Yeast strain having tolerance and ethanol production, and use thereof

Abstract

The present invention relates to the field of fermentation technology, and specifically provides a yeast strain having tolerance and ethanol production, and a use thereof. The strain is a Saccharomyces cerevisiae strain, said yeast strain having tolerance and the ability to produce ethanol when compared with a starting strain, and the yeast strain preferably being Saccharomyces cerevisiae M8; the sugar-to-ethanol conversion rate of the strain reaches 0.304 g / g, an increase of 38.18% compared with the original strain, and the growth and ethanol yield of the strain can maintain stable genetic characteristics.

Description

Yeast strains with tolerance and ethanol production and their applications

[0001] Related applications

[0002] This application claims priority to the prior application document with application number 202410033517.4 filed with the State Intellectual Property Office of China on January 9, 2024, and incorporates its entire contents into this document. Technical Field

[0003] The present application relates to the field of fermentation technology, and in particular to a yeast strain with tolerance and ethanol production and its application. Background Art

[0004] Bioethanol is a clean, renewable energy source with the advantages of high utilization efficiency and clean combustion. First-generation bioethanol is produced from starch through fermentation. However, it is greatly restricted by the problems of competing with people for food and competing with grain for land. The raw material for second-generation fuel ethanol is mainly lignocellulose. During the hydrolysis of lignocellulose, in addition to fermentable sugars, it also produces phenols, furans, and weak acids that are toxic to cells. As a eukaryotic model organism, Saccharomyces cerevisiae has a certain tolerance to its growth environment and is widely used in the industrial production of ethanol. Breeding highly tolerant Saccharomyces cerevisiae strains is key to the production of second-generation fuel ethanol.

[0005] Mutagenesis screening, a traditional, non-rational strain selection technique, primarily involves physical and chemical mutagenesis, and is widely used in strain selection. However, it is labor-intensive and complex. Ambient temperature and pressure plasma (ARTP) mutagenesis breeding has become a widely used breeding method in recent years. Due to its low jet temperature (25-35°C), uniform distribution of active particles, ease of operation, and high safety, it is widely used in fields such as bacteria and fungi. Summary of the Invention

[0006] In response to the technical problems existing in the above-mentioned prior art, the present application provides a yeast strain with tolerance and ethanol production and its application. The strain described in the present application is obtained by treating Saccharomyces cerevisiae with ARTP mutagenesis technology. After the mutated strain is cultured to a certain bacterial concentration, it is connected to a fully automatic high-throughput micro-droplet culture instrument (MMC). The high-throughput micro-droplet culture instrument uses a culture medium containing a hydrolyzate inhibitor for screening. The changes in bacterial concentration are observed at regular intervals to screen out strains with strong tolerance, and their genetic stability and ethanol production are studied, providing an excellent strain basis for subsequent fermentation culture optimization.

[0007] Specifically, this application proposes the following technical solutions.

[0008] The present application provides a yeast strain with tolerance and ethanol production, wherein the strain is a Saccharomyces cerevisiae strain. Compared with the starting strain, the yeast strain has tolerance and ethanol production capabilities.

[0009] Preferably, for the yeast strain described above, the yeast strain is Saccharomyces cerevisiae M8, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2023130.

[0010] The present application provides a bacterial agent comprising the yeast strain described above.

[0011] The present application provides the use of the above-mentioned yeast strain or the above-mentioned bacterial agent in fermentation to produce ethanol.

[0012] The present application provides the use of the yeast strain or the bacterial agent described above as a bioethanol fermentation agent to improve its acetic acid tolerance and / or furfural tolerance.

[0013] The present application provides a method for producing ethanol, comprising fermenting the yeast strain or the bacterial agent described above.

[0014] Preferably, in the above-mentioned method, the yeast strain or the bacterial agent is inoculated into a fermentation medium containing a composite inhibitor and fermented.

[0015] Preferably, for the above method, wherein the composite inhibitor comprises acetic acid, furfural and 5-hydroxymethylfurfural, preferably, the acetic acid is 0-5 g / L, the furfural is 0-2.5 g / L, and the 5-hydroxymethylfurfural is 0-0.5 g / L.

[0016] Preferably, for the above method, the fermentation medium contains glucose, peptone, yeast extract powder, urea, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and trehalose;

[0017] Preferably, glucose is 90-110 g / L, peptone is 15-25 g / L, yeast extract is 8-12 g / L, urea is 0.5-1.5 g / L, magnesium sulfate is 1.5-2 g / L, potassium dihydrogen phosphate is 0.5-1.0 g / L, calcium chloride is 0.5-1.5 g / L and trehalose is 0.2-1.0 g / L.

