CN114854607B - High-temperature-resistant saccharomyces cerevisiae, fermenting agent, application of high-temperature-resistant saccharomyces cerevisiae and fermenting agent, and ethanol production method - Google Patents
High-temperature-resistant saccharomyces cerevisiae, fermenting agent, application of high-temperature-resistant saccharomyces cerevisiae and fermenting agent, and ethanol production method Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention relates to the field of microorganisms, in particular to high-temperature resistant saccharomyces cerevisiae, a fermenting agent, application of the high-temperature resistant saccharomyces cerevisiae and the fermenting agent, and a production method of ethanol. The preservation number of the saccharomyces cerevisiae is CGMCC No.21567. The fermentation indexes of the high-temperature resistant saccharomyces cerevisiae strain provided by the invention at high temperature all meet the index requirements of the current industrial production using the commercial saccharomyces cerevisiae strain under the normal temperature control condition. Compared with a control strain, the saccharomyces cerevisiae strain has obviously improved ethanol yield and cell survival rate, and obviously reduced total sugar content in chromatography and total sugar content in titration filtration, so that the saccharomyces cerevisiae strain can ensure the stability of ethanol production of liquid fuel under the condition of not strictly controlling temperature, improve the ethanol production efficiency, reduce the production cost and is suitable for large-scale production of industrial ethanol.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to high-temperature resistant saccharomyces cerevisiae, a fermenting agent, application of the high-temperature resistant saccharomyces cerevisiae and the fermenting agent, and a production method of ethanol.
Background
Liquid fuel ethanol refers to ethanol which is obtained by taking biomass as a raw material through a biological fermentation way and the like and can be used as fuel. The denatured liquid fuel alcohol is mixed with gasoline in certain proportion to prepare alcohol gasoline for automobile. The fuel ethanol is usually fermented by Saccharomyces cerevisiae, and the optimal growth temperature of Saccharomyces cerevisiae is 30-32 ℃. In industrial production, metabolic heat, mechanical stirring heat, system heat and the like generated along with the production of the strain often cause the temperature of a fermentation system to rise (35-37 ℃ in time), and the production performance of the strain is greatly reduced due to high-temperature stress. The high temperatures typically cause changes in various components within the yeast cell, such as fatty acids, phospholipids, ergosterol, etc. These changes in composition in turn affect the normal physiological activity of the cell itself. Therefore, the high temperature is toxic to yeast cells, and the higher the temperature is, the more easily the yeast cells die. If the temperature can be well controlled and maintained at about 32 ℃, stable production can be maintained throughout the year. However, in the current industrial production, tap water is used for controlling the temperature of the fermentation tank in a circulating way, and the temperature is hardly controlled to be about 32 ℃ stably along with the influence of the change of the seasonal environmental temperature, so that the economic loss caused by unstable production due to high temperature is very huge. The development and application of the high-temperature-resistant saccharomyces cerevisiae can ensure the stability of fuel ethanol production on the premise of not strictly controlling the temperature, and avoid the economic loss.
The adaptability of the microorganism to the fermentation microenvironment is the result of the long-term evolution of the microorganism, and is also the comprehensive performance of the adaptation of the biochemical reaction and metabolic pathways of the microorganism to the fermentation microenvironment change, which is determined in cells. At present, intermittent fermentation is generally adopted in the fuel ethanol fermentation industry, so that the industrial saccharomyces cerevisiae loses the opportunity of long-term adaptive evolution to the fermentation microenvironment, and a forward mutant strain with stable genetic characters is difficult to obtain from the existing fermentation strain.
Disclosure of Invention
The invention aims to overcome the problems in the prior art, and provides the high-temperature-resistant saccharomyces cerevisiae, the leavening agent, the application of the high-temperature-resistant saccharomyces cerevisiae and the ethanol production method.
In order to achieve the purpose, the invention provides a high-temperature resistant saccharomyces cerevisiae (Saccharomyces cerevisiae), and the collection number of the saccharomyces cerevisiae is CGMCC No.21567.
In a second aspect the invention provides a starter comprising the high temperature resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) of claim 1.
