CN116731136B - Application of H3K23A histone point mutation in improving acetic acid tolerance and xylose fermentation performance of Saccharomyces cerevisiae - Google Patents

Application of H3K23A histone point mutation in improving acetic acid tolerance and xylose fermentation performance of Saccharomyces cerevisiae Download PDF

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CN116731136B
CN116731136B CN202310974394.XA CN202310974394A CN116731136B CN 116731136 B CN116731136 B CN 116731136B CN 202310974394 A CN202310974394 A CN 202310974394A CN 116731136 B CN116731136 B CN 116731136B
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histone
saccharomyces cerevisiae
acetic acid
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CN116731136A (en
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何德云
鲍晓明
杜臻
徐慧灵
张雪瑞
徐丽丽
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Qilu University of Technology
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
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    • 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
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
    • 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

Abstract

The application relates to an application of saccharomyces cerevisiae H3K23A histone point mutation in improving acetic acid tolerance and xylose fermentation performance of saccharomyces cerevisiae, belonging to the technical fields of genetic engineering, molecular biology and physiology. The adopted histone modification disturbance means respectively replaces amino acids which can be subjected to covalent modification in histone H3/H4 in saccharomyces cerevisiae with alanine which cannot be subjected to any modification, so that histone point mutants with different sites are constructed. And then screening histone mutation sites capable of improving acetic acid tolerance and xylose fermentation performance of the saccharomyces cerevisiae. The strain tolerance under the condition of adding inhibitor acetic acid culture and the strain fermentation performance under the condition of taking xylose as the sole carbon source are detected, so that the H3K23A histone point mutation can simultaneously improve the performances of the strain.

Description

Application of H3K23A histone point mutation in improving acetic acid tolerance and xylose fermentation performance of saccharomyces cerevisiae
Technical Field
The application relates to an application of saccharomyces cerevisiae H3K23A histone point mutation in improving acetic acid tolerance and xylose fermentation performance of saccharomyces cerevisiae, belonging to the technical fields of genetic engineering, molecular biology and physiology.
Background
The increasing scarcity of fossil fuels and the growing energy demand have prompted the development of renewable liquid fuels. As a novel renewable fuel additive, the ethanol has wide application prospect. The first-generation fuel ethanol is produced by taking sugar (such as sugarcane, beet and the like) and starch (such as corn, cassava and the like) agricultural raw materials as substrates and fermenting the substrates by microorganisms. The disadvantage of this production mode is obvious, namely the embarrassing situation of 'competing with people for grain and competing with land' caused by the massive consumption of agricultural raw materials, so the idea of producing second-generation fuel ethanol by taking cheap and easily available renewable lignocellulose as fermentation raw materials is favored.
The total amount of lignocellulose produced on the earth annually accounts for about 60% -80% of the total amount of photosynthetic synthetic biomass, and is widely distributed in forest trees, crop straws, urban household garbage and other fiber wastes. Lignocellulose consists of cellulose, hemicellulose and lignin, and because the internal chemical bonds of the high molecular polymer are complex, microorganisms are difficult to directly decompose and utilize the high molecular polymer, and therefore pretreatment (including physical method, chemical method, physicochemical method, subsection combination method, biological method and the like) and hydrolysis are needed, so that the high molecular polymer can be utilized by the microorganisms after being degraded into fermentable monosaccharides. However, in addition to releasing fermentable monosaccharides, primarily glucose and xylose, various fermentation inhibitors, including acetic acid, formic acid, furfural, hydroxymethylfurfural, peroxides, and phenols, etc., are produced, which inhibit the growth of microorganisms and the fermentation process, reducing ethanol yield. Therefore, microorganisms suitable for fermentation production of second-generation ethanol from lignocellulose have not only fermentation performance of hydrolyzing the whole sugar component by lignocellulose, but also strong inhibitor tolerance. Due to the saccharomyces cerevisiaeSaccharomyces cerevisiae) The strain has the advantages of strong tolerance to ethanol and low pH conditions, insensitivity to phage infection, clear study on physiological characteristics and genetic background, safety, environmental protection and the like, and becomes one of the most potential strains in the field of industrial production of the second-generation ethanol. However, saccharomyces cerevisiae also faces two major challenges in the production of secondary ethanol from lignocellulose: firstly, the saccharomyces cerevisiae has weak tolerance to inhibitors generated in the pretreatment process of lignocellulose; and secondly, the xylose produced by lignocellulose degradation cannot be utilized by the wild saccharomyces cerevisiae, and the xylose utilization capacity of the strain transformed by metabolic pathways is still lower. Thus, it is advantageous toImproving the tolerance of Saccharomyces cerevisiae to fermentation inhibitors and the fermentation performance of xylose by biotechnology means has become an effective way to solve the bottleneck of fermentation of lignocellulose ethanol.
