpressing a Cna1 gene, and a construction method and application thereof. Background Art

[0002] Biofilm, also known as biofilm, is a multicellular group formed spontaneously by the cooperation of microorganisms in nature. It is composed of extracellular polymers (EPS) secreted by microbial cells. In the life activities of microorganisms, biofilm plays a very important role. It can not only provide a stable internal environment for them to resist the harsh external environment, but also improve the tolerance of reaction substrates and products, so that the fermentation yield can be increased. This has certain development prospects in the industrial application of biofilm. At the same time, biofilm also undertakes the functions of transport and information transmission between cells and between cells and media. Therefore, improving the stability of biofilm is of great significance to the growth of microorganisms.

[0003] During the formation of microbial biofilms, they are regulated by various signaling pathways within the cells. Under different environmental conditions, these signaling pathways coordinate with each other to regulate the formation of biofilms. In fungi, there are reports on the effects of signaling pathways on biofilm formation, including the effects of the calcineurin signaling pathway on biofilms, but there are very few reports on this in Saccharomyces cerevisiae.

[0004] Traditional immobilized fermentation of Saccharomyces cerevisiae cells, such as Ghasem Najafpour, etc., uses sodium alginate gel to immobilize Saccharomyces cerevisiae cells, shortening the fermentation cycle to 7 hours, and increasing the ethanol yield by 8% compared with free fermentation. However, during the fermentation process, nutrients will diffuse into the gel, and the cells will also emit ethanol and CO2, and the stability of the gel beads themselves is poor and easy to break, resulting in the inability to achieve continuous fermentation efficiently for a long time, so it is not suitable for application in industrial immobilized fermentation to produce ethanol. Biofilm immobilized fermentation is a fermentation mode based on the clustering effect of microorganisms, which is significantly different from the traditional adsorption method. During the biofilm immobilized fermentation process, free cells are gradually adsorbed to the surface of the carrier to form a microbial clustering effect, and the biofilm density is improved, while the overall metabolic activity and resistance to the environment are improved. Therefore, the present invention promotes the formation of biofilm of the recombinant strain by constructing a recombinant Saccharomyces cerevisiae that overexpresses the Cna1 gene, so as to improve the biofilm amount of the recombinant strain in the production of ethanol by biofilm immobilized fermentation. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant brewer's yeast overexpressing the Cna1 gene in view of the deficiencies in the prior art.

[0006] The technical problem that the present invention needs to solve is to provide a method for constructing the above-mentioned recombinant Saccharomyces cerevisiae.

[0007] The final technical problem to be solved by the present invention is to provide the application of the above-mentioned recombinant Saccharomyces cerevisiae.

[0008] In order to solve the above-mentioned first technical problem, the present invention discloses a recombinant Saccharomyces cerevisiae overexpressing the Cna1 gene.

[0009] Among them, the nucleotide sequence of the Cna1 gene is shown as SEQ ID No.1.

[0010] The brewer's yeast is Saccharomyces cerevisiae S288c, which comes from the China Industrial Microbiological Culture Collection Center, and the strain collection number is CICC1308.

[0011] In order to solve the above second technical problem, the present invention discloses a method for constructing the above recombinant Saccharomyces cerevisiae, comprising the following steps:

[0012] (1) Using the genome of Saccharomyces cerevisiae S288c as a template, PCR amplification of the target gene Cna1 was performed to obtain the Cna1 gene fragment;

[0013] (2) connecting the Cna1 gene fragment obtained in step (1) with the purified linear plasmid pYX212 to obtain a recombinant plasmid pYX212-Cna1;

[0014] (3) The recombinant plasmid obtained in step (2) was transformed into E. coli DH5α competent cells, the recombinant plasmid pYX212-Cna1 was extracted and screened, and sequenced for verification.

[0015] (4) The recombinant plasmid extracted in step (3) is transferred into the competent cell of Saccharomyces cerevisiae S288c to construct a recombinant Saccharomyces cerevisiae strain S288c-pCna1 that overexpresses the Cna1 gene.

[0016] In step (1), the primers used for PCR amplification are Cna1-F and Cna1-R, and their primer sequences are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

[0017] In step (2), the linear plasmid pYX212 is a vector obtained by single restriction digestion of the plasmid pYX212 with BamHI enzyme, and its nucleotide sequence is shown in SEQ ID No.4.