In a third aspect the invention provides a high temperature resistant Saccharomyces cerevisiae as described above, or the use of a starter as described above in ethanol production.
In a fourth aspect, the present invention provides a process for producing ethanol, comprising: the high temperature resistant Saccharomyces cerevisiae as described above, or the starter as described above, is inoculated into a fermentation medium for ethanol fermentation.
The fermentation indexes of the high-temperature resistant saccharomyces cerevisiae strain provided by the invention at high temperature are obviously improved compared with the fermentation performance of the commercial saccharomyces cerevisiae strain used in the current industrial production. Compared with a control strain, the saccharomyces cerevisiae strain has obviously improved ethanol yield and cell survival rate, and obviously reduced total sugar content in chromatography and total sugar content in titration filtration, which indicates that the saccharomyces cerevisiae strain can ensure the stability of ethanol production of liquid fuel under the condition of not strictly controlling temperature, improve the ethanol production efficiency, reduce the production cost and is suitable for large-scale production of industrial ethanol.
Preservation of organisms
The Saccharomyces cerevisiae (Saccharomyces cerevisiae) is preserved in China general microbiological culture Collection center (address: north west way No. 1, no. 3, national academy of sciences microbiological research institute, china postal code: 100101) (preservation unit abbreviated as CGMCC) at 12 months and 29 days in 2020, and the preservation number is CGMCC No.21567, abbreviated as S.C7-2.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the fourth, fifth, sixth and seventh rounds of gradient dilution growth conditions for whole genome mutations of example 1;
FIG. 2 shows the results of the analysis of the wine at different temperatures in example 1.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In a first aspect, the invention provides a strain of high-temperature resistant saccharomyces cerevisiae (Saccharomyces cerevisiae), which is characterized in that the collection number of the saccharomyces cerevisiae is CGMCC No.21567.
The Saccharomyces cerevisiae CGMCC No.21567 is a high-temperature resistant Saccharomyces cerevisiae obtained by taking Saccharomyces cerevisiae of 5 generations of continuous fermentation of medium grain biochemical energy (Zhaodong) limited company as an initial strain, performing seven rounds of mutation, liquefied mash shaking fermentation verification, industrial material small scale verification and industrial material pilot scale verification by a whole genome mutation coupling laboratory training method.
The whole genome mutation coupling laboratory is trained to cut whole genome of Saccharomyces cerevisiae which is continuously fermented for 5 generations by Zhaodong, zhaodong and Zhaoning, through CRISPR editing technology, then random mutation is carried out through high mutation efficiency brought by knocking out the Rad52 gene of the strain, and after high-temperature culture and resistance screening at 40 ℃, the strain edited by the whole genome is obtained from the first round of mutation to carry out the second round of mutation. Wherein the specific conditions of the whole genome mutation method are as follows: after a background host is transferred into Cas9 plasmid of a CRISPR system, transferring into a sgRNA target library of a whole genome grade of 1.5 mug by using a yeast LiAc chemical conversion method, adding 30% and 50% YPD for resuscitation for 1-2 hours, directly transferring into a factory real fermentation material, fermenting and culturing at 38 ℃ for 24-36 hours, filtering the material by using seven layers of gauze to obtain turbid liquid, transferring 500 mug of turbid liquid into 50ml of stress YPD for 12-24 hours, transferring once every 12 hours, taking turbid liquid after transferring for 3 times, coating a HYB plate, verifying whether the sgRNA target library is lost, obtaining first-generation evolution bacterial liquid after the plasmid is lost cleanly, and carrying out next round of iterative evolution based on the material.
The shake flask fermentation of the liquefied mash is verified to be that 4 strains with better growth and fermentation performance after the fourth round, the fifth round, the sixth round and the seventh round of mutation are inoculated into the liquefied mash, and shake flask fermentation is performed at 38 ℃.
And (3) further carrying out industrial material small-scale test and industrial material pilot-scale test on the saccharomyces cerevisiae selected through the shake flask fermentation verification of the liquefied mash, comparing parameters such as ethanol yield, total chromatographic sugar content, total filtration sugar content, cell survival rate and the like in the fermentation liquor, and finally determining the saccharomyces cerevisiae strain S.C7-2 with high temperature resistance and high ethanol yield.
