Saccharomyces cerevisiae histone H3K18Q point mutation, point mutant and application

Abstract

The invention provides a saccharomyces cerevisiae histone H3K18Q point mutation, a point mutant and application, and belongs to the technical field of gene engineering. The saccharomyces cerevisiae histone H3K18Q point mutation is obtained by mutating 18-site lysine which can be acetylated at the N end of saccharomyces cerevisiae histone into glutamine, and a pJFE3-XI plasmid is transferred into the saccharomyces cerevisiae histone H3K18Q point mutation to form the saccharomyces cerevisiae histone H3K18Q point mutant. The Saccharomyces cerevisiae histone H3K18Q point mutant can significantly improve the xylose consumption rate and ethanol yield of recombinant Saccharomyces cerevisiae, can be directly used in performance optimization of lignocellulosic ethanol production strains, and provides reference for application of epigenetics in second-generation ethanol production. And a new theoretical basis and a new technical route are provided for strain optimization in industrial production of lignocellulosic ethanol.

Description

Technical Field

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

[0002] With the continuous growth of global energy demand and the increasingly serious environmental pollution problem, finding sustainable and environmentally friendly alternative energy has become a key issue that needs to be solved in today's society. As a clean and renewable energy source, second-generation bioethanol has received extensive attention in recent years. The development and utilization of second-generation fuel ethanol is considered to be one of the important ways to alleviate the energy crisis and reduce greenhouse gas emissions.

[0003] Different from the first generation of bioethanol, which uses fermentable sugars or starch crops as raw materials, the second generation of bioethanol uses lignocellulosic biomass as the core raw material. Among them, lignocellulosic biomass includes agricultural waste such as corn straw, rice and wheat straw, forestry processing residues such as sawdust, and energy crops such as Miscanthus. These non-grain raw materials have the characteristics of wide sources, low cost and renewable, which have promoted the development of second generation bioethanol. Lignocellulose is mainly composed of cellulose, hemicellulose and lignin. This type of high molecular polymer has a complex structure and needs to be degraded into small molecular monosaccharides through pretreatment and hydrolysis, and then it can be used for the fermentation production of second generation ethanol. Xylose is the second largest monosaccharide in lignocellulosic hydrolysate after glucose. However, microorganisms are generally weak in the ability to utilize xylose. Therefore, improving the xylose conversion efficiency of microorganisms has become one of the important directions to promote the development of second generation ethanol.

[0004] Saccharomyces cerevisiae has become the preferred strain for second-generation ethanol fermentation due to its clear genetic background, strong acetic acid tolerance and mature genetic manipulation technology. Natural Saccharomyces cerevisiae cannot utilize xylose, but by introducing exogenous genes, modifying xylose metabolism-related genes and xylose transporter genes, adaptive domestication and other genetic engineering methods, Saccharomyces cerevisiae can acquire a certain xylose metabolism ability. In addition, histone modification, as an important branch of epigenetics, can also affect the xylose metabolism performance of strains to a certain extent. For example, histone acetylation can loosen the chromatin structure by weakening the electrostatic interaction between histones and between histones and DNA, thereby affecting the expression of genes related to xylose metabolism. Therefore, changing the expression level of genes by perturbing histone modification has become an effective means to obtain excellent 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 the existing saccharomyces cerevisiae.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, the present application provides a Saccharomyces cerevisiae histone H3K18Q point mutation. The mutation site of the histone H3K18Q point mutation is that the 18th lysine of Saccharomyces cerevisiae histone H3 is mutated into glutamine, and the amino acid sequence of the histone H3K18Q point mutation is as shown in SEQ ID No.1.

[0007] In the present application, the amino acid sequence of Saccharomyces cerevisiae histone H3 is as shown in SEQ ID No.2.

[0008] In the 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 mutation to obtain a Saccharomyces cerevisiae histone H3K18Q point mutant carrying the pJFE3-XI plasmid.

[0009] In the third aspect, the Saccharomyces cerevisiae histone H3K18Q point mutant in the present application is used to improve the xylose utilization ability of Saccharomyces cerevisiae.

[0010] In the fourth aspect, the Saccharomyces cerevisiae histone H3K18Q point mutant in the present application is used to improve the efficiency of producing second-generation ethanol when Saccharomyces cerevisiae uses xylose as a carbon source.

[0011] In the fifth aspect, the Saccharomyces cerevisiae histone H3K18Q point mutant in the present application is used to improve the efficiency of producing ethanol when Saccharomyces cerevisiae uses lignocellulose as a carbon source.