[0018] Preferably, in the above method, the fermentation temperature is 28-34° C., and / or the fermentation time is 36-60 h.

[0019] The present application provides a culture medium for screening the yeast strain described above, which contains a composite inhibitor comprising acetic acid, furfural and 5-hydroxymethylfurfural.

[0020] Preferably, in the above-mentioned culture medium, the acetic acid is 0-5 g / L, the furfural is 0-2.5 g / L, and the 5-hydroxymethylfurfural is 0-0.5 g / L.

[0021] Preferably, for the above culture medium, wherein the culture medium further comprises glucose, peptone and yeast extract powder, preferably, the glucose is 15-25 g / L, the peptone is 15-25 g / L and the yeast extract powder is 5-15 g / L.

[0022] The present application provides a method for screening the yeast strain described above, comprising:

[0023] activating and culturing the yeast strain to obtain a seed solution;

[0024] The seed solution was subjected to ARTP mutagenesis to obtain a mutagenized strain;

[0025] The mutated strain is inoculated into a culture medium containing a composite inhibitor for cultivation, and high-throughput screening is performed to obtain the yeast strain.

[0026] In some embodiments, the culture medium containing the composite inhibitor is any one of the culture media described above.

[0027] Beneficial effects achieved by this application:

[0028] The strain described in the present application is a fast and efficient mutagenesis of Saccharomyces cerevisiae by combining ARTP technology with MMC high-throughput microbial droplet culture instrument and lignin hydrolysis simulation liquid screening strategy. In the process, acetic acid can cause cytoplasmic acidification, forcing cell death, and furfural and 5-hydroxymethylfurfural can inhibit the activity of important enzymes related to the carbon metabolism of the cell, leading to cell death. Therefore, acetic acid, furfural and 5-hydroxymethylfurfural are added to the culture medium for screening to obtain a high-tolerance yeast strain. The sugar alcohol conversion rate of the obtained strain reaches 0.304g / g, which is 38.18% higher than that of the original strain (further optimization reaches 0.4358g / g, reaching 85.45% of the theoretical conversion rate). Genetic stability experiments show that the growth of the induced strain and ethanol production can maintain stable genetic characteristics. As can be seen from this, the mutagenesis method and high-throughput screening have the advantages of simplicity, efficiency and good stability, and are suitable for strain improvement of high-tolerance yeast.

[0029] This application combines ARTP random mutagenesis technology with the MMC high-throughput micro-droplet culture instrument and the directional inhibition of cell growth by lignin hydrolysis simulation liquid, thereby increasing the probability of the induced strain mutating in the direction of high-tolerance growth, increasing the probability of screening excellent strains, reducing the workload of screening, and providing a beneficial method for screening dominant microbial strains.

[0030] Strain deposit information

[0031] The strain Saccharomyces cerevisiae M8 used in this application was deposited in the China Center for Type Culture Collection on February 16, 2023, with the deposit number CCTCC NO: M 2023130, and the deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: (027) 68754052. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a schematic diagram showing the relationship between the lethality of Saccharomyces cerevisiae and time.

[0033] FIG2 is a schematic diagram showing the effects of different concentrations of composite inhibitors on Saccharomyces cerevisiae.

[0034] FIG3 is a schematic diagram showing the relationship between the selected mutagenic strains and the original strains.

[0035] FIG4 is a schematic diagram of the genetic stability of the mutagenized strain. DETAILED DESCRIPTION

[0036] The present application provides a yeast strain with tolerance and ethanol production, wherein the yeast strain is a Saccharomyces cerevisiae strain. Compared with the starting strain, the yeast strain has tolerance and ethanol production capabilities.

[0037] The yeast strain is obtained through ARTP mutagenesis technology and high-throughput screening, and has excellent tolerance and high ethanol production capacity.

[0038] In the present application, the high-throughput screening is to screen the strains obtained by ARTP mutagenesis using a composite inhibitor.

[0039] In the present application, the composite inhibitor comprises acetic acid, furfural and 5-hydroxymethylfurfural. Preferably, the acetic acid is 0-5 g / L, the furfural is 0-2.5 g / L, and the 5-hydroxymethylfurfural is 0-0.5 g / L.