The high-temperature resistant saccharomyces cerevisiae strain S.C7-2 is preserved in China general microbiological culture collection center (CGMCC) of China Committee for culture Collection of microorganisms on 12 months 29 days 2020, the preservation center address is China national academy of sciences of China, national institute of sciences of North Chen, lu 1, no. 3, and the preservation number is CGMCC No.21567.
In a second aspect, the invention provides a starter comprising a high temperature resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) as described above.
According to the invention, the starter contains living bacteria of high temperature resistant Saccharomyces cerevisiae CGMCC No.21567, and the content of the high temperature resistant Saccharomyces cerevisiae is preferably 10 per gram of the starter 5 -10 10 CFU, more preferably 10 7 -10 9 CFU。
Wherein CFU (Colony-Forming Units) refers to the number of viable bacteria. In the case of viable bacteria culture counting, colonies formed by growth and propagation of a single cell or a plurality of cells aggregated into a group on a solid medium are called colony forming units, which represent the number of viable bacteria. The culture broth after the culture may be diluted to an appropriate level and plated to obtain the culture broth.
According to the present invention, the starter can be prepared according to a conventional method, for example, fermentation culture can be performed according to the method described above in the first aspect to obtain cells of high temperature resistant Saccharomyces cerevisiae CGMCC No.21567, and then the starter can be prepared according to a conventional method.
The fermentation agent may be in a liquid state or a solid state, and is usually in a solid state. Specifically, when the fermentation culture is completed, if the fermentation culture can be used within a relatively short period of time, for example, within 1 month, the obtained fermentation liquid can be directly used or the fermentation liquid can be properly concentrated to be used as a fermentation agent, and in this case, the fermentation agent can be in a liquid state; however, if the solid fermentation medium is required to be stored for a long time and then used, the solid fermentation medium can be prepared, for example, fermentation liquid is subjected to solid-liquid separation to obtain bacterial cell sediment, and then the bacterial cell sediment is mixed with a freeze-drying protective agent and freeze-dried to obtain the solid fermentation medium.
The freeze-drying protective agent can be at least one of skim milk powder, maltodextrin, trehalose, dextran, glycerol and the like.
The starter culture may be obtained by inoculating, for example, cells in a logarithmic growth phase into glycerol, which usually contains 25 to 35% by volume, for example, 30% by volume of glycerol.
The inventor of the invention adopts a secondary liquefied mash pilot-scale test of a Zhaodong limited company to verify the fermentation effect of the high-temperature resistant saccharomyces cerevisiae CGMCC No.21567 strain, and experiments show that various indexes of fermentation of the saccharomyces cerevisiae CGMCC No.21567 strain at high temperature all meet the index requirements of Angel super wine purchased by Angel company under the normal temperature control condition. Compared with a control strain, the saccharomyces cerevisiae CGMCC No.21567 strain has obviously improved ethanol yield and cell survival rate at high temperature, and the total sugar content of chromatography and the total sugar content of titration filtration are obviously reduced, namely the saccharomyces cerevisiae CGMCC No.21567 strain can ensure the stability of ethanol production under the condition of not strictly controlling the temperature, improve the ethanol production efficiency, reduce the production cost and is suitable for large-scale production of industrial liquid fuel ethanol. Therefore, the invention provides the application of the saccharomyces cerevisiae with the preservation number of CGMCC No.21567 in the fermentation production of industrial liquid fuel ethanol.
Thus, in a third aspect, the present invention provides a high temperature resistant Saccharomyces cerevisiae as described above, or the use of a starter as described above in ethanol production.
In a fourth aspect, the present invention provides a process for the production of ethanol, the process comprising: the high temperature resistant Saccharomyces cerevisiae as described above, or the starter as described above, is inoculated into a fermentation medium for ethanol fermentation.
According to the present invention, the conditions for the fermentation may be conventional conditions for preparing ethanol by yeast fermentation, and as described above, the high temperature resistant Saccharomyces cerevisiae provided by the present invention does not require strict control of low temperature environment (30-32 ℃), and thus the fermentation temperature thereof may be changed within a wide range, preferably, the conditions for the fermentation include: the temperature is 25-45 ℃.