Classical genetics mainly uses gene function and expression level change caused by gene sequence change as research core, such as gene mutation, gene heterozygous loss, microsatellite instability and the like. Epigenetic is the concept corresponding to classical genetics, and uses the change of gene function and expression level caused by non-gene sequence change as the main research content, such as the generation and erasure of DNA and histone modification, nucleosome translocation, non-coding RNA interference, etc. As one of the important branches of epigenetic science, histone modification mainly occurs in the free N short "tail" of histone, and the modification types include more than 20 kinds of methylation, acetylation, phosphorylation, ubiquitination, thresh, proline isomerization, propionylation, butyrylation, and the like. In addition to directly affecting the interaction between nucleosomes and the structure of chromatin, histone modifications are involved in the physiological metabolic processes of cells in an indirect manner, such as activating or inhibiting effector proteins associated with upstream and downstream signaling pathways, mediating the binding of chromatin remodeling proteins or transcription factors to chromatin, and the like. Currently, there is little research on improving Saccharomyces cerevisiae inhibitor tolerance or xylose fermentation performance by means of epigenetic means, and as a leading edge discipline, the application space of the method in solving the bottleneck of lignocellulose ethanol fermentation is very large.
Disclosure of Invention
Aiming at the problems that the prior saccharomyces cerevisiae strain for producing second-generation ethanol has weak acetic acid tolerance and low xylose fermentation performance in the pretreatment process of lignocellulose, the application discloses a construction method of a histone point mutant, and the obtained H3K23A point mutation can obviously improve the acetic acid tolerance and the xylose fermentation performance of the strain.
The technical scheme of the application is as follows:
a histone point mutation, which is an H3K23A histone point mutation; the H3K23A is formed by mutating lysine at the 23 rd position of saccharomyces cerevisiae histone H3 into alanine;
the amino acid sequence of the H3 histone is shown as SEQ ID No. 1;
the amino acid sequence of the histone after the histone point mutation is shown as SEQ ID No. 2.
Another object of the application is to protect a histone point mutant; the histone point mutant has the H3K23A histone point mutation; then transferring pJFE3-XI plasmid into the plasmid, and screening positive clone by using a flat plate to obtain the plasmid.
The application of the histone point mutant in improving the acetic acid tolerance of saccharomyces cerevisiae.
The application of the histone spot mutant in improving the xylose fermentation performance of saccharomyces cerevisiae.
The application of the histone point mutant in improving the production efficiency of the saccharomyces cerevisiae lignocellulose ethanol is provided.
The construction and screening of the mutant are as follows:
(1) Construction of histone H3/H4 Point mutant
Amino acid sites capable of modification in 19H 3/H4 histones were collected; then BSPC039 strain (available from the university of Shandong Bao Xiaoming teacher, see for details Metabolic Engineering (2012) 9-18) for a set of copies of the H3/H4 encoding geneHHT1-HHF1By usingNatMX4The resistance marker gene was replaced and the modified strain was designated BSPC040. The BSPC040 is taken as an initial strain, and the amino acids at 19 selected sites are respectively replaced by alanine which cannot be subjected to any modification, so that 19 histone H3/H4 point mutants are constructed. The histone point mutant used in the application is constructed based on a Dai Junbiao laboratory presented histone H3/H4 mutant library (Cell 134, 1066-1078, september 19, 2008), and pJFE3-XI plasmid capable of over-expressing Xylose Isomerase (XI) is transferred into 19 histone H3/H4 point mutants and BSPC040 strain serving as a starting strain, so that Saccharomyces cerevisiae has certain xylose metabolism capability, and the 19 histone point mutants and a control strain (BSPC 040 strain transferred into pJFE3-XI plasmid) are obtained for acetic acid tolerance and xylose fermentation performance detection.
