A saccharomyces cerevisiae histone H3K18Q point mutation, point mutant and application thereof

Abstract

The present application provides a Saccharomyces cerevisiae histone H3K18Q point mutation, a point mutant and an application, which belongs to the field of genetic engineering technology. The present application obtains a Saccharomyces cerevisiae histone H3K18Q point mutation by mutating the 18th lysine at the N-terminus of the Saccharomyces cerevisiae histone, which can be acetylated, into glutamine, and transfers the pJFE3‑XI plasmid into the Saccharomyces cerevisiae histone H3K18Q point mutation to form a Saccharomyces cerevisiae histone H3K18Q point mutant. The Saccharomyces cerevisiae histone H3K18Q point mutant can significantly increase the xylose consumption rate and ethanol yield of recombinant Saccharomyces cerevisiae, and can be directly used in the performance optimization of lignocellulosic ethanol production strains, providing a reference for the application of epigenetics in second-generation ethanol production, and providing a new theoretical basis and technical route for strain optimization in the industrial production of lignocellulosic ethanol.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and relates to a saccharomyces cerevisiae histone H3K18Q point mutation, a point mutant and applications. Background Art

[0002] With the continuous growth of global energy demand and the increasing severity of environmental pollution, the search for sustainable and environmentally friendly alternative energy sources has become a critical issue that society urgently needs to address. Second-generation bioethanol, as a clean, renewable energy source, has garnered widespread attention in recent years. The development and utilization of second-generation fuel ethanol is considered a key approach to alleviating the energy crisis and reducing greenhouse gas emissions.

[0003] Unlike first-generation bioethanol, which uses fermentable sugars or starchy crops as its raw materials, second-generation bioethanol utilizes lignocellulosic biomass as its core raw material. Lignocellulosic biomass includes agricultural waste such as corn straw and rice and wheat straw, forestry processing residues such as sawdust, and energy crops such as Miscanthus. These non-grain feedstocks are widely available, low-cost, and renewable, driving the development of second-generation bioethanol. Lignocellulose, primarily composed of cellulose, hemicellulose, and lignin, is a complex polymer that requires pretreatment and hydrolysis to be broken down into small monosaccharides for fermentation and production of second-generation ethanol. Xylose is the second most abundant monosaccharide in lignocellulosic hydrolysate, after glucose. However, microbial utilization of xylose is generally poor. Therefore, improving microbial xylose conversion efficiency has become a key area of ​​focus for the development of second-generation ethanol.

[0004] Saccharomyces cerevisiae is the preferred strain for second-generation ethanol fermentation due to its clear genetic background, strong acetic acid tolerance, and mature genetic manipulation techniques. Naturally, Saccharomyces cerevisiae cannot utilize xylose. However, through genetic engineering methods such as introducing exogenous genes, modifying genes related to xylose metabolism and xylose transporter genes, and adaptive domestication, Saccharomyces cerevisiae can acquire a certain degree of xylose metabolism ability. Furthermore, histone modification, a key branch of epigenetics, can also influence a strain's xylose metabolism performance to a certain extent. For example, histone acetylation weakens electrostatic interactions between histones and between histones and DNA, loosening chromatin structure and thus affecting the expression of genes involved in xylose metabolism. Therefore, altering gene expression levels by perturbing histone modifications is an effective means of obtaining strains with improved xylose fermentation performance. Summary of the Invention

[0005] The purpose of the present invention is to provide a saccharomyces cerevisiae histone H3K18Q point mutation, a point mutant and an application thereof, so as to solve the problem of low xylose utilization ability of existing saccharomyces cerevisiae.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present application provides a Saccharomyces cerevisiae histone H3K18Q point mutation, wherein the mutation site of the histone H3K18Q point mutation is that the 18th lysine of the Saccharomyces cerevisiae histone H3 is mutated to glutamine, and the amino acid sequence of the histone H3K18Q point mutation is shown in SEQ ID No. 1.

[0008] In the present application, the amino acid sequence of Saccharomyces cerevisiae histone H3 is shown as SEQ ID No. 2.

[0009] In a second aspect, the present application provides a Saccharomyces cerevisiae histone H3K18Q point mutant, which is obtained by transferring the pJFE3-XI plasmid into the above-mentioned Saccharomyces cerevisiae histone H3K18Q point mutant to obtain a Saccharomyces cerevisiae histone H3K18Q point mutant carrying the pJFE3-XI plasmid.

[0010] In a third aspect, the Saccharomyces cerevisiae histone H3K18Q point mutant in the present application is used to improve the xylose utilization ability of Saccharomyces cerevisiae.