Preferably, the fermentation time is 65-80 hours.
According to a preferred embodiment of the present invention, in order to further increase the yield of ethanol, the conditions of the fermentation include: fermentation starts for 12-16 hours, and the fermentation temperature is 25-32 ℃ (first stage fermentation); fermenting for 12-16 hr to 20-28 hr at 32-38deg.C (second stage fermentation); fermenting for 20-28 hr to 45-50 hr at 38-45deg.C (third stage fermentation); fermenting for 45-50 hr until the fermentation is completed, wherein the fermentation temperature is 35-40deg.C (fourth stage fermentation).
The temperatures of the first-stage fermentation and the second-stage fermentation are sequentially increased, for example, when the temperature of the first-stage fermentation is 32 ℃, the temperature of the second-stage fermentation is > 32 ℃.
Wherein the temperature of the fermentation in the third stage is more than or equal to that of the fermentation in the fourth stage, and the temperature of the fermentation in the fourth stage is less than or equal to that of the fermentation in the second stage.
According to a more preferred embodiment of the invention, the conditions of the fermentation comprise: fermentation starts to 16 hours of fermentation, and the temperature of fermentation is 32 ℃; fermentation is carried out for 16 hours to 24 hours at a temperature of 32-38deg.C (second stage fermentation, e.g., 38deg.C); 24-48 hours of fermentation, the temperature of fermentation is 38-45 ℃ (third stage fermentation, e.g., 38 ℃); and (3) fermenting for 48 hours until the fermentation is finished, wherein the fermentation temperature is 38 ℃ (fermentation in the fourth stage).
According to the present invention, the fermentation medium may be a conventional medium for ethanol fermentation using yeast, for example, may be a conventional liquefied mash, which may be obtained by sequentially pulverizing, slurrying, and liquefying biomass raw materials, and the specific preparation process is well known to those skilled in the art, and the present invention is not repeated here.
According to the invention, preferably, the liquefied mash has a reducing sugar concentration of 4-6, a total sugar concentration of 20-25 and a dry matter content of 20-30 wt.%.
Preferably, the reducing sugars in the liquefied mash have a DE value of 20-23 calculated by refraction and 17-20 calculated as dry matter.
Preferably, the viscosity of the liquefied mash is 120-140mpa.s (4.5-6 cp), pH3-5.
The biomass raw material may be a cellulosic biomass raw material, for example, straw, or a starchy raw material, for example, corn.
According to the present invention, the content of the high temperature resistant s.cerevisiae in the fermentation medium after inoculation is not particularly limited, and for example, the initial concentration of the high temperature resistant s.cerevisiae living cells after inoculation is 15 to 25 hundred million CFU with respect to 1L of the fermentation medium, based on the high temperature resistant s.cerevisiae living cells.
According to a preferred embodiment of the present invention, before inoculating the high temperature resistant saccharomyces cerevisiae or the starter culture to the fermentation medium, activating the same, specifically inoculating the high temperature resistant saccharomyces cerevisiae as described above or the starter culture as described above to a seed medium for expansion culture to obtain expanded culture seeds; and then inoculating the expanded seed into a fermentation medium for ethanol fermentation.
Wherein, the seed culture medium can be YPD liquid culture medium with pH value of 7-7.2;
the extent of the expansion culture in the seed medium is not particularly limited, and for example, the strain may be cultured to a high-temperature-resistant Saccharomyces cerevisiae concentration of 0.125 hundred million/ml.
The number of the expansion culture stages is not limited, and may be, for example, 1 to 3 stages.
Wherein the seed of the expanded culture is inoculated in an amount of 12-20% by volume, for example, 16% by volume, based on the concentration of the high temperature resistant Saccharomyces cerevisiae after the expanded culture of 0.125 hundred million/ml.