(2) Screening of high acetate tolerance histone Point mutants
Firstly, adopting a relatively simple flat plate titration experiment to detect the tolerance of the inhibitor acetic acid of 19H 3/H4 histone point mutants introduced into XI xylose metabolic pathway. The growth of the H3K9A, H K23A and H3K36A mutants on solid media supplemented with 100 mM and 125 mM acetic acid, respectively, was found to be significantly better than that of the control strain. The three histone point mutants and the control bacteria are subjected to shake flask culture in a liquid culture medium taking glucose as a carbon source, and a growth curve is drawn, so that the growth trend of the mutants is nearly consistent with that of the control bacteria. The above strain was cultured in glucose liquid medium supplemented with 50 mM acetic acid, and the obtained growth curve results were consistent with the drop plate experiment results. The H3K9A, H3K23A and H3K36A mutants were shown to be more acetic acid tolerant than the control strain.
(3) Screening of histone point mutants with high xylose fermentation performance
In order to obtain mutant strains with simultaneously improved acetic acid tolerance and xylose fermentation performance, three high acetic acid tolerance histone point mutants obtained by screening are subjected to detection of xylose fermentation performance. After the strain is cultured in a culture medium taking xylose as a sole carbon source and xylose consumption and ethanol yield are detected every 12H samples, only the xylose consumption rate and the ethanol yield of the H3K23A point mutant are higher than those of a control strain, and the growth conditions of other mutant strains are almost consistent with those of the control strain. Through the experiment, the acetic acid tolerance and xylose fermentation performance of the H3K23A point mutant obtained by screening from 19 histone mutants are obviously better than those of a control strain.
The beneficial effects of the application are that
The application obtains the H3K23A mutation site capable of improving the xylose fermentation performance and acetic acid tolerance of the strain for the first time, and the research thinking widens the application value of epigenetic science in the field of second-generation ethanol, and provides technical reference and basis for optimizing the special saccharomyces cerevisiae performance of the second-generation ethanol.
The improvement of the xylose fermentation performance of the saccharomyces cerevisiae can improve the production efficiency of the lignocellulose ethanol of the strain. The improvement of the acetic acid tolerance of the saccharomyces cerevisiae can obviously improve the tolerance of the strain to lignocellulose hydrolysis small molecule inhibitors. The experimental scheme can effectively solve the bottleneck problem of lignocellulose ethanol fermentation.
Drawings
The marker loci in FIG. 1 are the mutation sites of histone H3/H4;
FIG. 2 shows the genes encoding histones H3 and H4HHT1-HHF1AndHHT2-HHF2is a model of the transformation. "four" represents the amino acid mutation site,hhtSrepresents the mutated H3-encoding gene,HHFSrepresents the H4 coding gene which is not mutated,HHT2the gene encoding the histone H3 is provided,HHF2a gene encoding histone H4;
FIG. 3 shows the growth of control strain and 19 histone spot mutants on glucose solid medium without acetic acid and with 100 mM and 125 mM acetic acid, respectively;
FIG. 4 is a plot of growth of control strain and H3K9A, H K23A, H K36A histone spot mutant in glucose medium (20 g/L glucose) without addition of acetic acid and with addition of 50 mM acetic acid, wherein A: strain growth curves without acetic acid addition; b: strain growth curve under culture conditions with addition of 50 mM acetic acid;
FIG. 5 shows the oxygen limiting fermentation capacity of control strain and H3K9A, H K23A, H K36A histone point mutation in liquid medium with xylose as sole carbon source (20 g/L xylose); in the figure, the solid line represents the xylose consumption rate, and the broken line represents the ethanol production rate.
Detailed Description
The types and formulations of the culture media used in this example
YPD medium: 10 g/L yeast powder, 20 g/L peptone, 20 g/L agar powder (solid medium), sterilizing at 115deg.C for 15 min, and adding 20 g/L sterile glucose solution before use to obtain YPD medium.