[0011] In a fourth aspect, the Saccharomyces cerevisiae histone H3K18Q point mutant in the present application is used to improve the efficiency of Saccharomyces cerevisiae in producing second-generation ethanol when xylose is used as a carbon source.

[0012] In a fifth aspect, the Saccharomyces cerevisiae histone H3K18Q point mutant in the present application is used to improve the efficiency of ethanol production by Saccharomyces cerevisiae using lignocellulose as a carbon source.

[0013] The present invention has the following beneficial effects:

[0014] This application uses a Saccharomyces cerevisiae histone H3K18Q point mutation, mutating the acetylated lysine 18 at the N-terminus of histones to glutamine. This point mutation is then transferred into the Saccharomyces cerevisiae histone H3K18Q point mutant using the pJFE3-XI plasmid. This Saccharomyces cerevisiae histone H3K18Q point mutant significantly increases the xylose consumption rate and ethanol yield of the recombinant Saccharomyces cerevisiae and can be directly used to optimize the performance of lignocellulosic ethanol production strains. This provides a reference for the application of epigenetics in second-generation ethanol production and offers a new theoretical basis and technical approach for strain optimization in the industrial production of lignocellulosic ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The growth of 14 histone point mutants and the background strain BSPZ042 in the medium with xylose as the carbon source;

[0016] Figure 2Figure 2 shows the xylose utilization and ethanol production of the H3K18Q point mutant and the background strain BSPZ042, ​​where the solid line represents the xylose consumption rate and the dotted line represents the ethanol yield. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0018] The culture media used in this application include YPD medium, Sc-Ura+X medium with xylose as the carbon source, and Sc-Ura+G medium with glucose as the carbon source. Among them, YPD medium is used for the transformation, activation, culture and strain preservation of Saccharomyces cerevisiae; Sc-Ura+X medium is used for plate titration experiments and xylose fermentation performance detection experiments; Sc-Ura+G medium is mainly used for plate titration experiments.

[0019] The specific composition of the above culture medium is:

[0020] YPD medium: 20 g / L peptone, 10 g / L yeast powder, 20 g / L sterile glucose solution, 20 g / L agar powder added to the solid medium, sterilized at 115°C for 15 min.

[0021] Sc-Ura+X medium: 1.7 g / L Yeast Nitrogen Base, 5 g / L ammonium sulfate, 0.77 g / L CSM-Ura, then add 40% sterile xylose solution to a xylose concentration of 20 g / L. Add 20 g / L agar powder to the solid medium and sterilize at 115°C for 15 min.

[0022] Sc-Ura+G medium: 1.7 g / L Yeast Nitrogen Base, 5 g / L ammonium sulfate, 0.77 g / L CSM-Ura, then add 40% sterile glucose solution to a glucose concentration of 20 g / L. Add 20 g / L agar powder to the solid medium and sterilize at 115°C for 15 min.

[0023] Example 1 - Construction of 14 histone point mutants

[0024] 1. Extraction of genomic DNA of histone point mutants in the S288C background strain

[0025] To a 1.5 mL EP tube, add 200 μL of DNA extraction buffer (pH 8.0, consisting of 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl, and 1 mM EDTA). Resuspend the cells of a histone point mutant strain (Cell 134, 1066–1078, September 19, 2008) in the DNA extraction buffer. Add 0.4 g of acid-washed glass beads and 200 μL of DNA extraction buffer II (phenol: chloroform: isoamyl alcohol in a 25:24:1 ratio by volume). Vortex for 1 minute, centrifuge at 12,000 g for 10 minutes, and aspirate the supernatant. Add 1 mL of anhydrous ethanol to the supernatant, mix well, let stand for 10 minutes, and centrifuge again at 12,000 g for 10 minutes. Discard the supernatant, and air-dry the pellet. Add 40 μL of deionized water to the precipitate to dissolve the DNA to obtain the genomic DNA solution.

[0026] 2. Use PCR amplification to obtain DNA fragments carrying mutation sites

[0027] Using the extracted genomic DNA solution as a template, primers P1 and P2 were used to amplify DNA sequences T1-T14 containing the histone mutation sites via PCR. The base sequence of primer P1 is shown in SEQ ID No. 3, specifically 5'-CTTGGTACTAATTCCGGAAG-3', and the base sequence of primer P2 is shown in SEQ ID No. 4, specifically 5'-TGGTGGATTTTGGAAGG-3'. Table 1 lists the PCR amplification procedure, and Table 2 lists the PCR amplification conditions.

