Yeast high temperature responsive synthetic promoter and its application

Abstract

The present invention discloses a yeast high-temperature responsive synthetic promoter and its application. The synthetic promoter is UX-Y-Z-pTDH3-core, wherein X, Y, and Z are any one of UAS-104, UAS-26, and UAS-30, respectively. The synthetic promoter is an inducible / responsive promoter, which can have a lower expression level in the early stage under simulated industrial gradient temperature increase conditions without cooling water, and a higher expression level after heating, and different synthetic promoters have different expression levels and have good response multiples. Therefore, the early growth burden can be effectively reduced, and the expression of downstream genes can be increased after heating in response to high-temperature fermentation conditions. The high-response synthetic promoter constructed by the present invention, which has different strengths after heating, can be used in various fields to increase the expression level of specific genes during high-temperature fermentation and reduce the early growth burden, laying the foundation for realizing the industrial application of recombinant strains.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology and relates to a synthetic promoter and application thereof, and in particular to a yeast high temperature responsive synthetic promoter and application thereof. Background Art

[0002] As the first model eukaryote to have its entire genome sequenced, Saccharomyces cerevisiae boasts advantages such as a clear genetic background, strong genetic manipulation, rapid growth and reproduction, and excellent fermentation performance. Since its inception in production, it has been one of the most important foundational cells in metabolic engineering. As a cell factory for the production of a variety of raw materials, Saccharomyces cerevisiae is commonly used in the production of pharmaceuticals such as human insulin, hepatitis vaccines, and human papillomavirus vaccines; bulk chemicals such as ethanol, lactic acid, and succinic acid; biofuels such as fatty acids and hydrocarbons; and fine chemicals such as terpenes and polyketides.

[0003] The promoter sequence determines the expression intensity and expression pattern of downstream genes. It is a core regulatory element of synthetic biology that drives gene expression, regulates gene circuits, and constructs high-performance cell factories. The endogenous promoters of Saccharomyces cerevisiae can be divided into two categories: constitutive and inducible / responsive. The expression level of constitutive promoters is relatively constant, while inducible / responsive promoters respond to certain specific physical and chemical conditions such as high temperature, oxidative stress, chemical toxic substances, osmotic pressure, etc., or are related to the growth stage of the strain, and start the expression of downstream genes during a specific growth period. Currently, most promoters used to improve the high-temperature fermentation ability of Saccharomyces cerevisiae are constitutive promoters, but constitutive promoters also have a high expression level in the early stage of fermentation. Using constitutive promoters to express some genes may affect the normal growth of the strain in the early stage. Compared with traditional constitutive promoters, inducible / responsive promoters have low expression in the early stage of fermentation when growth is rapid, and the expression increases under stress conditions after the fermentation temperature rises, which can effectively reduce the metabolic burden of early growth and improve the fermentation ability of the strain. In view of this, the present invention is proposed. Summary of the Invention

[0004] To overcome the shortcomings and deficiencies of the prior art, the present invention provides a synthetic yeast high-temperature-responsive promoter and its application. This synthetic promoter is UX-YZ-pTDH3-core (abbreviated as pUX-YZ), where X, Y, and Z are UAS sequences at positions 1, 2, and 3, respectively. The UAS sequences are UAS-104, UAS-26, and UAS-30. UAS-104 is the upstream activator element located in the -334 to -140 region of the native Saccharomyces cerevisiae promoter pHSP104; UAS-26 is the upstream activator element located in the -509 to -291 region of pHSP26; and UAS-30 is the upstream activator element located in the -588 to -255 region of pHSP30. pTDH3-core is the core promoter element located in the -149 to -1 region of the pTDH3 promoter. This synthetic promoter is used to increase ethanol production in Saccharomyces cerevisiae under simulated industrial conditions without a cooling water gradient.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a yeast high-temperature-responsive synthetic promoter, named UX-YZ-pTDH3-core, abbreviated as pUX-YZ, wherein X is any one of UAS-104, UAS-26 and UAS-30, Y is any one of UAS-104, UAS-26 and UAS-30, and Z is any one of UAS-104, UAS-26 and UAS-30; UAS-104 is shown as bp 1-195 in SEQ ID NO: 1, UAS-26 is shown as bp 186-404 in SEQ ID NO: 2, UAS-30 is shown as bp 423-756 in SEQ ID NO: 3, and pTDH3-core is shown as bp 825-973 in SEQ ID NO: 4.