[0018] In order to solve the third technical problem mentioned above, the present invention discloses the use of the above recombinant Saccharomyces cerevisiae in promoting biofilm formation.

[0019] Furthermore, the present invention discloses the use of the recombinant Saccharomyces cerevisiae in the fermentation production of ethanol.

[0020] Wherein, the fermentation is separation fermentation or immobilized fermentation, preferably immobilized fermentation.

[0021] The fermentation medium formula is as follows: 60 g / L glucose, 4 g / L peptone, 0.5 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 3 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.05 g / L ferrous sulfate heptahydrate, 0.05 g / L zinc sulfate heptahydrate, and the solvent is water.

[0022] Wherein, the fermentation comprises the following steps:

[0023] (a) 10 μL of glycerol culture S288c (original bacteria) and S288c-pCna1 were added to 5 mL of sterilized YPD liquid medium and cultured overnight for activation;

[0024] (b) transferring the bacterial solution obtained in step (1) to 100 mL of YPD liquid medium at an inoculation ratio of 1% by volume, and continuing to culture at 30° C. and 200 r / min until the OD600 of the bacterial solution is between 0.8 and 1.2;

[0025] (c) The seed solution of step (b) is transferred to 100 mL of fermentation medium at a ratio of 10% of the inoculation volume, which is divided into free fermentation and immobilized fermentation.

[0026] When performing immobilized fermentation: add cotton fiber material as immobilized material to the fermentation medium, add 4g of cotton fiber medium to each shaking flask; ferment at 30°C and 200r / min, and the reaction ends after the glucose is exhausted. Use a sugar meter to measure the residual sugar content in the fermentation liquid at each time period, and use a high-performance gas chromatograph to measure the alcohol content in the fermentation liquid.

[0027] When performing separation fermentation: except that no immobilized material is added, the rest is the same as immobilized fermentation.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] 1. The present invention constructs a recombinant Saccharomyces cerevisiae that overexpresses the Cna1 gene, which promotes the formation of biofilm of the recombinant strain and increases the amount of biofilm of the recombinant strain.

[0030] 2. This invention is the first to utilize fungal signaling pathway genes in the production of ethanol based on biofilm immobilized fermentation.

[0031] 3. The recombinant brewer's yeast constructed by the present invention has the advantages of shortening the fermentation cycle, increasing the ethanol fermentation yield, and improving the fermentation efficiency through biofilm immobilization fermentation, and has good application prospects and value in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0033] Figure 1 Schematic diagram of the construction of the recombinant expression plasmid pYX212-Cna1.

[0034] Figure 2 This is the electrophoresis result of Cna1 gene amplification.

[0035] Figure 3 This is the result of vector linearization electrophoresis.

[0036] Figure 4 Electrophoresis results of recombinant plasmid construction.

[0037] Figure 5 The recombinant bacteria were verified by colony PCR.

[0038] Figure 6 These are the results of the strain growth ability experiment (the top one is the recombinant bacteria, the bottom one is the original bacteria).

[0039] Figure 7 The results of the plate invasion experiment (the left is the original bacteria, and the right is the recombinant bacteria).

[0040] Figure 8 The results of the crystal violet staining experiment in a 96-well plate (the original bacteria are on the left and the recombinant bacteria are on the right).

[0041] Fig. 9 Read the crystal violet plate reader for the 96-well plate.

[0042] Fig.10 The residual sugar data of the original bacteria S288c (W) and the Cna1 gene overexpression bacteria (pCna1) in free fermentation and immobilized fermentation. Among them: W-wild bacteria free fermentation, WI-wild bacteria immobilized fermentation, PCna1-overexpression bacteria free fermentation, pCna1I-overexpression bacteria immobilized fermentation.

[0043] Fig.11 The data of ethanol fermentation products of the original bacteria S288c (W) and the bacteria overexpressing the Cna1 gene (pCna1) in free fermentation and immobilized fermentation. Among them: W-free fermentation of wild bacteria, WI-immobilized fermentation of wild bacteria, PCna1-free fermentation of overexpressed bacteria, pCna1I-immobilized fermentation of overexpressed bacteria. DETAILED DESCRIPTION

[0044] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0045] The S288c-pCna1, pCna1 and +Cna1 mentioned in the present invention have the same meaning, and all refer to a recombinant Saccharomyces cerevisiae strain that overexpresses the Cna1 gene.