According to the technical scheme, the invention provides a high-temperature-resistant saccharomyces cerevisiae strain and application thereof. The high-temperature resistant saccharomyces cerevisiae strain S.C7-2 has various indexes of fermentation at high temperature, and meets the index requirements of Angel super wine purchased by Angel company under the normal temperature control condition. Compared with a control strain, the saccharomyces cerevisiae strain has obviously improved ethanol yield and cell survival rate, and obviously reduced total sugar content in chromatography and total sugar content in titration filtration, so that the saccharomyces cerevisiae strain can ensure the stability of fuel ethanol production under the condition of not strictly controlling temperature, improve the ethanol production efficiency, reduce the production cost and is suitable for large-scale production of industrial liquid fuel ethanol.
The present invention will be described in detail by examples. In the following examples, all reagents used in the examples of the present invention are commercially available products, and are commercially available, unless otherwise specified. The control strains are Angel super wine purchased by Angel company. Chemicals and reagents used in the experiments, such as urea, penicillin, su Hong saccharifying enzyme, etc., are all of industrial grade.
Example 1
For illustrating genome-wide mutant coupling laboratory adaptation training
1. And (3) inoculating a certain amount of bacteria-retaining liquid into a proper amount of YPD liquid culture medium, adding corresponding penicillin, and performing shake culture at 30 ℃ and 200rpm overnight. Inoculating the activated bacterial liquid into fresh YPD culture medium at 10% volume ratio, adding penicillin, culturing at 30deg.C and 200rpm for 4-5 hr until cell concentration is 2×10 7 About one/ml. 1ml of the bacterial liquid is taken and centrifuged at 4000rpm at 4 ℃ for 5min. The supernatant was discarded, resuspended in 1ml of sterile water and centrifuged at 4000rpm for 5min at 4 ℃. The supernatant was discarded, resuspended in 1ml of 100mM LiAc and allowed to stand for 3min. Centrifuge at 4000rpm for 3min and discard supernatant. The transformation solution (a.74-x. Mu.l of sterile water b.x. Mu.l of DNA (500-1000 ng) c.10. Mu.l of ss-DNA (boiled for 5min, rapidly cooled on ice and then used) d.240. Mu.l of 50% PEG3350 (added to the bottom of the tube) e.36. Mu.l of 1M LiAc (added to the bottom of the tube, in PEG to minimize contact with the cells)) was added in sequence, and then vortexed and mixed rapidly, and incubated in a 30℃incubator, allowed to stand for 30min, and thermally shocked at 42℃for 20-30min. Centrifugation at 4000rpm for three minutes, the supernatant was discarded and YPD was added for resuscitation for 2 hours. The solution was washed once with sterile water. Centrifuge at 4000rpm for three minutes, discard supernatant and wash once with sterile water. Resuspension with sterile water, plating a proper amount of bacterial liquid, culturing for 48 hours, collecting the plate, verifying single colony with colony PCR to obtain background host containing Cas9 plasmid, and preserving.
2. And (3) inoculating a certain amount of bacteria-retaining liquid into a proper amount of YPD liquid culture medium, adding corresponding antibiotics, and performing shake culture at 30 ℃ and 200rpm overnight. The target sgRNA of the Rad52 gene is transformed again according to the previous step, and a homologous template used for knockout is added. And (3) re-suspending the obtained product by using sterile water after the conversion is finished, coating a proper amount of bacterial liquid on a plate, culturing the plate for 48 hours, collecting the plate, and verifying a single colony by using colony PCR to obtain a background host for knocking out the Rad52 gene. Selecting positive monoclonal, adding G418 resistance culture in YPD liquid culture medium for 12-24h, transferring once every 12-24h, taking bacterial liquid to coat a HYB plate after transferring for 3 times to see whether growth exists, verifying whether sgRNA is lost clean (no growth is lost clean), obtaining a sensitive chassis host after plasmid is lost clean, preserving, and carrying out whole genome mutation and adaptability training on the host.