SC-Ura medium: 1.7 g/L Yeast Nitrogen Base,5 g/L ammonium sulfate, 0.77 g/L CSM-Ura,20 g/L agar powder (solid Medium), and sterilizing at 115℃for 15 min. Adding 20G/L glucose to prepare Sc-Ura+G culture medium, or adding 20G/L xylose to prepare Sc-Ura+X culture medium.
EXAMPLE 1 construction of histone Point mutants Using BSPZ040 as starting Strain
The specific experimental method involved in the histone point mutation construction scheme of the application is as follows:
(1) Extraction of histone Point mutant DNA of S288C as background Strain
To a 1.5 mL EP tube was added 200. Mu.L of DNA extract (2% Triton X-100,1% SDS,100 mM NaCl,10 mM Tris-Cl,1 mM EDTA, pH 8.0). Histone point mutant cells (Cell 134, 1066-1078, september 19, 2008) with S288C as background strain were taken into an EP tube to which 200 μl of DNA extract had been added (fig. 1), while 1.0 g glass beads were added to the EP tube, and 200 μl of phenol was added: chloroform: isoamyl alcohol (25:24:1) solution, vortex shaking, and centrifuging at 12000 and g for 5 min after uniform mixing. Transferring the supernatant to an EP tube containing 1 mL absolute ethanol, mixing, standing for more than 10 min, centrifuging for 10 min at 12000 g, removing supernatant, drying, and adding 35 μl ddH 2 O was prepared as a template DNA solution.
(2) PCR amplification to obtain target fragments H1 and H2
One copy of the gene encoding H3/H4HHT1-HHF1Gene useNatMX4Gene substitution, another copyHHT2-HHF2S288C strain DNA carrying group protein point mutation is used as a template, and PCR amplification is carried out to obtain the DNA containing the left sideHHT1- HHF1The upstream homologous sequence of the gene contains on the right sideHHT1-HHF1Homologous sequence downstream of the gene, comprisingNatMX4The long DNA fragment H1 of the gene uses HHT1F1-A (base sequence shown in SEQ ID No.3, specifically 5'-GTTCTTCATCTCCGGTTCTG-3') and HHT1F1-B (base sequence shown in SEQ ID No.4, specifically 5'-ATTCCGTA ACTCT TCTACCTTC-3'). Also as described aboveHHT2-HHF2The DNA of S288C strain carrying group protein point mutation is used as a template, and the primers P3 (the base sequence is shown as SEQ ID No.5, and 5'-CTTGGTACTAATTCCGGAAG-3' in particular) and P4 (the base sequence is shown as SEQ ID No.6, and 5'-TGGTGGATTTTGGAAGG-3' in particular) are used for PCR amplification to obtainHHT2-HHF2DNA sequence H2 containing histone codon mutation, whereinHHT2-HHF2Gene non-coding region carryingURA4A selectable marker gene. Table 1 shows PCR amplificationThe procedure, table 2, shows PCR amplification conditions.
TABLE 1 PCR amplification procedure
TABLE 2 KOD PCR amplification System
(3) Transfer of H1 fragment into Saccharomyces cerevisiae strain and PCR verification of transformant
Activating BSPC040 original strain, inoculating into YPD liquid culture medium, and adjusting to OD 600 0.2. 30. Culturing cells at temperature of 5000g, centrifuging for 5 min to collect thallus, removing supernatant, adding water to resuspend thallus, centrifuging for 1min at 5000g, and removing supernatant. After re-suspending the cells with 1 mL of 0.1M LiAC, the bacterial suspension was transferred to a 1.5 mL EP tube, centrifuged at 5000g for 1min, the supernatant was removed, then 240 μl of 50% PEG was added to the EP tube, after vortexing, 36 μl of 1M LiAC solution, 30 μl of 2 mg/mL salmon sperm DNA (ssDNA), boiled for 5 min before use, 30 μl of H1 fragment, and vortexing. The EP tube with the mixed solution is placed in a water bath kettle at 30 ℃ for incubation for 30 min, and then transferred to the water bath kettle at 42 ℃ for placement for 20 min.5000g centrifugation for 1min, collecting the cells, removing the supernatant, adding 200. Mu.L of sterile water to resuspend the cells, plating 100. Mu.L of the bacterial liquid onto a YPD solid plate added with 100 mg/L Nat antibiotics, culturing at 30 ℃ for 3 days, picking single colonies from the screening plate, shaking the bacteria by using YPD liquid medium, controlling the shaking speed at 200 rpm and controlling the temperature at 30 ℃. Collecting 2 mL bacteria liquid, extracting genome DNA, and extracting according to the method for extracting histone point mutant DNA. The obtained genomic DNA was used as a template, and HHT1F1-C (base sequence shown as SEQ ID No.7, specifically 5'-GACCAATGTTACTTTGCCTG-3') and HHT1F1-D (base sequence shown as SEQ ID No.8, specifically 5'-GCTCTCAGATATTAATGCCG-3') were used as primers to verify whether the H1 fragment was integrated at the correct site by PCR (FIG. 2), thereby obtaining a positive clone with the genome correctly inserted into the H1 fragment.