[0040] For example, the acetic acid may be 0.1 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, or contain acetic acid within a numerical range consisting of any two of the above specific values ​​as endpoints; and / or

[0041] The furfural may be 0.1 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, or contain furfural within a numerical range consisting of any two of the above specific numerical values ​​as endpoints; and / or

[0042] The 5-hydroxymethylfurfural can be 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, or contain 5-hydroxymethylfurfural within a numerical range consisting of any two of the above specific values ​​as endpoints.

[0043] In the present application, the concentrations of acetic acid, furfural and 5-hydroxymethylfurfural are all not 0, that is, they are all greater than 0.

[0044] In some embodiments, the ARTP mutagenesis technique step comprises:

[0045] The saccharomyces cerevisiae seed suspension is coated on a metal slide, and the metal slide is placed in a mutagenesis chamber for mutagenesis treatment to obtain mutated bacteria.

[0046] In the present application, the brewer's yeast seed suspension refers to the suspension of Angel Super Brewer's yeast, which is a commonly used strain in the art.

[0047] In some embodiments, the concentration of the Saccharomyces cerevisiae seed suspension is OD 600 =0.7-1.0.

[0048] For example, the OD 600 It can be 0.7, 0.8, 0.9, 1.0, or a numerical range consisting of any two of the above specific values ​​as endpoints.

[0049] In some embodiments, the temperature of the ARTP is 18-22° C., the power is 100-200 W, and the flow rate of helium is 0.8-1.0 L / min.

[0050] For example, the temperature of ARTP can be 18°C, 19°C, 20°C, 21°C, 22°C, or a range of values ​​including any two of the above specific values ​​as endpoints;

[0051] The power is 100W, 110W, 120W, 130W, 140W, 150W, 160W, 170W, 180W, 190W, 200W, or a numerical range consisting of any two of the above specific values ​​as endpoints;

[0052] The flow rate of helium gas can be 0.8 L / min, 0.9 L / min, 1.0 L / min, or a numerical range containing any two of the above specific values ​​as endpoints.

[0053] In some embodiments, before applying the Saccharomyces cerevisiae seed suspension onto the metal slide, the metal support needs to be burned for at least 30-40 seconds.

[0054] In some embodiments, the distance between the emitter in the mutagenesis chamber and the bacterial solution is 1.5-3 nm, preferably 2 nm.

[0055] In some embodiments, the mutagenesis time is 120-140 s.

[0056] In some embodiments, the mutagenized strain is inoculated into a YPD medium containing a composite inhibitor and cultured to an appropriate bacterial concentration. A high-throughput micro-droplet culture apparatus (MMC) is used for culture, and the bacterial concentration is observed every day to select the mutagenized strain with good growth.

[0057] The composite inhibitor is the composite inhibitor described above.

[0058] In some embodiments, the bacterial solution concentration OD 600 It is 0.8-1.0.

[0059] For example, the bacterial concentration OD 600 It can be 0.8, 0.9, 1.0, or a numerical range consisting of any two of the above specific values ​​as endpoints.

[0060] In some embodiments, the YPD medium further comprises glucose, peptone and yeast extract powder. Preferably, the glucose is 15-25 g / L, the peptone is 15-25 g / L and the yeast extract powder is 5-10 g / L.

[0061] For example, the glucose may be 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, or a range of glucose containing any two of the above specific values ​​as endpoints; and / or

[0062] The peptone may be 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, or a peptone within a numerical range consisting of any two of the above specific values ​​as endpoints; and / or

[0063] The yeast extract can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or a yeast extract within a numerical range consisting of any two of the above specific values ​​as endpoints.

[0064] In some embodiments, during the culture process using a high-throughput micro-droplet culture apparatus (MMC), the number of droplets is 50-100, the number of passages is 1-3, the passage time is 18-22 hours, and the temperature is 28-32°C.

[0065] The MMC is a fully automatic high-throughput micro-droplet culture instrument (MMC) jointly developed by Tsinghua University and Beijing Siqingyuan Biotechnology Co., Ltd. It has the characteristics of simple operation and direct effect.

[0066] In some embodiments, the superior strains are fermented and cultured, that is, the screened strains are inoculated into seed liquid for culture to obtain seed liquid, and then the seed liquid is inoculated into fermentation medium for culture to obtain the strains.