[0028] Table 1: PCR amplification program

[0029]

[0030] Table 2: PCR amplification conditions

[0031]

[0032] 3. Transform the DNA fragment T1-T14 carrying the mutation site into the background strain BSPC040

[0033] The T1-T14 fragment obtained by PCR amplification was transformed into the background strain BSPC040 using the LiAC transformation method. For the construction of the background strain BSPC040, please refer to the patent application number 202310974394.X. The specific operation is as follows:

[0034] The background strain BSPC040 was cultured in YPD solid medium for two days and then inoculated into YPD liquid medium. The cells were cultured in a shake flask at 30°C until the logarithmic growth phase. The cells were harvested by centrifugation at 5000g for 1 minute, the supernatant removed, and the cells were resuspended in sterile water. The cells were centrifuged at 5000g for 1 minute, and the supernatant removed. The cells were resuspended in 1 mL of 0.1 M Lithium Acetate (LiAC) and transferred to a 1.5 mL EP tube. The cells were centrifuged at 5000g for 1 minute, and the supernatant removed. 240 μL of 50% PEG was added to the EP tube, vortexed to mix, and 36 μL of 1 M LiAC solution was added. Heat the salmon sperm DNA in boiling water for 5 minutes and place it on ice. Take 10 μL of 10 mg / mL salmon sperm DNA and add it to the EP tube. PCR amplify and obtain 30 μL of each T1-T14 DNA fragment. Vortex and mix. Incubate the 1.5 mL EP tube containing the mixture at 30°C for 30 minutes, then transfer it to 42°C and incubate it for 20 minutes. Collect the bacteria by centrifugation at 5000g for 1 minute, remove the supernatant, add 1 mL of YPD liquid medium, incubate at 30°C for 2 hours, collect the bacteria by centrifugation at 5000g for 1 minute, add 200 μL of sterile water to resuspend the bacteria, and aspirate 100 μL of the bacterial suspension to Sc-Ura+G solid plate and culture at 30°C for 3 days. Since the insertion sequence carries loxp- URA4 -loxp fragment, so it can grow in a medium lacking uracil. The background strain BSPC040 lacks URA4 Positive clones were obtained because they could not grow in this medium.

[0035] Using DNA from the positive clone strain and the background strain BSPC040 as templates, PCR amplification was performed using primers upstream of the insertion site, P3, and downstream of the insertion site, P4. The base sequence of upstream primer P3 is shown in SEQ ID No. 5, specifically 5'-GAGATATACCGTAGCAGTTTCCC-3', and the base sequence of downstream primer P4 is shown in SEQ ID No. 6, specifically 5'-CTGGAGTAATTTTGAGATTGCG-3'. If DNA fragments T1-T14 carrying the mutation site are correctly inserted into the predicted site, the DNA fragment amplified using the positive clone strain as a template will be 1176 bp longer than the DNA fragment amplified using the background strain BSPC040 as a template, verifying that fragments T1-T14 are correctly inserted into the genomic DNA. Table 3 shows the PCR amplification program used in PCR verification, and Table 4 shows the PCR amplification conditions used in PCR verification.

[0036] Table 3: PCR amplification program used in PCR validation

[0037]

[0038] Table 4: PCR amplification conditions used in PCR validation

[0039]

[0040] 4. Transfer the histone point mutation into the pJFE3-XI plasmid

[0041] The histone point mutation was placed in YPD liquid medium and cultured overnight at 30°C and 200 rpm. 10 mL of the bacterial solution was collected at 5000 rpm for 1 minute and then plated onto a solid plate supplemented with 1 g / L 5-FOA. Strains carrying the URA4 gene were unable to grow in the medium supplemented with 5-FOA. Positive clones obtained by screening were those carrying the histone point mutation and missing loxp- URA4 -loxp fragment strain. Take the positive clone and background strain BSPC040 and culture them at 30℃ for 2 days. Use LiAc transformation method to transform URA4 The pJFE3-XI plasmid tagged for screening was transformed into the above strain. After transformation, positive clones were screened using Sc-Ura+G plates. Only strains carrying the pJFE3-XI plasmid could grow on the plates, resulting in the BSPC040 strain with 14 histone point mutants.

[0042] Example 2 - Plate titration experiment of strains

[0043] To identify mutants with superior xylose fermentation performance compared to the background strain, 14 H3 / H4 point mutants introduced into the XI metabolic pathway were screened using a relatively simple plate titration method. The background strain BSPC040, transformed with the plasmid pJFE3-XI, served as a control strain, while the BSPC040 strain transformed with the 14 histone point mutants from the plasmid pJFE3-XI served as the experimental strain. The growth of the control and experimental strains was tested in media containing glucose and xylose as carbon sources, respectively.