[0012] The present invention has the following beneficial effects: In the present application, the 18th lysine at the N-terminus of Saccharomyces cerevisiae histone, which can be acetylated, is mutated into glutamine to obtain a Saccharomyces cerevisiae histone H3K18Q point mutation, and the pJFE3-XI plasmid is transferred into the Saccharomyces cerevisiae histone H3K18Q point mutation to form a Saccharomyces cerevisiae histone H3K18Q point mutant. This Saccharomyces cerevisiae histone H3K18Q point mutant can significantly improve the xylose consumption rate and ethanol yield of recombinant Saccharomyces cerevisiae, and can be directly used for the performance optimization of lignocellulose ethanol production strains, providing a reference for the application of epigenetics in second-generation ethanol production and a new theoretical basis and technical route for strain optimization in lignocellulose ethanol industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Growth of 14 histone point mutants and the background strain BSPZ042 in a medium with xylose as the carbon source; Figure 2For 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 dashed line represents the ethanol production rate. Detailed implementation mode

[0014] The technical solution of the present invention will be further explained and illustrated below through specific examples.

[0015] The culture media used in this application include YPD culture medium, Sc-Ura+X culture medium with xylose as the carbon source, and Sc-Ura+G culture medium with glucose as the carbon source. Among them, YPD culture medium is used for the transformation, activation, culture and strain preservation of Saccharomyces cerevisiae; Sc-Ura+X culture medium is used for plate titration experiments and xylose fermentation performance detection experiments; Sc-Ura+G culture medium is mainly used for plate titration experiments.

[0016] The specific compositions of the above culture media are as follows: YPD culture medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L sterile glucose solution, and 20 g / L agar powder is added to the solid culture medium, sterilized at 115 °C for 15 min.

[0017] Sc-Ura+X culture medium: 1.7 g / L Yeast Nitrogen Base, 5 g / L ammonium sulfate, 0.77 g / L CSM-Ura, and then 40% sterile xylose solution is added until the xylose concentration is 20 g / L. 20 g / L agar powder is added to the solid culture medium, sterilized at 115 °C for 15 min.

[0018] Sc-Ura+G culture medium: 1.7 g / L Yeast Nitrogen Base, 5 g / L ammonium sulfate, 0.77 g / L CSM-Ura, and then 40% sterile glucose solution is added until the glucose concentration is 20 g / L. 20 g / L agar powder is added to the solid culture medium, sterilized at 115 °C for 15 min.

[0019] Example 1 - Construction of 14 histone point mutants 1. Extraction of genomic DNA of histone point mutants with S288C as the background strain Add 200 μL of DNA extraction solution with a pH of 8.0 and a composition including 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl, and 1 mM EDTA to an EP tube with a volume of 1.5 mL. Suspend the cells of the histone point mutant with S288C as the background strain (Cell 134, 1066–1078, September 19, 2008) in the DNA extraction solution; add 0.4 g of acid-washed glass beads and 200 μL of DNA extraction solution II with a volume ratio of phenol:chloroform:isoamyl alcohol of 25:24:1. After vortexing for 1 min, centrifuge at 12,000 g for 10 min and aspirate the supernatant. Add 1 mL of absolute ethanol to the supernatant, mix well and let stand for 10 min, then centrifuge at 12,000 g for 10 min again. Pour out the supernatant and let the precipitate dry. Add 40 μL of deionized water to the precipitate to dissolve the DNA, which is the genomic DNA solution.

[0020] 2. Obtain the DNA fragment carrying the mutation site by PCR amplification Using the extracted genomic DNA solution as a template, use primers P1 and P2 to obtain the DNA sequence T1-T14 containing the histone mutation site by PCR amplification. Among them, 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 shows the PCR amplification program, and Table 2 shows the PCR amplification conditions.