3. A certain amount of sensitive chassis host SC-Rad52 delta bacteria-preserving liquid is inoculated into a proper amount of YPD (2% glucose, 2% peptone, 1% yeast powder, 2% agar powder, pH 7.0) liquid culture medium, and shake cultivation is carried out at 30 ℃ and 200rpm for overnight activation. The activated bacterial liquid is inoculated in a fresh YPD culture medium in a volume ratio of 10%, and is cultured for 4-5 hours at 30 ℃ and 200rpm after the corresponding antibiotics are added until the cell concentration is about 2X 107 cells/ml. ) 1ml of the bacterial liquid is taken and centrifuged at 4000rpm at 4 ℃ for 5min. The supernatant was discarded, resuspended in 1ml of sterile water and centrifuged at 4000rpm for 5min at 4 ℃. The supernatant was discarded, resuspended in 1ml of 100mM LiAc and allowed to stand for 3min. Centrifuge at 4000rpm for 3min and discard supernatant. The conversion solutions are added in sequence and then rapidly and uniformly vortex. a.74-x. Mu.l sterile water b.x. Mu.l Cas9 plasmid (500-1500 ng) c.10. Mu.l ss-DNA (boiled for 5min, immediately after ice-micro-cooling) d.240. Mu.l 50% PEG3350 (added to the bottom of the tube) e.36. Mu.l 1M LiAc (added to the bottom of the tube in PEG, with minimal contact with the cells) 30℃incubator, standing for 30min, and heat-shock at 42℃for 20-30min. Centrifugation at 4000rpm for three minutes, the supernatant was discarded, and stressed YPD (30% Glu, 6 Eth) was added for resuscitation at 38℃for 1h. Directly transferring into actual fermentation materials after recovery, and fermenting and culturing at 40deg.C for 24-36 hr.
4. After fermentation, filtering materials by using seven layers of gauze to obtain turbid liquid, transferring 500 mul of turbid liquid to 50ml of stressed YPD for culturing for 12-24 hours, transferring once every 12 hours, transferring 3 times, taking turbid liquid, coating a HYB plate, verifying whether a sgRNA target library is lost completely, obtaining an evolution bacterial liquid of the next generation after the plasmid is lost completely, and carrying out iterative evolution based on the obtained bacterial liquid.
5. Dominant strain screening: after four rounds of mutation, YPD solid medium was prepared with factory process water, and was high Wen Shaixuan at 40 ℃. Seven rounds of mutation were performed in total, and the second round of mutation was performed on the mixed bacteria after discarding sgrnas from the previous round of mutation (fig. 1).
5. And selecting dominant strains obtained by mixed bacteria screening after fourth, fifth, sixth and seventh generations of mutation to perform shake flask fermentation verification of liquefied mash. The results were verified using medium grain biochemical energy (culprit) company three-phase liquefied mash, shake flask fermentation at 38 ℃ for 66 h. The results of shake flask fermentation of the 4 strains at 38deg.C are shown in Table 1.
TABLE 1
| Sample name | Cellobiose | Glucose | Xylose | Arabinose (Arabic sugar) | Xylitol | Lactic acid | Glycerol | Acetic acid | Ethanol |
| 4-3 | 5.114 | 41.377 | 2.585 | 0.673 | 0.000 | 0.218 | 12.248 | 1.879 | 100.849 |
| 5-2 | 5.320 | 27.150 | 2.251 | 0.640 | 0.000 | 0.066 | 11.978 | 2.380 | 104.584 |
| 6-5 | 4.572 | 54.275 | 0.000 | 0.287 | 0.564 | 0.000 | 13.115 | 0.953 | 107.757 |
| 7-2 | 3.997 | 43.509 | 0.000 | 0.084 | 0.665 | 0.000 | 11.592 | 0.935 | 109.467 |
| Control strain | 5.501 | 48.610 | 0.000 | 0.201 | 0.598 | 0.000 | 11.634 | 1.158 | 102.735 |
Note that: the control strain is Angel super wine purchased by Angel company
The results show that the yield of 7-2 (named S.C 7-2) ethanol is significantly higher than that of the control strain, and the glucose residue, glycerol and ethanol are all superior to those of the control strain.