[0044] Specifically, the background strain BSPC040 transformed with the pJFE3-XI plasmid and the 14 histone point mutants transformed with the pJFE3-XI plasmid were inoculated into Sc-Ura+G liquid medium and cultured overnight at 30°C and 200 rpm. 600 After the logarithmic growth period of 0.5-1.0, the bacterial solution was diluted with Sc-Ura+G medium to an OD of 600 The bacterial solution of each bacterium was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4The original bacterial solution and the diluted bacterial solution were spotted onto the Sc-Ura+G and Sc-Ura+G solid plates from left to right in descending order of concentration. 5 μL of bacterial solution was added to each sample. After standing at 30°C for 3 days, the samples were photographed. Figure 1 .

[0045] By the attached Figure 1 As can be seen, the Saccharomyces cerevisiae histone H3K18Q point mutant grew significantly better than the control strain in xylose medium, while its growth rate in glucose medium was almost the same as that of the control strain. The growth of other histone point mutations in glucose and xylose medium was almost the same as that of the control strain. This indicates that the H3K18Q point mutant in the BSPZ040 strain can grow on xylose as a carbon source.

[0046] Example 3 - Xylose fermentation performance test of strains

[0047] The xylose fermentation performance of the Saccharomyces cerevisiae histone H3K18Q point mutant and the BSPZ040 strain was tested, with the BSPZ040 strain serving as the control group.

[0048] The Saccharomyces cerevisiae histone H3K18Q point mutant carrying the pJFE3-XI plasmid and the BSPZ040 strain were cultured in Sc-Ura+G liquid medium at 30°C and 200 rpm overnight. 600 When the logarithmic growth phase is 0.5-1.0, the bacteria are washed twice with sterile water and then diluted with Sc-Ura+X liquid medium with xylose as the carbon source to an OD of 600 The fermentation liquid was centrifuged at 12000 g for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane and injected into a sample bottle. The xylose and ethanol contents of the supernatant in the sample bottle were determined by high performance liquid chromatography and Aminex HPX-87H ion exchange column. Figure 2 The column temperature of the ion exchange column was controlled at 45°C, 5 mM H2SO4 was used as the mobile phase, the flow rate was set to 0.6 mL / min, and a differential refractometer was used for parameter determination. The following is the calculation formula for the sample consumption or generation rate.

[0049]

[0050] Where r is the specific utilization or generation rate of the detection object during the period from sampling point m to n; A, B and t are the metabolite concentration, biomass concentration and time at sampling time points n, i and m, respectively.

[0051] By the attached Figure 2 It can be seen that under xylose culture conditions, when fermentation reached 96 h, the Saccharomyces cerevisiae histone H3K18Q point mutant almost consumed all the xylose. At this time, the xylose consumption rate of the Saccharomyces cerevisiae histone H3K18Q point mutant was 0.20±0.01 g L -1 h -1 The ethanol yield was 0.60 ± 0.02 g L -1 h -1 The xylose consumption rate of the control strain BSPZ040 was 0.17±0.01 g L -1 h -1 The ethanol yield was 0.05 ± 0.03 g L -1 h -1 The Saccharomyces cerevisiae histone H3K18Q point mutant showed a 17.65% higher xylose consumption rate and a 20% higher ethanol yield than the control. This indicates that the Saccharomyces cerevisiae histone H3K18Q point mutation significantly increases the xylose consumption rate and ethanol yield of the BSPZ040 strain.

[0052] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Use of a Saccharomyces cerevisiae histone H3K18Q point mutant in improving the xylose utilization ability of Saccharomyces cerevisiae. The Saccharomyces cerevisiae histone H3K18Q point mutant is obtained by transferring the pJFE3-XI plasmid into the BSPZ040 strain carrying the Saccharomyces cerevisiae histone H3K18Q point mutation. The mutation site of the histone H3K18Q point mutation is a mutation of lysine 18 of the Saccharomyces cerevisiae histone H3 to glutamine. The amino acid sequence of the histone H3K18Q point mutation is shown in SEQ ID No.

1.

2. Use of a Saccharomyces cerevisiae histone H3K18Q point mutant in improving the efficiency of second-generation ethanol production in Saccharomyces cerevisiae using xylose as a carbon source. The Saccharomyces cerevisiae histone H3K18Q point mutant is obtained by transferring the pJFE3-XI plasmid into the BSPZ040 strain carrying the Saccharomyces cerevisiae histone H3K18Q point mutation. The mutation site of the histone H3K18Q point mutation is the mutation of lysine 18 of the Saccharomyces cerevisiae histone H3 to glutamine. The amino acid sequence of the histone H3K18Q point mutation is shown in SEQ ID No. 1.

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