[0046] The original bacteria S288c (W), the original strain WT of Saccharomyces cerevisiae, and the glycerol bacteria S288c (original bacteria) mentioned in the present invention have the same meaning, and all refer to the original strain S288c of Saccharomyces cerevisiae.

[0047] Example 1: Molecular transformation of target strains

[0048] 1. Amplify the target gene

[0049] The target gene Cna1 was amplified using the Saccharomyces cerevisiae S288c genome as a template. Based on the NCBI gene sequence, the primers designed for amplification are shown in Table 1. Among them, the primers contain a BamHI restriction enzyme site for subsequent connection with plasmid pYX212.

[0050] Table 1 Experimental primer sequences used for amplification

[0051]

[0052] The PCR used a 25 μL reaction system, 1.5 μL each of primers Cna1-F and Cna1-R; 1.0 μL of template (genome); 1.0 μL of KOD enzyme; 10 μL of dNTP (150 mM); 10 μL of sterile water, each filled into a 25 μL tube, and purified by gel recovery after PCR.

[0053] 2. Construction of recombinant plasmid

[0054] 2.1 Plasmid extraction

[0055] (1) Inoculate E. coli DH5α glycerol stock (containing plasmid pYX212) into liquid LB (kanamycin concentration: 50 μg / mL) and culture at 37°C for 12 hours.

[0056] (2) Collect the cells cultured in the previous step in a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm for 2 min, and remove the supernatant.

[0057] (3) Plasmid pYX212 was extracted according to the instructions of the AxyPrep plasmid extraction kit from Corning Life Sciences, Inc.

[0058] 2.2 Linearization of vector

[0059] In order to connect the target gene Cna1 to the plasmid pYX212, the vector needs to be digested by enzymes. The single enzyme digestion reaction system is (20μL) 0.1% BSA 2μL, 1*M buffer 2μL, BamHI enzyme 1μL, plasmid pYX212 10μL, sterile water 5μL. The digestion reaction is carried out at 37℃ for 30min. After digestion, gel recovery is performed for subsequent experiments.

[0060] 2.3 Construction of plasmid pYX212-Cna1

[0061] The above Cna1 fragment and the purified linear vector were cloned according to Vazyme II One-step cloning kit instructions to obtain the recombinant plasmid pYX212-Cna1. The one-step cloning reaction system is shown in Table 2. After 30 minutes in a 37°C water bath, immediately ice bath for 5 minutes and store at -20°C. The schematic diagram of constructing the recombinant expression plasmid is as follows Figure 1 shown.

[0062] Table 2 One-step cloning reaction system

[0063]

[0064] 3. Transformation and screening of recombinant plasmids

[0065] Take out the competent cells and thaw them on ice until liquid. Add competent cells and plasmid in a ratio of 10:1 to a pre-cooled centrifuge tube. Place on ice for 30 minutes and heat shock in a 42°C water bath for 90 seconds. Cool in an ice box for 3 minutes, add 1 ml of LB and culture on a shaker at 37°C and 200 rpm for 1 hour. Centrifuge to remove most of the supernatant, resuspend the bacterial solution and apply it to a kanamycin resistance plate.

[0066] A single spot on the plate was picked and placed in a shaking tube (50 μg / mL kanamycin), cultured overnight in a 37°C incubator, and then the plasmid pYX212-Cna1 was extracted. The sequencing primers were designed as shown in Table 3.

[0067] Table 3 Experimental primer sequences used for sequencing

[0068]

[0069] After successful sequencing verification, construct the overexpression strain S288c-pCna1. Transform the constructed plasmid into the competent yeast S288c by the same transformation method. Pick a single colony for colony PCR and extract the plasmid for sequencing verification. The primers are the same as in the table above.

[0070] result:

[0071] (1) Figure 2 This is the electrophoresis result of Cna1 amplification: the first lane is a 2000bp DNA marker, the length of gene Cna1 is 1702bp, corresponding to the first 2000bp bright band of the marker. It can be seen that gene Cna1 is amplified successfully and the next step of the experiment can be carried out.

[0072] (2) Figure 3 This is the electrophoresis result of vector linearization: Lane 1 is 10000bp marker, pYX212 plasmid is 9990bp, and after BamHⅠ digestion, it is 9990bp. There are multiple bands before digestion, and a single band after digestion, which corresponds to the first band of 10000bp maker, so pYX212 linearization is successful.