(4) Transfer of H2 fragment into Saccharomyces cerevisiae strain and PCR verification of transformant
Transferring H2 fragment into Saccharomyces cerevisiae strain inserted with H1 fragment by LiAC transformation method, screening positive clone by using Ura-deficient plate, picking single colony from the screening plate, shaking by using YPD liquid culture medium, and controlling shaking rotation speed at 200 rpm and temperature at 30deg.C. Collecting 2 mL bacteria liquid, extracting genome DNA, and extracting according to the method for extracting histone point mutant DNA. Using the obtained genomic DNA as a template, the correct integration of the H2 fragment was verified by using P5 (base sequence shown as SEQ ID No.9, specifically 5'-GAGATATACCGTAGC AG TTTCCC-3') and P6 (base sequence shown as SEQ ID No.10, specifically 5'-CTGGAGTAATTTTGAGATTGCG-3') as primers (FIG. 2). Wherein the amino acid sequence before H3 mutation is shown as SEQ ID No.1, specifically MARTKQTARKSTGGKAPRKQLASKAARKSAPSTGGVKKPHRYKPGTVALR EIRRFQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAIGALQESVEAYLVSLFEDTNLAAIHAKRVTIQKKDIKLARRLRGERS, the amino acid sequence of H3K23A histone obtained after point mutation is shown as SEQ ID No.2, specifically MARTKQTARKSTGGKAPRKQL ASaAARKSAPSTGGVKKPHRYKPGTVALREIRRFQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAIGALQESVEAYLVSLFEDTNLAAIHAKRVTIQKKDIKLARRLRGERS, wherein lower case letters are mutation sites.
Table 3 shows the PCR amplification procedure used in PCR verification, and Table 4 shows the PCR amplification conditions used in PCR verification.
TABLE 3 PCR amplification procedure
TABLE 4 PCR amplification System
(5) Transfer of pJFE3-XI plasmid into histone Point mutant
Inoculating proper amount of correctly identified histone spot mutant bacteria into YPD liquid culture medium at 30deg.C under shaking at 200 rpmThe culture was carried out overnight in the bed. Collecting 10 mL bacteria liquid, plating onto solid plate with 1 g/L5-FOA to obtain genome inserted H2 fragment and missing loxp-URA4The strain of loxp was cultured at 30℃for 3 days. Positive clones were grown overnight at 30℃in a 200 rpm shaker and transformed with LiAcURA4For screening the marked pJFE3-XI plasmid, transferring the plasmid into a correct histone point mutant, screening positive clones by using a plate of Sc-Ura+G after transformation, and completing the construction of the histone point mutant.