[0021] Table 1: PCR amplification program Table 2: PCR amplification conditions 3. Transfer the DNA fragment T1-T14 carrying the mutation site into the background strain BSPC040 Transfer the T1-T14 fragment obtained by PCR amplification into the background strain BSPC040 using the LiAC transformation method. Among them, the construction of the background strain BSPC040 can refer to the patent with the application number 202310974394.X. The specific operation is as follows: The background strain BSPC040 was cultured in YPD solid medium for two days, then inoculated into YPD liquid medium and cultured in a shaker at 30 °C until the logarithmic growth phase. The cells were collected by centrifugation at 5000g for 1 min, the supernatant was removed, and the cells were resuspended with sterile water. After centrifugation at 5000g for 1 min, the supernatant was removed. After resuspending the cells with 1 mL of 0.1 M LiAC (Lithium acetate), the cell suspension was transferred to a 1.5 mL EP tube and centrifuged at 5000g for 1 min to remove the supernatant. Subsequently, 240 μL of 50% PEG was added to the EP tube, and after vortexing and mixing, 36 μL of 1 M LiAC solution was added. The salmon sperm DNA was heated in boiling water for 5 min and then placed on ice. 10 μL of 10 mg / mL salmon sperm DNA was added to the EP tube. 30 μL of each of the T1 - T14 DNA fragments was obtained by PCR amplification, vortexed and mixed. The 1.5 mL EP tube containing the mixture was incubated at 30 °C for 30 min and then transferred to 42 °C for 20 min. The cells were collected by centrifugation at 5000g for 1 min, the supernatant was removed, 1 mL of YPD liquid medium was added, and after incubation at 30 °C for 2 h, the cells were collected by centrifugation at 5000g for 1 min and resuspended with 200 μL of sterile water. 100 μL of the cell suspension was pipetted onto a Sc-Ura+G solid plate and cultured at 30 °C for 3 days. Since the inserted sequence carries the loxp- URA4 -loxp fragment, it can grow in the medium lacking uracil. The background strain BSPC040 cannot grow in this medium because it lacks URA4 and thus positive clone strains were obtained.

[0022] Using the DNA of the positive clone strain and the background strain BSPC040 as templates, PCR amplification was carried out with the upstream primer P3 at the insertion site and the downstream primer P4 at the insertion site as primers. Among them, the base sequence of the upstream primer P3 is shown in SEQ ID No.5, specifically 5’ -GAGATATACCGTAGCAGTTTCCC- 3’, and the base sequence of the downstream primer P4 is shown in SEQ ID No.6, specifically 5’ -CTGGAGTAATTTTGAGATTGCG- 3’. If the DNA fragments T1 - T14 carrying the mutation sites are correctly inserted into the predicted sites, the DNA fragment amplified using the positive clone strain as the template should be 1176 bp longer than the DNA fragment amplified using the background strain BSPC040 as the template, so as to verify whether the fragment T1 - T14 is correctly inserted into the genomic DNA. Among them, Table 3 shows the PCR amplification program used in the PCR verification, and Table 4 shows the PCR amplification conditions used in the PCR verification.

[0023] Table 3: PCR amplification program used in PCR verification Table 4: PCR amplification conditions used in PCR verification 4. Transfer the pJFE3-XI plasmid into the histone point mutation Place the histone point mutation in YPD liquid medium and culture overnight at 30 °C and 200 rpm. Take 10 mL of the bacterial solution, centrifuge at 5000 rpm for 1 min to collect the bacteria, and then spread it on a solid plate supplemented with 1 g / L 5-FOA. Strains carrying the URA4 gene cannot grow in the medium supplemented with 5-FOA. The positive clones obtained by screening are strains carrying the histone point mutation and lacking the loxp- URA4 -loxp fragment. Incubate the positive clones and the background strain BSPC040 at 30 °C for 2 days. Using the LiAc transformation method, transfer the pJFE3-XI plasmid with URA4 as the screening marker into the above strains. After transformation, use the Sc-Ura+G plate to screen for positive clones. Only the strains carrying the pJFE3-XI plasmid can grow on the plate, and 14 BSPC040 strains with histone point mutations are obtained.

[0024] Example 2 - Plate titration experiment of strains To screen for mutants with better xylose fermentation performance than the background strain, a relatively simple plate titration method was first used to screen 14 H3 / H4 point mutants introduced with the XI metabolic pathway. The background strain BSPC040 transfected with the plasmid pJFE3-XI was used as the control strain, and the 14 BSPC040 strains with histone point mutations transfected with the plasmid pJFE3-XI were used as the experimental strains. The growth of the control strain and the experimental strains in the media with glucose as the carbon source and xylose as the carbon source was detected.

[0025] Specifically, inoculate the background strain BSPC040 transfected with the pJFE3-XI plasmid and the 14 histone point mutants transfected with the plasmid pJFE3-XI into the Sc-Ura+G liquid medium respectively, and culture overnight at 30 °C and 200 rpm. After the bacterial solution grows to the logarithmic growth phase with an OD 600 of 0.5 - 1.0, dilute the bacterial solution with the Sc-Ura+G medium to an OD 600 of 0.5. Dilute the bacterial solution of each bacterium to 10 -1 、10 -2 、10 -3 、10 -4 respectively. Spot the original bacterial solution and the diluted bacterial solutions onto the Sc-Ura+G and Sc-Ura+G solid plates from left to right according to the concentration from high to low, and add 5 μL of the bacterial solution for each sample. After placing at 30 °C for 3 days, take pictures respectively to obtainFigure 1 。

[0026] As shown in the appendix Figure 1 It can be seen that the growth of the histone H3K18Q point mutant of Saccharomyces cerevisiae in the medium with xylose as the carbon source is significantly better than that of the control strain, while the growth rate in the glucose medium is almost indistinguishable from that of the control strain. For other histone point mutants, there is little difference in growth between the glucose and xylose media compared to the control strain. This indicates that the H3K18Q point mutant of strain BSPZ040 can grow using xylose as the carbon source.