[0007] Further, the yeast high temperature responsive synthetic promoter is pU104-30-26, pU104-30-30, pU26-26-30, pU26-30-26, pU26-30-30, pU30-104-26, pU30-104-30, pU30-26-30, pU30-30-26, pU30-30-30, pU104-104-26, pU104-104-30, pU104-26-26, pU26-104-26, pU26-104-30, pU26-26-104, pU26-26-26 or pU30-26-26.

[0008] The present invention also provides a biomaterial related to the yeast high-temperature-responsive synthetic promoter, which is any one or more combinations of the following biomaterials:

[0009] (a) an expression cassette containing the yeast high-temperature-responsive synthetic promoter;

[0010] (b) a recombinant vector containing the yeast high-temperature-responsive synthetic promoter;

[0011] (c) a recombinant vector containing the expression cassette described in (a);

[0012] (d) a recombinant strain containing the above yeast high temperature responsive synthetic promoter;

[0013] (e) a recombinant strain containing the expression cassette described in (a);

[0014] (f) A recombinant strain containing the recombinant vector described in (b) or (c).

[0015] Furthermore, the expression cassette in (a) further contains a target gene, and the target gene is operably linked to the yeast high temperature responsive synthetic promoter.

[0016] Furthermore, the recombinant vectors in (b) and (c) can be any yeast expression plasmid, preferably any Saccharomyces cerevisiae expression plasmid.

[0017] Furthermore, the host strain corresponding to the recombinant strains in (d), (e), and (f) is selected from yeast, etc.; the yeast includes Saccharomyces cerevisiae; specifically, Saccharomyces cerevisiae BY4741 or industrial Saccharomyces cerevisiae CY.

[0018] The present invention provides the use of the yeast high-temperature responsive synthetic promoter or its related biological materials in inducing the expression of a target gene in yeast under high-temperature high-response induction. Furthermore, the use of the yeast high-temperature responsive synthetic promoter or its related biological materials in inducing the expression of a target gene in yeast under high-temperature high-response induction in Saccharomyces cerevisiae.

[0019] Furthermore, the yeast high temperature responsive synthetic promoter or its related biological materials are used in the preparation of polypeptides and proteins.

[0020] Furthermore, the protein includes fluorescent protein, etc., preferably includes green fluorescent protein.

[0021] Furthermore, the yeast high temperature responsive synthetic promoter or its related biological materials are used to improve the ethanol fermentation ability of yeast; preferably, they are used to improve the ethanol fermentation ability of Saccharomyces cerevisiae.

[0022] The present invention has the following advantages and effects compared to the prior art:

[0023] (1) The synthetic promoter constructed by the present invention can have a low expression level in the early stage under the conditions of simulated industrial gradient temperature increase without cooling water, and a higher expression level after the temperature is increased. Different synthetic promoters have different expression levels and have good response multiples.

[0024] (2) Compared with existing constitutive promoters, the synthetic promoter improved by the present invention is an inducible / responsive promoter, which can effectively reduce the early growth burden, respond to high temperature fermentation conditions, and increase the expression of downstream genes after the temperature is raised. In a 96-well deep-well plate, YPD (20% Glucose) was heated at a gradient of 1°C / h, 30→37°C from 16h to characterize the transcription factor gene containing the artificial heat-responsive promoter pU30-30-26 under anaerobic conditions. Srb2 Compared to a control strain containing an empty plasmid, the industrial Saccharomyces cerevisiae CY strain harboring a single-copy plasmid showed a 20% increase in sugar consumption and a 55% increase in ethanol production. The highly responsive synthetic promoter constructed by this invention, which exhibits varying strengths upon temperature increase, can be used in various fields to increase the expression of specific genes during high-temperature fermentation and reduce the burden of early growth, laying the foundation for the industrial application of recombinant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthetic promoter constructed in the present invention.

[0026] Figure 2 The fluorescence ratio OD of each promoter in the haploid model of Saccharomyces cerevisiae BY4741 16 hours before and 24 hours after heating 600 The numbers between the bars represent the fluorescence ratio OD before and after heating. 600 The ratio of values.