[0073] (3) Figure 4 The electrophoresis results of the recombinant plasmid construction: the first lane is a 10000bp DNA marker, and the third is the electrophoresis diagram of the recombinant plasmid. The size of the recombinant plasmid is 11658bp. It can be seen from the figure that the electrophoresis result is greater than 10000bp, which shows that the recombinant plasmid was successfully constructed.

[0074] (4) Figure 5 Verification of the recombinant bacterial colony PCR: Lane 1 is a 2000bp DNA marker, lanes 2, 3, 4 and 5 are the electrophoresis results of the transformed bacteria using Cna1-check primer colony PCR. If the transformation is successful, the check primer PCR band size will be 1862bp. It can be seen from the figure that the transformed bacteria colony PCR band size is correct, and the S288c-pCna1 strain has been successfully transformed.

[0075] Example 2: Biofilm characterization experiment

[0076] 1. Strain growth ability experiment

[0077] Pick the original strain WT and the single colony of the overexpressed strain of Saccharomyces cerevisiae into 5mL / 50mL YPD, and place the centrifuge tube in a shaker at 30℃ and 200rpm for overnight culture. Dilute the overnight culture strain with sterile ultrapure water to a bacterial concentration (OD600) of 1 and 1×10 -1 , 1×10 -2 , 1×10 -3, take 2 μL of bacterial solution and spot it on the YPD plate, let it stand for 10 minutes, then place it in a 30℃ incubator for 12 hours, and observe the growth ( Figure 6 ).

[0078] 2. Plate invasion assay

[0079] Dilute the cultured strains with sterilized ultrapure water to an OD600nm of 1, then drop 5 μL onto the YPD plate, let it stand for 10 minutes, and then culture it in a 30°C incubator for 5 days. Then rinse the colonies on the YPD plate with a uniform water flow until no cells can be washed off, and observe the residual colonies on the plate ( Figure 7 ).

[0080] 3. Crystal Violet Staining

[0081] In this experiment, crystal violet staining was used to compare the relative expression of biofilms of the two target bacteria. Therefore, we performed biofilm formation verification on these two strains in a 96-well plate ( Figure 8 and Fig. 9 ).

[0082] (1) We cultured the two strains at 30°C and 220 rpm until they reached the logarithmic phase (OD600 was approximately 0.6).

[0083] (2) The bacterial solution was diluted to an OD600 of 0.1, 180 μL of YPD liquid culture medium was added to a 96-well plate, and then 20 μL of the diluted bacterial solution was inoculated into the culture medium. The plates were incubated at 30°C for 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, and 42 h, respectively, to allow the E. coli to form a film on the bottom of the 96-well plate.

[0084] (3) Pour off the YPD liquid culture medium, rinse with PBS 2-3 times, fix the biofilm with methanol for 15 minutes, pour off the methanol and dry it in an oven, and finally add 0.1% crystal violet to stain for 20 minutes.

[0085] (4) Pour off the crystal violet, rinse with PBS, add 33% glacial acetic acid and shake gently on an oscillator for 30 minutes to decolorize and dissolve the crystal violet.

[0086] (5) Read the sample at OD570 using an ELISA reader.

[0087] (6) Repeat the above experimental process and observe the differences in film formation between the two strains in the fermentation medium.

[0088] The original bacteria and the recombinant bacteria constructed by the present invention were subjected to a series of characterization experiments, such as Figure 8 and Fig. 9 The crystal violet staining results showed that the OD of the recombinant bacteria 570The reading was 1.87, while the reading of the original strain was 1.12. The amount of biofilm increased by about 70%. Figure 6 Strain growth ability results and Figure 7 The invasion of the strain plate also clearly showed that the amount of biofilm of the recombinant bacteria increased significantly, which was a manifestation of the increase in the amount of biofilm of the recombinant bacteria compared to the original bacteria.

[0089] Example 3: Fermentation experiment

[0090] (1) Take 10 μL of glycerol bacteria S288c (original bacteria) and S288c-pCna1 and add them to 5 mL of sterilized YPD liquid medium for overnight culture and activation;

[0091] (2) Transfer the bacterial solution obtained in step (1) to 100 mL of YPD liquid medium at an inoculation ratio of 1% by volume, and continue culturing at 30° C. and 200 r / min until the OD600 of the bacterial solution is between 0.8 and 1.2;

[0092] (3) The seed solution of step (2) was transferred to 100 mL of fermentation medium at an inoculation volume ratio of 10%, which was divided into free fermentation and immobilized fermentation.