Example 2
For illustrating small test certificate of industrial materials
And (3) taking S.C7-2 further temperature-regulated industrial material pilot scale verification of S.C7-2 screened by secondary liquefied mash of Zhaodong Limited company on whole genome mutation. The process conditions are as follows: the second liquefied mash is used as a substrate, dry powder of a control strain (Angel super wine purchased by Angel Co.) is added after being activated at 35 ℃, S.C7-2 screened by whole genome mutation is inoculated into a seed culture medium (YPD liquid culture medium), a certain number of yeasts are obtained through secondary culture, the liquid mash is subjected to centrifugation and water washing for one time according to the conversion amount of 0.125 hundred million/ml, physiological saline is used for suspending, 350g of the liquid mash is added into a 1L shaking bottle, and the expanded yeast is inoculated according to the inoculation amount of 16 vol%. The test groups of the test strains of the fermentation process are subjected to temperature adjustment (0-16 hours at 32 ℃, then 0.5-2 ℃ is sequentially increased per hour according to the set temperature, the temperature is raised to the set temperature until the final fermentation is finished), two batches of fermentation are carried out, the final temperatures of the first batch are respectively 34.5 ℃,39.5 ℃ and the second batch are respectively 35 ℃, the final temperatures of the second batch are respectively 35 ℃, wherein the basic data of the liquefied mash of the second period of the material are shown in table 2, the detection data of the fermentation chemical titration of each group are shown in table 3, and the HPLC data of each group of fermentation are shown in tables 4 and 5.
Table 2 material second-phase liquefied mash base data:
TABLE 3 chemical titration assay data
Table 4 s.c7-2 strain and control strain first batch fermentation HPLC data:
table 5 second batch fermentation HPLC data for strain C-7-2 and control strain:
the results in tables 3, 4 and 5 show that the indexes of the strain S.C-7-2 in the fermentation at low temperature are not significantly different from those of the Angel super wine yeast used at present, but the indexes of the strain S.C-7-2 in the fermentation at high temperature are significantly improved from those of the Angel super wine yeast used at present, and the strain S.C-7-2 has extremely high application value.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (12)
1. A strain of high-temperature resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) is characterized in that the collection number of the saccharomyces cerevisiae is CGMCC No.21567.
2. A starter culture, characterized in that it comprises the high temperature resistant saccharomyces cerevisiae (Saccharomyces cerevisiae) according to claim 1.
3. The starter culture according to claim 2, wherein the content of the high temperature resistant saccharomyces cerevisiae per gram of the starter culture is 10 5 -10 10 CFU。
4. A starter according to claim 3, wherein the high temperature resistant saccharomyces cerevisiae is present in an amount of 10 per gram of starter 7 -10 9 CFU。
5. Use of the high temperature resistant saccharomyces cerevisiae of claim 1 or the starter of claim 2 or 3 in ethanol production.
6. A process for producing ethanol, comprising: inoculating the high temperature resistant saccharomyces cerevisiae of claim 1 or the starter of claim 2 or 3 into a fermentation medium for ethanol fermentation.
7. The method of claim 6, wherein the method further comprises: inoculating the high-temperature-resistant saccharomyces cerevisiae described in claim 1 or the starter culture described in claim 2 or 3 into a seed culture medium for expansion culture to obtain expansion culture seeds; and then inoculating the expanded seed into a fermentation medium for ethanol fermentation.
8. The method of claim 6 or 7, wherein the fermentation conditions comprise: the temperature is 25-45 ℃ and the time is 65-80 hours.
9. The method of claim 8, wherein the fermentation conditions comprise: fermenting for 12-16 hours at 25-32deg.C; fermenting for 12-16 hours to 20-28 hours, wherein the fermentation temperature is 32-38 ℃; fermenting for 20-28 hours to 45-50 hours at 38-45 ℃; fermenting for 45-50 hours until the fermentation is finished, wherein the fermentation temperature is 35-40 ℃.
10. The method of claim 6 or 7, wherein the fermentation medium is a biomass feedstock liquefaction broth.
11. The method of claim 7, wherein the seed medium is YPD liquid medium and has a pH of 7-7.2.
12. The method of claim 7, wherein the seed is inoculated in an amount of 12-20% by volume based on a high temperature resistant s.cerevisiae concentration of 0.125 million/ml.
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