[0027] Figure 3 The fluorescence ratio OD of each promoter in the industrial Saccharomyces cerevisiae CY 16 hours before and 24 hours after heating 600 The numbers between the bars represent the fluorescence ratio OD before and after heating. 600 The ratio of values.

[0028] Figure 4 The ethanol production and sugar consumption of each recombinant strain in industrial Saccharomyces cerevisiae CY are shown. DETAILED DESCRIPTION

[0029] The present invention is described in further detail below with reference to the examples and accompanying drawings, but the present invention is not limited thereto. Experimental methods in the following examples, where specific experimental conditions are not specified, generally followed conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0030] Example 1 Preparation of recombinant plasmid

[0031] The schematic diagram of the construction of synthetic promoter is shown in Figure 1 As shown, the synthetic promoter is UX-YZ-pTDH3-core (abbreviated as pUX-YZ), where X, Y, and Z are UAS sequences at positions 1, 2, and 3, respectively, and pTDH3-core (pTDH3 core promoter) is the -149 to -1 core promoter element in the promoter pTDH3. The upstream activating element UAS-104 of the natural promoter pHSP104 of Saccharomyces cerevisiae located in the -334 to -140 region is shown as 1-195 bp in SEQ ID NO: 1, the upstream activating element UAS-26 located in the -509 to -291 region of pHSP26 is shown as 186-404 bp in SEQ ID NO: 2, the upstream activating element UAS-30 located in the -588 to -255 region of pHSP30 is shown as 423-756 bp in SEQ ID NO: 3, and the -149 to -1 core promoter element pTDH3-core in the promoter pTDH3 is shown as 825-973 bp in SEQ ID NO: 4.

[0032] The preparation of the recombinant plasmid containing the synthetic promoter UX-YZ-pTDH3-core comprises the following steps:

[0033] (1) Recombinant plasmid p414_Tcyc1-pTDH3-yeGFP-Tpgk1, the nucleotide sequence of which is shown in SEQ ID NO: 4, wherein 1-275 bp: cyc1 terminator (Tcyc1), 276-973 bp: TDH3 promoter (pTDH3), 974-1702 bp: green fluorescent protein gene (yeGFP), 1703-2002 bp: pgk1 terminator (Tpgk1), 2003-7591 bp: p414-Vector.

[0034] (2) The pTDH3 in the recombinant plasmid p414_Tcyc1-pTDH3-yeGFP-Tpgk1 was replaced with the promoters pHSP104 (SEQ ID NO: 1), pHSP26 (SEQ ID NO: 2), and pHSP30 (SEQ ID NO: 3) by Gibson assembly, respectively. The recombinant plasmids p414_Tcyc1-pHSP104-yeGFP-Tpgk1, p414_Tcyc1-pHSP26-yeGFP-Tpgk1, and p414_Tcyc1-pHSP30-yeGFP-Tpgk1 were obtained and used to determine the activities of the promoters pHSP104, pHSP26, and pHSP30.

[0035] (3) The pTDH3 in the recombinant plasmid p414_Tcyc1-pTDH3-yeGFP-Tpgk1 was replaced with the synthetic promoter UX-YZ-pTDH3-core (abbreviated as pUX-YZ) by Golden Gate assembly to obtain the recombinant plasmid p414_Tcyc1-pUX-YZ-yeGFP-Tpgk1, which was used to determine the activity of the synthetic promoter. The synthetic promoter pU30-30-26 is used as an example, which is the abbreviation of U30-30-26-pTDH3-core, where 30 represents UAS-30 and 26 represents UAS-26; the same applies to the others.

[0036] Example 2 Preparation of recombinant strains

[0037] The recombinant plasmid containing the promoter prepared in Example 1 was transformed into Saccharomyces cerevisiae competent cells by electroporation to obtain a recombinant strain. The specific implementation method is as follows:

[0038] Preparation of Saccharomyces cerevisiae competent cells: Pick a single clone of haploid model Saccharomyces cerevisiae BY4741 or industrial Saccharomyces cerevisiae CY strain, inoculate it into 10 mL YPD liquid medium, and culture it in a shaker at 30°C and 220 rpm for 15 hours; inoculate the activated strain into 50 mL YPD liquid medium so that the starting OD 600The value is between 0.1-0.2, and then placed in a 30℃ shaker at 220 rpm for 4 hours to allow the strain to complete at least two divisions and improve the electroporation efficiency; centrifuge at 5000 rpm for 5 minutes in a centrifuge precooled to 4℃, and remove the supernatant; add 25 mL of precooled sterile water, resuspend the cells, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and repeat once; add 2 mL of precooled sorbitol, resuspend the cells, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and repeat once; add 400 μL of precooled sorbitol, resuspend the cells, and prepare Saccharomyces cerevisiae competent cells (haploid model Saccharomyces cerevisiae BY4741 competent cells and industrial Saccharomyces cerevisiae CY competent cells).

[0039] Among them, industrial S. cerevisiae CY is disclosed in the document “Xu K, Qin L,Bai W, Wang X, Li F, Ren S, GaoX, Chen B, Tong Y, Li J, Li BZ, Yuan YJ, Li C.Multilevel defense system (MDS) relieves multiple stresses for economicallyboosting ethanol production ofindustrial Saccharomyces cerevisiae. ACS Energy Letters 2020, 5:572-582.”

[0040] Preparation of recombinant strains: Take 50 μL of the prepared Saccharomyces cerevisiae competent cells in a 1.5 mL sterile centrifuge tube, add 5 μL of the recombinant plasmid, mix with the competent cells, transfer the mixture to a pre-cooled electroporation cup, and gently tap the tube wall to mix; place the electroporation cup into the electroporator and perform electroporation with the electroporation program of 1.5 kV, 200 W, 25 uF, and 5 ms; immediately after the electroporation, add 1 mL of YPD liquid medium, transfer all the liquid to a 1.5 mL sterile centrifuge tube, and incubate at 30°C in a shaker at 220 rpm for 2 hours; centrifuge at 4000 rpm for 5 min to remove the medium, resuspend with 100 μL of 1M sorbitol and coat the plate; incubate upside down at 30°C for 48 hours to obtain recombinant strains (haploid model Saccharomyces cerevisiae BY4741 recombinant strain and industrial Saccharomyces cerevisiae CY recombinant strain).

[0041] Example 3 Activity test in haploid yeast BY4741

[0042] The activity test method of the synthetic promoter in the haploid model of Saccharomyces cerevisiae BY4741 is as follows: take a 96-well deep-well plate, add 900 μL of YPD liquid medium containing nourseothricin (80 mg / L) and glucose (20 g / L) to each well, and then pick three (three parallel experiments) monoclonal strains in the deep-well plate containing the medium and activate them for 24 hours. 600 Each strain was transferred to a fresh 900 μL YPD liquid medium containing nourseoin (80 mg / L) and glucose (20 g / L) in a deep-well plate at 0.1 and activated again for 24 hours. The activated yeast was centrifuged at 4000 rpm for 4 minutes, the supernatant was removed, and the yeast was resuspended in sterile water to measure the OD. 600 The cells were centrifuged at 4000 rpm for 4 minutes, the supernatant was removed, and an appropriate amount of YPD liquid medium containing glucose (40 g / L) was added to resuspend the cells. 600 Each strain was inoculated at 0.1 into a fresh 900 μL YPD liquid medium containing nourseoin (80 mg / L) and glucose (40 g / L) in a 96-well deep-well plate. An airtight sealing film was used to simulate anaerobic ethanol fermentation conditions. Starting from the 16th hour of culture, the shaking table temperature was increased by 1°C every hour to simulate the gradient temperature increase conditions of industrial fermentation without cooling water. When the temperature was increased from 30°C to 37°C, the culture was continued for 24 hours. Before the 16-hour temperature rise and after the 24-hour temperature rise, appropriate amounts of bacterial culture were collected and centrifuged at 4000 rpm for 4 minutes. The supernatant was removed and the cells were resuspended in 200 μL of sterile purified water and transferred to a 96-well Corning® 96-well transparent flat-bottom black microplate. The OD value of the bacterial culture was determined using a Spark® multi-function microplate reader (enzyme reader) with the temperature set to 30°C, the filter excitation wavelength of 485 nm, the excitation bandwidth of 20 nm, the emission wavelength of 535 nm, the emission bandwidth of 25 nm, and the gain of 50. 600 The OD value and fluorescence value were calculated by 200 μL sterile purified water. 600 The OD value and fluorescence value were used as reference to determine the OD value of the strain. 600 Value and fluorescence value, OD of strain 600 The values ​​were multiplied by the same proportional coefficient, based on the fluorescence values / OD measured at 16 hours and 24 hours. 600 The experimental results are shown in the following figure. Figure 2 As shown in the figure, the natural constitutive promoter pADH1 (from Saccharomyces cerevisiae) and the natural responsive promoters pHSP104, pHSP26, and pHSP30 were used as controls in this experiment. Figure 2 As can be seen in the fluorescence value / OD 600The expression strength of the promoter was evaluated by the expression value. The expression strength of the constitutive promoter pADH1 was lower after heating than before heating, while the expression strength of the natural responsive promoters pHSP104, pHSP26, and pHSP30 was higher after heating than before heating, which were 1.08, 3.12, and 2.63 times higher than before heating, respectively. The expression strength of the artificial promoters after heating was higher than before heating, with different multiples, including pU104-30-26, pU104-30-30, pU26-26-30, and pU26- The response multiples of pU30-26, pU26-30-30, pU30-104-26, pU30-104-30, pU30-26-30, pU30-30-26, and pU30-30-30 were 3.50, 4.44, 3.03, 6.36, 4.00, 3.65, 3.41, 4.23, 5.32, and 6.02, respectively. They have good response capabilities and can be used to efficiently start the expression of downstream genes of model Saccharomyces cerevisiae BY4741 after heating.