[0093] When performing immobilized fermentation: add cotton fiber material as immobilized material to the fermentation medium, add 4g of cotton fiber medium to each shaking bottle; ferment at 30°C and 200r / min, and the reaction ends after the glucose is exhausted. Use a sugar meter to measure the residual sugar content of the fermentation liquid at each time period, and use a high-performance gas chromatograph to measure the alcohol content in the fermentation liquid;

[0094] When performing separation fermentation: except that no immobilized material is added, the rest is the same as immobilized fermentation;

[0095] The fermentation medium formula is as follows: 60 g / L glucose, 4 g / L peptone, 0.5 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 3 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.05 g / L ferrous sulfate heptahydrate, 0.05 g / L zinc sulfate heptahydrate, and the solvent is water.

[0096] result:

[0097] The fermentation results are as follows Fig.10 and Fig.11As shown, it can be seen from the fermentation data that immobilized fermentation has a faster sugar consumption rate and higher ethanol yield than free fermentation; the immobilized fermentation cycles of the original strain S288c and the genetically modified strain S288c-pCna1 are 28h and 25h, respectively, the fermentation cycle of the genetically modified strain is shortened by about 3h, and the sugar consumption rate is increased by about 11%; at the end of immobilized fermentation, the ethanol production of the original strain S288c and the genetically modified strain S288c-pCna1 are 23.31g / L and 26.13g / L, respectively, the ethanol production of the genetically modified strain is increased by about 3g / L, and the fermentation efficiency is increased by about 12.10%.

[0098] The present invention provides a recombinant cerevisiae strain overexpressing the Cna1 gene and the ideas and methods for constructing the same. There are many methods and approaches for implementing the technical solution. 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A recombinant Saccharomyces cerevisiae strain overexpressing the Cna1 gene, characterized in that: The recombinant Saccharomyces cerevisiae overexpresses the Cna1 gene; Wherein, the nucleotide sequence of the Cna1 gene is shown in SEQ ID No.1; Wherein, the brewer's yeast is Saccharomyces cerevisiae S288c.

2. The method for constructing the recombinant Saccharomyces cerevisiae according to claim 1, characterized in that: The following steps are involved: (1) Using the genome of Saccharomyces cerevisiae S288c as a template, PCR amplification of the target gene Cna1 was performed to obtain the Cna1 gene fragment; (2) connecting the Cna1 gene fragment obtained in step (1) with the purified linear plasmid pYX212 to obtain the recombinant plasmid pYX212-Cna1; (3) The recombinant plasmid obtained in step (2) was transformed into E. coli DH5α competent cells, the recombinant plasmid pYX212-Cna1 was extracted and screened, and sequenced for verification; (4) The recombinant plasmid extracted in step (3) is transferred into Saccharomyces cerevisiae S288c competent cells to construct a recombinant Saccharomyces cerevisiae strain S288c-pCna1 that overexpresses the Cna1 gene.

3. The method for constructing a recombinant Saccharomyces cerevisiae according to claim 2, characterized in that: In step (1), the primers used for PCR amplification are Cna1-F and Cna1-R, wherein the sequence of Cna1-F is shown in SEQ ID No.2, and the sequence of Cna1-R is shown in SEQ ID No.

3.

4. The method for constructing a recombinant Saccharomyces cerevisiae according to claim 2, characterized in that: In step (2), the linear plasmid pYX212 is a vector obtained by single restriction digestion of the plasmid pYX212 with BamH Ⅰ enzyme, and its nucleotide sequence is shown in SEQ ID No.

4.

5. Use of the recombinant Saccharomyces cerevisiae according to claim 1 in promoting biofilm formation.

6. Use of the recombinant Saccharomyces cerevisiae according to claim 1 in fermentation production of ethanol.

7. The use according to claim 6, characterized in that: The recombinant brewer's yeast is a fermentation strain, and ethanol is prepared by immobilized fermentation at a fermentation temperature of 30° C. and a fermentation speed of 200 r / min.

8. The use according to claim 7, characterized in that: The immobilized fermentation uses cotton fiber material as a medium.

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