[0067] In some embodiments, the screened strain is inoculated into a seed solution and cultured for 12-14 hours, and then the seed solution is inoculated into a fermentation medium and cultured at a temperature of 28-32° C. and a rotation speed of 180-220 r / min for 26-48 hours.

[0068] The strain prepared by the above method in the present application has good tolerance and high ethanol production ability.

[0069] In some embodiments, the yeast strain is Saccharomyces cerevisiae M8, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 2023130.

[0070] The present application provides a bacterial agent comprising the yeast strain described above.

[0071] The present application provides the use of the above-mentioned yeast strain or the above-mentioned bacterial agent in fermentation to produce ethanol.

[0072] The present application also provides the use of the yeast strain or the bacterial agent as a bioethanol fermentation agent to improve its acetic acid tolerance and / or furfural tolerance.

[0073] The present application provides a method for producing ethanol, comprising fermenting the yeast strain or the bacterial agent described above. In some embodiments, the yeast strain or the bacterial agent described above is inoculated into a fermentation medium containing a composite inhibitor for fermentation. In some embodiments, the composite inhibitor comprises acetic acid, furfural, and 5-hydroxymethylfurfural at a concentration of 0-5 g / L acetic acid, 0-2.5 g / L furfural, and 0-0.5 g / L 5-hydroxymethylfurfural.

[0074] In some embodiments, the fermentation medium contains glucose, peptone, yeast extract powder, urea, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and trehalose;

[0075] Preferably, glucose is 90-110 g / L, peptone is 15-25 g / L, yeast extract is 8-12 g / L, urea is 0.5-1.5 g / L, magnesium sulfate is 1.5-2 g / L, potassium dihydrogen phosphate is 0.5-1.0 g / L, calcium chloride is 0.5-1.5 g / L and trehalose is 0.2-1.0 g / L.

[0076] For example, the glucose content may be 90 g / L, 91 g / L, 92 g / L, 93 g / L, 94 g / L, 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, 101 g / L, 102 g / L, 103 g / L, 104 g / L, 105 g / L, 106 g / L, 107 g / L, 108 g / L, 109 g / L, or 110 g / L, or a range of glucose containing any two of the above-mentioned specific values ​​as endpoints; and / or

[0077] The peptone may be 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, or a peptone within a numerical range consisting of any two of the above specific values ​​as endpoints; and / or

[0078] The yeast extract may be 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, or a yeast extract within a numerical range consisting of any two of the above specific values ​​as endpoints; and / or

[0079] The urea content may be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, or a content within a numerical range consisting of any two of the above specific values ​​as endpoints; and / or

[0080] The magnesium sulfate may be 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, or a range of values ​​between any two of the above-mentioned specific values ​​as endpoints; and / or

[0081] The potassium dihydrogen phosphate may be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, or a range of potassium dihydrogen phosphate containing any two of the above specific values ​​as endpoints; and / or

[0082] The calcium chloride may be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, or a calcium chloride content within a numerical range consisting of any two of the above specific values ​​as endpoints; and / or

[0083] The trehalose content may be 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, or a range of trehalose containing any two of the above specific values ​​as endpoints.

[0084] In some embodiments, the fermentation temperature is 28-34° C. and / or the fermentation time is 36-60 h.

[0085] For example, the fermentation temperature may be 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or a range of values ​​containing any two of the above specific values ​​as endpoints;

[0086] The fermentation time can be 36h, 38h, 40h, 42h, 44h, 46h, 48h, 50h, 52h, 54h, 56h, 58h, 60h, or a numerical range containing any two of the above specific values ​​as endpoints.

[0087] The present application provides a culture medium for screening the yeast strain described above, comprising a composite inhibitor comprising acetic acid, furfural, and 5-hydroxymethylfurfural. In some embodiments, the acetic acid is present at 0-5 g / L, the furfural is present at 0-2.5 g / L, and the 5-hydroxymethylfurfural is present at 0-0.5 g / L.

[0088] In some embodiments, the culture medium further comprises glucose, peptone and yeast extract powder. Preferably, the glucose is 15-25 g / L, the peptone is 15-25 g / L and the yeast extract powder is 5-10 g / L.

[0089] The present application provides a method for screening the yeast strain described above, comprising:

[0090] activating and culturing the yeast strain to obtain a seed solution;

[0091] The seed solution was subjected to ARTP mutagenesis to obtain a mutagenized strain;

[0092] The mutated strain is inoculated into a culture medium containing a composite inhibitor for cultivation, and high-throughput screening is performed to obtain the yeast strain.