[0027] Example 3 - Detection of Xylose Fermentation Performance of Strains The xylose fermentation performance of the histone H3K18Q point mutant of Saccharomyces cerevisiae and strain BSPZ040 was detected, with strain BSPZ040 as the control group. Specifically: The histone H3K18Q point mutant of Saccharomyces cerevisiae carrying the pJFE3-XI plasmid and strain BSPZ040 were respectively cultured overnight at 30 °C and 200 rpm in Sc-Ura+G liquid medium. When the bacterial liquid was cultured to the logarithmic growth phase with an OD 600 of 0.5 - 1.0, it was washed twice with sterile water, and then the bacterial liquid was diluted to an OD 600 of 0.2 with Sc-Ura+X liquid medium using xylose as the carbon source, and continued to be cultured in a shaker at 200 rpm and 30 °C. Samples were taken every 12 h, 1 mL of fermentation broth was taken each time, centrifuged at 12000 g for 15 min, the supernatant after centrifugation was filtered through a 0.22 μm filter membrane and injected into a sample bottle, and the xylose and ethanol contents in the supernatant in the sample bottle were determined using a high-performance liquid chromatograph and an Aminex HPX-87H ion exchange column, obtaining Figure 2 。Among them, 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 at 0.6 mL / min, and a differential refractometer was used for parameter determination. The following is the calculation formula for the consumption or production rate of the sample.

[0028] Among them, r is the specific utilization or production rate of the detection object at the time between sampling points m and n; A, B, and t are the metabolite concentration, biomass concentration, and time at sampling time points n, i, and m, respectively.

[0029] As shown in the appendix Figure 2 It can be seen that under xylose culture conditions, when fermented for 96 h, the histone H3K18Q point mutant of Saccharomyces cerevisiae almost consumed all the xylose. At this time, the xylose consumption rate of the histone H3K18Q point mutant of Saccharomyces cerevisiae was 0.20 ± 0.01 g L -1 h-1 , the ethanol yield was 0.60 ± 0.02 g L -1 h -1 ; while the xylose consumption rate of the control group BSPZ040 strain was 0.17 ± 0.01 g L -1 h -1 , and the ethanol yield was 0.05 ± 0.03 g L -1 h -1 , the xylose consumption rate of the Saccharomyces cerevisiae histone H3K18Q point mutant was 17.65% higher than that of the control group, and the ethanol yield was 20% higher than that of the control group. This indicates that the Saccharomyces cerevisiae histone H3K18Q point mutation significantly improves the xylose consumption rate and ethanol yield of the BSPZ040 strain.

[0030] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications and changes can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A Saccharomyces cerevisiae histone H3K18Q point mutation, characterized in that, The mutation site of the histone H3K18Q point mutation is the 18th lysine of Saccharomyces cerevisiae histone H3 mutated into glutamine, and the amino acid sequence of the histone H3K18Q point mutation is shown as SEQ ID No.

1.

2. The Saccharomyces cerevisiae histone H3K18Q point mutation according to claim 1, characterized in that, The amino acid sequence of the Saccharomyces cerevisiae histone H3 is shown as SEQ ID No.

2.

3. A Saccharomyces cerevisiae histone H3K18Q point mutant, characterized in that, The point mutant is obtained by transferring the pJFE3-XI plasmid into the Saccharomyces cerevisiae histone H3K18Q point mutation described in claim 1 or 2.

4. The Saccharomyces cerevisiae histone H3K18Q point mutant described in claim 3 is used to improve the xylose utilization ability of Saccharomyces cerevisiae.

5. The Saccharomyces cerevisiae histone H3K18Q point mutant described in claim 3 is used to improve the efficiency of producing second-generation ethanol when Saccharomyces cerevisiae uses xylose as a carbon source.

6. The Saccharomyces cerevisiae histone H3K18Q point mutant described in claim 3 is used to improve the efficiency of producing ethanol when Saccharomyces cerevisiae uses lignocellulose as a carbon source.

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