[0043] Example 4 Activity test of industrial yeast CY

[0044] The activity test method of the synthetic promoter in industrial Saccharomyces cerevisiae CY was as follows: 900 μL of YPD liquid medium containing nourseothricin (80 mg / L) and glucose (20 g / L) was added to each well of a 96-well deep-well plate. Three (three parallel experiments) monoclonal strains were then picked and placed in the deep-well plate containing the medium. The strains were activated for 24 hours and then the starting OD was used to determine the activity of the synthetic promoter. 600 Each strain was transferred to a fresh 900 μL YPD liquid medium containing nourseoin (80 mg / L) and glucose (20 g / L) in a deep-well plate at 0.1 and activated again for 24 hours. The activated yeast was centrifuged at 4000 rpm for 4 minutes, the supernatant was removed, and the yeast was resuspended in sterile water to measure the OD. 600 The cells were centrifuged at 4000 rpm for 4 minutes, the supernatant was removed, and an appropriate amount of YPD liquid medium containing glucose (100 g / L) was added to resuspend the cells. 600Each strain was transferred to a fresh 900 μL YPD liquid medium containing nourseoin (80 mg / L) and glucose (100 g / L) in a 96-well deep-well plate at a concentration of 0.1. An airtight sealing film was used to simulate anaerobic ethanol fermentation conditions. Starting from the 16th hour of culture, the shaking table temperature was increased by 1°C every hour to simulate the gradient temperature increase conditions of industrial fermentation without cooling water. When the temperature was increased from 30°C to 37°C, the culture was continued for 24 hours. Before the 16-hour temperature rise and after the 24-hour temperature rise, appropriate amounts of bacterial suspension were collected and centrifuged at 4000 rpm for 4 minutes. The supernatant was removed and the cells were resuspended in 200 μL of sterile purified water and transferred to a 96-well Corning® 96-well transparent flat-bottom black microplate. The OD value of the bacterial suspension was determined using a Spark® multi-function microplate reader (enzyme reader) with the temperature set to 30°C, the filter excitation wavelength of 485 nm, the excitation bandwidth of 20 nm, the emission wavelength of 535 nm, the emission bandwidth of 25 nm, and the gain of 50. 600 The OD value and fluorescence value were calculated by 200 μL sterile purified water. 600 The OD value and fluorescence value were used as reference to determine the OD value of the strain. 600 Value and fluorescence value, OD of strain 600 The values ​​were multiplied by the same proportional coefficient, based on the fluorescence values / OD measured at 16 hours and 24 hours. 600 The experimental results are shown in the following figure. Figure 3 As shown in the figure, the activity test results of the haploid model Saccharomyces cerevisiae BY4741 are similar. In this experiment, the natural constitutive promoter pADH1 was used as a control. Figure 3 As can be seen in the fluorescence value / OD 600The value was used as an indicator of promoter expression strength. The strength of the constitutive promoter pADH1 after heating was lower than that before heating, while the strength of the artificial synthetic promoters after heating was higher than that before heating, with different fold increases, including pU104-104-26, pU104-104-30, pU104-26-26, pU104-30-26, pU104-30-30, pU26-104-26, pU26-104-30, pU26-26-104, pU26-26-26, pU26-26-30, pU26-30-26, and pU26-30-30. The response multiples of pU30-104-26, pU30-104-30, pU30-26-26, pU30-26-30, pU30-30-26, and pU30-30-30 were 4.02, 3.02, 5.98, 4.96, 4.86, 4.37, 5.97, 3.41, 7.82, 5.02, 8.37, 3.56, 3.13, 3.23, 12.66, 9.52, 7.07, and 6.19, respectively. They have good response capabilities and can be used to efficiently start the expression of industrial Saccharomyces cerevisiae CY downstream genes after warming.