[0093] In some embodiments, the yeast strain is Angel Super Winemaking Highly Active Dry Yeast (referred to as Angel Super Winemaking Yeast strain).

[0094] In some embodiments, the culture medium containing the composite inhibitor is any one of the culture media described above.

[0095] In some embodiments, the seed solution is diluted to an OD of 600 It is a bacterial suspension of 0.5-0.8.

[0096] In some embodiments, when ARTP mutagenesis is used, it is performed under conditions conventional in the art, for example, at a temperature of 10-30° C., a power of 110-130 W, and a helium flow rate of 5-15 L / min.

[0097] Example

[0098] The following describes the manufacturers of the raw materials and equipment used in this example, as well as the equipment and analysis methods used for product analysis. The chemical substances not otherwise specified are of the chemical purity level of conventional reagents.

[0099] Example 1 Preparation of strains

[0100] (1) ARTP mutagenesis

[0101] Angel Yeast Co., Ltd.'s "Angel Super Brewing Highly Active Dry Yeast" was used as a seed culture to the mid-to-late logarithmic period and diluted with physiological saline to an OD 600 The value is 0.7, and the bacterial suspension is obtained. The suspension is resuspended by oscillation 2-3 times, and 5% glycerol is added for standby use. The temperature of ARTP is maintained at 20 ° C, the power is adjusted to 120W, and the helium (He) flow rate is 10L / min. 10 μL of bacterial suspension is evenly applied on a sterile metal slide, and the slide is placed in the mutagenesis room. The emission source is 2mm away from the bacterial solution, and different time periods (0s, 20s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, 180s) are used to induce the bacterial solution. The treated bacteria are inoculated into 1mL of normal saline and shaken for 1min, and the gradient dilution is made to 107 times. The dilution factor is 10 7 Spread 100 μL of the diluted bacterial solution on the surface of YPD solid culture medium and culture at 30°C for 2 days. Count the colonies and calculate the lethality to determine the optimal mutagenesis time. The calculation formula is as follows:

[0102] Lethality rate = (number of viable bacteria in the control group - number of viable bacteria in the induced group) / number of viable bacteria in the control group × 100%

[0103] The relationship between the lethality and time of Saccharomyces cerevisiae is shown in Figure 1. As can be seen, longer mutagenesis times increase the lethality of the strain. When the mutagenesis time is between 0 and 100 seconds, the lethality increases exponentially; at a mutagenesis time of 160 seconds, the lethality approaches 100%. To maintain a certain lethality and to obtain strains with enhanced viability and fermentation capacity, a mutagenesis treatment time of 120 seconds was chosen for this study.

[0104] (2) Screening of composite inhibitors

[0105] The overnight cultured seed solution (the seed solution of the yeast sold by Angel Yeast Co., Ltd. under the name of "Angel Super Brewing Highly Active Dry Yeast") was inoculated into YPD medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L) so that the OD 600 The culture medium was 0.2, and composite inhibitors with concentrations of 0%, 20%, 40%, 60%, 80% and 100% were respectively inoculated. The culture was carried out at 30°C and 200r / min for 24h. The uninoculated culture medium was used as a control. The strain was inoculated into the culture medium containing the composite inhibitor and cultured. The effects of different concentrations of composite inhibitors on the growth of the starting strain were measured. The composite inhibitor was a series of composite inhibitors with different contents of acetic acid, furfural and hydroxymethylfurfural. The results are shown in Figure 2.

[0106] As can be seen from Figure 2, when the composite inhibitor concentration was 0-40% (containing 2 g / L acetic acid, 1 g / L furfural, and 0.2 g / L 5-HMF), yeast growth was almost unaffected. When the composite inhibitor concentration was 80% (4 g / L acetic acid, 2 g / L furfural, and 0.4 g / L 5-HMF), yeast growth was significantly inhibited in the first 12 hours, and the growth rate was subsequently lower than that during low-concentration culture. However, when the composite inhibitor concentration was 100% (5 g / L acetic acid, 2.5 g / L furfural, and 0.5 g / L 5-HMF), yeast growth was completely inhibited. Therefore, YPD medium containing 5 g / L acetic acid, 2.5 g / L furfural, and 0.5 g / L 5-HMF at a composite inhibitor concentration of 100% was selected as the liquid screening medium.