Example 2 detection of Yeast tolerance to acetic acid Using plate titration
Control strain CK: BSPC040 starting strain transformed into pJFE3-XI plasmid;
and inoculating a proper amount of constructed 19 histone point mutation bacteria into Sc-Ura+G liquid culture medium, and culturing overnight in a shaking table at 30 ℃ and 200 rpm. Culturing the bacterial liquid to logarithmic phase (OD) 600 0.5-1.0), the bacterial liquid is diluted to OD by the culture medium 600 At 0.5, the concentration of the bacterial liquid at this time was 10 0 Diluting bacterial liquid from left to right ten times to 10 -1 、10 -2 、10 -3 、10 -4 . And (3) dripping 5 mu L of bacterial liquid from each sample on the diluted bacterial liquid from a point plate from left to right to Sc-Ura+G and a Sc-Ura+G solid flat plate added with 100 Mm and 125 mM acetic acid according to the concentration. The plates were left at 30℃for 3 days and the photographed results showed that the growth of the H3K9A, H K23A and H3K36A point mutants on acetic acid solid medium was significantly better than the control strain (FIG. 3).
Example 3 detection of acetic acid tolerance of strains Using growth curves
An appropriate amount of BSPC040 (control strain CK) transferred into pJFE3-XI plasmid and point mutant yeasts H3K9A, H K23A and H3K36A were inoculated into Sc-Ura+G liquid medium and cultured overnight in a shaking table at 30℃and 200 rpm. Culturing the bacterial liquid to logarithmic phase (OD) 600 0.5-1.0), the bacterial liquid is diluted to OD by Sc-Ura+G culture medium 600 Each strain was equally divided into two equal parts after 0.2, acetic acid was added to one part to 50 mM, the two bacterial solutions were continued to be cultured overnight, and samples were taken every 3 h to measure OD 600 After being plotted into a growth curve, it was found that, in glucose medium,the mutant showed a nearly identical trend to the control bacteria (FIG. 4A). Whereas in glucose broth supplemented with 50 mM acetate, the growth of the H3K9A, H K23A and H3K36A mutants was significantly better than the control strain (FIG. 4B).
Example 4 Saccharomyces cerevisiae xylose fermentation Performance detection
Control strain CK: BSPC040 starting strain transformed into pJFE3-XI plasmid;
a proper amount of yeast is inoculated into Sc-Ura+G liquid culture medium and cultured overnight at 30 ℃ in a shaking table at 200 rpm. Culturing the bacterial liquid to logarithmic phase (OD) 600 0.5-1.0), sc-Ura+X is used to dilute the bacterial solution to OD 600 Culturing at 30deg.C and 200 rpm with shaker at 30deg.C, sampling every 12H, centrifuging at 12000 g for 15 min, filtering the supernatant with 0.22 μm filter membrane, and testing the content of xylose and ethanol in the supernatant by high performance liquid chromatograph and Aminex HPX-87H ion exchange column. The column temperature of the ion exchange column was controlled at 45℃using 5 mM H 2 SO 4 As a mobile phase, the flow rate was set to 0.6. 0.6 mL/min, and the measurement of parameters was performed by using a differential refractometer, and the following was a calculation formula of the sample consumption or generation rate.
Note that: r is the utilization or generation rate of the detection object ratio at the time of sampling points m to n; A. b and t are the metabolite concentration, biomass concentration and time at sampling time points n, i and m, respectively.
After examining the xylose consumption and ethanol yield, only the H3K23A mutant showed higher xylose consumption rate and ethanol yield than the control strain, and the other mutant strains showed almost the same growth as the control strain (FIG. 5).

Claims (3)

1. Use of a histone point mutant with a H3K23A histone point mutation for increasing saccharomyces cerevisiae acetate tolerance; then transferring pJFE3-XI plasmid into the plasmid, and screening positive clones by using a flat plate to obtain the plasmid; the amino acid sequence of the histone after the histone point mutation is shown as SEQ ID No. 2.
2. An application of a histone point mutant in improving the xylose fermentation performance of saccharomyces cerevisiae, wherein the histone point mutant has H3K23A histone point mutation; then transferring pJFE3-XI plasmid into the plasmid, and screening positive clones by using a flat plate to obtain the plasmid; the amino acid sequence of the histone after the histone point mutation is shown as SEQ ID No. 2.
3. An application of a histone point mutant in improving the production efficiency of saccharomyces cerevisiae lignocellulose ethanol, wherein the histone point mutant has H3K23A histone point mutation; then transferring pJFE3-XI plasmid into the plasmid, and screening positive clones by using a flat plate to obtain the plasmid; the amino acid sequence of the histone after the histone point mutation is shown as SEQ ID No. 2.
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