[0045] Example 5 Ethanol fermentation test in industrial Saccharomyces cerevisiae CY

[0046] The ethanol fermentation test method of industrial Saccharomyces cerevisiae CY is as follows: 900 μL of YPD liquid medium containing nourseoin (80 mg / L) and glucose (20 g / L) was added to each well of a 96-well deep-well plate. Three (three parallel experiments) monoclonal strains were picked and placed in the deep-well plate containing the medium. The strains were activated for 24 hours and then the starting OD was used to determine the strain size. 600 Each strain was transferred to a fresh 900 μL YPD liquid medium containing nourseoin (80 mg / L) and glucose (20 g / L) in a deep-well plate at 0.1 and activated again for 24 hours. The activated yeast was centrifuged at 4000 rpm for 4 minutes, the supernatant was removed, and the yeast was resuspended in sterile water to measure the OD. 600 The cells were centrifuged at 4000 rpm for 4 minutes, the supernatant was removed, and an appropriate amount of YPD liquid medium containing glucose (200 g / L) was added to resuspend the cells. 600Each strain was transferred to a fresh 900 μL YPD liquid medium containing nourseoin (80 mg / L) and glucose (200 g / L) in a 96-well deep-well plate at 0.1. An airtight sealing membrane was used to simulate anaerobic high-sugar ethanol fermentation conditions. Starting from the 16th hour of culture, the shaking temperature was increased by 1°C every hour to simulate the gradient temperature increase conditions of industrial fermentation without cooling water from 30°C to 37°C. Culture was continued for 48 hours. After 48 hours of fermentation, the bacterial liquid was collected and centrifuged at 4000 rpm for 4 minutes. The supernatant was collected and filtered through a 0.22 μm polyethersulfone (PES) filter membrane. The residual glucose content and ethanol content were determined by high-performance liquid chromatography. The high-performance liquid chromatography instrument was an Agilent 1260 Infinity II, the detector was a differential refractive index detector (RID), and the chromatographic column was a Bio-Rad Aminex HPX-87H (300 mm×7.8 mm). mmol / L H2SO4 solution was used as the mobile phase, the flow rate was 0.5 mL / min, and the column temperature was 45°C. Gan et al. found that transcription factors Srb2 It is involved in the regulation of long-term heat tolerance of Saccharomyces cerevisiae and is a potential target for improving the ethanol fermentation capacity of industrial Saccharomyces cerevisiae (Gan YM, Qi XN, Lin YP, et al. AHierarchicalTranscriptional Regulatory Network Required for Long-Term ThermalStress Tolerance in an Industrial Saccharomyces cerevisiae Strain [J]. Frontiers in Bioengineering and Biotechnology, 2021, 9: 826238.). Therefore, this experiment used a vector containing the plasmid p414-Vector empty vector (containing only the nourseothricin resistance gene and the single cloning replication site, the nucleotide sequence is shown in SEQ ID NO: 4, 2003-7591 bp) and p414_Tcyc1-pSrb2-Srb2-Tpgk1 (expressed with the Saccharomyces cerevisiae natural promoter pSrb2). Srb2 The recombinant industrial Saccharomyces cerevisiae CY strain containing the nourseothricin resistance gene and the monoclonal replication site was used as a control. The experimental results are shown in Figure 2. Figure 4As shown, it can be seen that the recombinant industrial Saccharomyces cerevisiae CY strain (denoted as pU30-30-26-Srb2) containing the recombinant plasmid p414_Tcyc1-pU30-30-26-Srb2-Tpgk1 (using the Saccharomyces cerevisiae synthetic promoter pU30-30-26 to express the Srb2 gene, containing the nourseothricin resistance gene and the single cloning replication site) has an ethanol yield of 82.7 g / L and a sugar consumption of 194.5 g / L, which are 55% higher than the ethanol yield (53.1 g / L) and 20% higher than the control strain containing the recombinant plasmid p414-Vector (denoted as Vector) and the ethanol consumption (162.0 g / L) are increased. Compared with the control strain containing the recombinant plasmid p414_Tcyc1-pSrb2-Srb2-Tpgk1 (denoted as OP-Srb2), the ethanol yield (62.0 g / L) and the sugar consumption (159.5 g / L) increased by 22%.