[0107] (3) High-throughput screening

[0108] Since there are many mutants produced by mutagenesis, a high-throughput rapid screening method was established. After mutagenesis using ARTP at the optimal mutagenesis time, the bacterial solution was inoculated into YPD medium (glucose is 20g / L, the peptone is 20g / L and the yeast extract is 10g / L), and YPD medium containing simulated hydrolyzate (80% concentration of composite inhibitor, i.e. 4g / L acetic acid, 2g / L furfural and 0.4g / L 5-hydroxymethylfurfural) was prepared and sterilized for use. The bacterial solution was cultured until the OD 600 After the OD value was 0.1, the bacterial solution, YPD medium containing low concentration simulated hydrolyzate (the low concentration refers to 80% concentration of composite inhibitor), and YPD medium containing high concentration simulated hydrolyzate were respectively inoculated into No. 2 bacterial solution bottle, No. 4 medium bottle, and No. 6 high concentration chemical factor bottle (the high concentration refers to the composite inhibitor including acetic acid 6g / L, furfural 3g / L and 5-hydroxymethylfurfural 0.6g / L), and adaptive evolution was selected. The number of droplets was set to 50, the number of passages was set to 3, and the passage time was set to 22h. The influencing factor gradient was set, that is, the system automatically mixed out several gradients of composite inhibitors with different concentrations. The mutant strains were screened and acclimated using a fully automatic high-throughput micro-droplet culture instrument (MMC), and the bacterial solution in the corresponding numbered microplate or micro-droplet was gradiently diluted to OD 600 The concentration of the diluted bacterial solution was 1.0, and then diluted 3000-5000 times. 100 μL of the diluted bacterial solution was spread on a resistant plate medium and cultured at 30°C for 2 days. Strains with good growth were selected and preserved, and their colonies were plump. Figure 3 shows the relationship between the selected mutagenized strains and the original strains, which are Saccharomyces cerevisiae M1-M8, Saccharomyces cerevisiae K1-K10, and Saccharomyces cerevisiae P1-P10.

[0109] As can be seen from Figure 3, the bacterial concentrations of most of the mutagenized strains (Saccharomyces cerevisiae M1-M8, Saccharomyces cerevisiae K1-K10 and Saccharomyces cerevisiae P1-P10) were higher than those of the original strains. The high-yield strain M8 was selected as the starting strain for the rescreening.

[0110] (4) Fermentation culture

[0111] The original strain and the screened mutagenic strain were inoculated into seed liquid, respectively, and cultured at 30°C and 200 r / min to the mid-to-late logarithmic stage. They were then inoculated into YPD high-sugar medium (fermentation medium) at a 10% inoculum size and inoculated with a lethal concentration of a composite inhibitor (5 g / L acetic acid, 2.5 g / L furfural, and 0.5 g / L 5-hydroxymethylfurfural). The strains were cultured at 30°C and 200 r / min for 2 days. Samples were taken every 12 hours, and the ethanol production of the strains was detected by headspace gas chromatography (chromatographic conditions were a conventional chromatographic column, column temperature 80°C, heating for 30 min, injection temperature 160°C, detector (FID) temperature 155°C, initial 1 min; heating at 20°C / min to 85°C, holding for 2 min; heating at 25°C / min to 160°C, holding for 2 min). The residual sugar content was determined by the DNS method. The results are shown in Table 1.

[0112] Table 1 Results of rescreening of mutagenized yeast strains of Saccharomyces cerevisiae

[0113] It can be seen from the table that under non-resistant fermentation culture conditions, the ethanol production and sugar alcohol conversion rate of the induced strain are lower than those of the parent strain, but under resistant culture conditions, the induced strain can metabolize normally to produce ethanol, and the ethanol production and sugar alcohol conversion rate are also close to the ethanol production and sugar alcohol conversion rate of the parent strain (that is, the starting strain or original strain, also called strain W) under non-resistant culture conditions. It can be seen that the mutant strain has stronger high tolerance than the original strain.

[0114] (5) Genetic stability

[0115] The mutagenized strain was serially passaged seven times on an agar medium (glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, and agar powder 20 g / L) and then fermented (fermentation temperature 28-32°C, rotation speed 180-220 r / min). Headspace gas chromatography (chromatographic conditions were the same as above) was used to detect the ethanol production of the strain and study its genetic stability. The results are shown in FIG4 .

[0116] As can be seen from Figure 4, the ethanol production of the strains from the 1st to the 7th generation (M8) remained stable, indicating that the mutagenized strains have good genetic stability and can stably genetically resist toxicity.