[0047] Among them, pTDH3-yeGFP in the recombinant plasmid p414_Tcyc1-pTDH3-yeGFP-Tpgk1 was replaced with pSrb2-Srb2 (SEQ ID NO: 5, wherein, 1-500 bp: pSrb2, 501-1234 bp: Srb2 gene, 515-615 bp: intron) by the Gibson assembly method to obtain the recombinant plasmid p414_Tcyc1-pSrb2-Srb2-Tpgk1; then, pSrb2 in the recombinant plasmid p414_Tcyc1-pSrb2-Srb2-Tpgk1 was replaced with the synthetic promoter pU30-30-26 to obtain the recombinant plasmid p414_Tcyc1-pU30-30-26-Srb2-Tpgk1.

[0048] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A yeast high temperature responsive synthetic promoter, characterized in that: The synthetic promoter is named UX-YZ-pTDH3-core, abbreviated as pUX-YZ, wherein X, Y, Z, and pTDH3-core are connected in sequence, X is any one of UAS-104, UAS-26, and UAS-30, Y is any one of UAS-104, UAS-26, and UAS-30, and Z is any one of UAS-104, UAS-26, and UAS-30; UAS-104 is as shown in SEQ ID NO: 1, bp 1-195, UAS-26 is as shown in SEQ ID NO: 2, bp 186-404, UAS-30 is as shown in SEQ ID NO: 3, bp 423-756, and pTDH3-core is as shown in SEQ ID NO: 4, bp 825-973; The yeast high temperature responsive synthetic promoter is pU104-30-26, pU104-30-30, pU26-30-26, pU26-30-30, pU30-104-26, pU30-104-30, pU30-26-30, pU30-30-26, pU30-30-30, pU104-26-26, pU26-104-30, pU26-26-26 or pU30-26-26.

2. The biomaterial related to the yeast high temperature responsive synthetic promoter according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette comprising the yeast high-temperature-responsive synthetic promoter of claim 1; (b) a recombinant vector containing the yeast high temperature responsive synthetic promoter according to claim 1; (c) a recombinant vector containing the expression cassette described in (a); (d) a recombinant strain containing the yeast high temperature responsive synthetic promoter according to claim 1; (e) a recombinant strain containing the expression cassette described in (a); (f) a recombinant strain containing the recombinant vector described in (b) or (c); The host strain corresponding to the recombinant strains in (d), (e) and (f) is selected from Saccharomyces cerevisiae.

3. The biomaterial according to claim 2, characterized in that: The expression cassette in (a) further contains a target gene, which is operably linked to the yeast high-temperature-responsive synthetic promoter.

4. The biomaterial according to claim 2 or 3, characterized in that: The brewer's yeast is brewer's yeast BY4741 or industrial brewer's yeast CY.

5. Use of the yeast high-temperature responsive synthetic promoter according to claim 1 or the biomaterial according to any one of claims 2 to 4 in high-temperature responsive induction and initiation of target gene expression in Saccharomyces cerevisiae.

6. Use of the yeast high-temperature responsive synthetic promoter according to claim 1 or the biomaterial according to any one of claims 2 to 4 in improving the ethanol fermentation capacity of Saccharomyces cerevisiae, characterized in that: The target gene induced by the synthetic promoter is a transcription factor Srb2 .

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