[0117] Example 2: Strain Sequencing and Identification

[0118] Strain M8 genomic DNA was crudely extracted using a DNA extraction kit (purchased from Beijing Qingke Biotechnology Co., Ltd.). PCR amplification was performed using ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′ (SEQ ID NO. 1) and ITS4: 5′-TCCTCCGCTTATTGATATGC-3′ (SEQ ID NO. 2) as upstream and downstream primers. The PCR reaction parameters were: 25 μL PCR Mix; 1 μL sample; 2 μL ITS1 (10 μM); 2 μL ITS4 (10 μM); 20 μL ddH2O; reaction conditions were: 98°C pre-denaturation for 5 min; 98°C denaturation for 10 s; 57°C annealing for 10 s; 72°C extension for 30 s; 35 cycles of extension at 72°C for another 5 min, and storage at 4°C. After the PCR product was qualified, ITS sequencing was performed to identify the sequence as shown in SEQ ID NO: 3. Homology comparison analysis was performed in the database to identify the species of the sample, and the result showed that it was Saccharomyces cerevisiae.

[0119] Strain M8 was named Saccharomyces cerevisiae M8 and deposited. Saccharomyces cerevisiae M8 was deposited with the China Center for Type Culture Collection on February 16, 2023, with a deposit number of CCTCC NO: M 2023130. The sequence listing of Saccharomyces cerevisiae M8 is shown in SEQ ID NO. 3.

[0120] The sequencing identification steps of strain M1 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain M1 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae M1, and the sequence table is shown in SEQ ID NO.4.

[0121] The sequencing identification steps of strain M2 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain M2 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae M2, and the sequence table is shown in SEQ ID NO.5.

[0122] The sequencing identification steps of strain M3 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain M3 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae M3, and the sequence table is shown in SEQ ID NO.6.

[0123] The sequencing identification steps of strain M4 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain M4 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae M4, and the sequence table is shown in SEQ ID NO.7.

[0124] The sequencing identification steps of strain M5 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain M5 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae M5, and the sequence table is shown in SEQ ID NO.8.

[0125] The sequencing identification steps of strain M6 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain M6 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae M6, and the sequence table is shown in SEQ ID NO.9.

[0126] The sequencing identification steps of strain M7 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain M7 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae M7, and the sequence listing is shown in SEQ ID NO.10.

[0127] The sequencing identification steps of strain K1 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K1 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K1, and the sequence listing is shown in SEQ ID NO.11.

[0128] The sequencing identification steps of strain K2 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K2 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K2, and the sequence listing is shown in SEQ ID NO.12.

[0129] The sequencing identification steps of strain K3 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K3 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K3, and the sequence listing is shown in SEQ ID NO.13.

[0130] The sequencing identification steps of strain K4 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K4 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K4, and the sequence listing was the same as that of strain K1, as shown in SEQ ID NO.11.

[0131] The sequencing identification steps of strain K5 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K5 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K5, and the sequence listing was the same as that of strain K2, as shown in SEQ ID NO.12.

[0132] The sequencing identification steps of strain K6 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K6 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K6, and the sequence listing was the same as that of strain K1, as shown in SEQ ID NO.11.

[0133] The sequencing identification steps of strain K7 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K7 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K7, and the sequence listing was the same as that of strain M8, as shown in SEQ ID NO.3.

[0134] The sequencing identification steps of strain K8 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K8 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K8, and the sequence listing was the same as that of strain K1, as shown in SEQ ID NO.11.

[0135] The sequencing identification steps of strain K9 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K9 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K9, and the sequence listing is shown in SEQ ID NO.14.

[0136] The sequencing identification steps of strain K10 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain K10 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae K10, and the sequence listing is shown in SEQ ID NO.15.

[0137] The sequencing identification steps of strain P1 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P1 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P1, and the sequence listing is shown in SEQ ID NO.16.

[0138] The sequencing identification steps of strain P2 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P2 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P2, and the sequence listing was the same as that of strain P1, as shown in SEQ ID NO.16.

[0139] The sequencing identification steps of strain P3 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P3 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P3, and the sequence listing was the same as that of strain P1, as shown in SEQ ID NO.16.

[0140] The sequencing identification steps of strain P4 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P4 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P4, and the sequence listing was the same as that of strain K1, as shown in SEQ ID NO.11.

[0141] The sequencing identification steps of strain P5 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P5 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P5, and the sequence listing was the same as that of strain M8, as shown in SEQ ID NO.3.

[0142] The sequencing identification steps of strain P6 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P6 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P6, and the sequence listing is shown in SEQ ID NO.17.

[0143] The sequencing identification steps of strain P7 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P7 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P7, and the sequence listing was the same as that of strain M8, as shown in SEQ ID NO.3.

[0144] The sequencing identification steps of strain P8 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P8 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P8, and the sequence listing was the same as that of strain K2, as shown in SEQ ID NO.12.

[0145] The sequencing identification steps of strain P9 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P9 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P9, and the sequence listing was the same as that of strain M8, as shown in SEQ ID NO.3.

[0146] The sequencing identification steps of strain P10 were the same as those of M8. Homology comparison analysis was performed in the database, and the species of strain P10 was identified as Saccharomyces cerevisiae. It was named Saccharomyces cerevisiae P10, and the sequence listing is shown in SEQ ID NO.18.

[0147] Table 2 Sequence Listing

[0148] The above is only a preferred embodiment of the implementation of this application and does not limit this application in any form. Any modifications, equivalent replacements and improvements made within the spirit and principles of this application shall be included in the scope of protection of this application.

Claims

1. A yeast strain with tolerance and ethanol production ability, said strain being a Saccharomyces cerevisiae strain, and having tolerance and the ability to produce ethanol compared with the starting strain.

2. The yeast strain according to claim 1, wherein, The yeast strain is Saccharomyces cerevisiae M8, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2023130.

3. A bacterial agent, which comprises the yeast strain according to claim 1 or 2.

4. Use of the yeast strain according to claim 1 or 2 or the bacterial agent according to claim 3 in the fermentation production of ethanol.

5. Use of the yeast strain according to claim 1 or 2 or the bacterial agent according to claim 3 as a bioethanol fermenting agent in enhancing its acetic acid tolerance and / or furfural tolerance.

6. A method for producing ethanol, which includes fermenting the yeast strain according to claim 1 or 2 or the bacterial agent according to claim 3.

7. The method according to claim 6, wherein Inoculating the yeast strain according to claim 1 or 2 or the bacterial agent according to claim 3 into a fermentation medium containing a composite inhibitor for fermentation.

8. The method according to claim 7, wherein, The composite inhibitor comprises acetic acid, furfural and 5-hydroxymethylfurfural. Preferably, the acetic acid is 0 - 5 g / L, the furfural is 0 - 2.5 g / L and the 5-hydroxymethylfurfural is 0 - 0.5 g / L.

9. The method according to claim 7 or 8, wherein The fermentation medium contains glucose, peptone, yeast extract powder, urea, magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and trehalose; Preferably, the glucose is 90 - 110 g / L, the peptone is 15 - 25 g / L, the yeast extract powder is 8 - 12 g / L, the urea is 0.5 - 1.5 g / L, the magnesium sulfate is 1.5 - 2 g / L, the potassium dihydrogen phosphate is 0.5 - 1.0 g / L, the calcium chloride is 0.5 - 1.5 g / L and the trehalose is 0.2 - 1.0 g / L.

10. The method according to any one of claims 6-9, wherein, The fermentation temperature is 28 - 34 °C, and / or The fermentation time is 36 - 60 h.

11. A medium for screening the yeast strain according to claim 1 or 2, which comprises a composite inhibitor, and the composite inhibitor comprises acetic acid, furfural and 5-hydroxymethylfurfural.

12. The culture medium according to claim 11, wherein, The acetic acid is 0 - 5 g / L, the furfural is 0 - 2.5 g / L and the 5-hydroxymethylfurfural is 0 - 0.5 g / L.

13. The culture medium according to claim 11 or 12, wherein, The medium further comprises glucose, peptone and yeast extract powder. Preferably, the glucose is 15 - 25 g / L, the peptone is 15 - 25 g / L and the yeast extract powder is 5 - 15 g / L.

14. A method for screening the yeast strain according to claim 1 or 2, which includes: Activating and culturing the yeast strain to obtain a seed solution; Performing ARTP mutagenesis on the seed solution to obtain a mutagenized strain; Inoculating the mutagenized strain into a medium containing a composite inhibitor for culturing, and performing high-throughput screening to obtain the yeast strain.

15. The method according to claim 14, wherein The medium containing a composite inhibitor is the medium according to any one of claims 11 - 13.

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