A met2 gene knockout saccharomyces cerevisiae strain, a construction method and application thereof

Abstract

The application provides a MET2 gene knockout Saccharomyces cerevisiae strain, a construction method and application, and belongs to the technical field of genetic engineering. The MET2 gene knockout Saccharomyces cerevisiae strain is obtained by replacing the MET2 gene in the Saccharomyces cerevisiae strain BSPC040 with a hygromycin B resistance gene and then introducing the pJFE3-XI plasmid. The MET2 gene knockout Saccharomyces cerevisiae strain can significantly improve the glycol alcohol conversion rate of the Saccharomyces cerevisiae BSPC040 strain carrying a xylose metabolic pathway. In the case of consuming the same amount of xylose, compared with the Saccharomyces cerevisiae strain without the MET2 gene knockout, the ethanol yield of the MET2 gene knockout Saccharomyces cerevisiae strain is increased by 23.1%, which provides a new theoretical basis and gene editing strategy for strain optimization in the industrial production of lignocellulosic ethanol.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a MET2 gene knockout Saccharomyces cerevisiae strain, its construction method, and its application. Background Technology

[0002] Biofuels, especially fuel ethanol, are considered an important way to replace fossil fuels and alleviate the energy crisis due to their renewable and low-carbon emission advantages. Current first-generation fuel ethanol production relies on food crops as raw materials, which competes with human and livestock industries for food resources, making long-term sustainable development difficult. Therefore, research on second-generation fuel ethanol is gradually shifting towards utilizing lignocellulosic raw materials such as corn stalks, sugarcane bagasse, rice straw, and sawdust. These resources are widely available, inexpensive, and renewable, possessing the potential for large-scale industrial application.

[0003] Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin, and it must undergo pretreatment and enzymatic hydrolysis to release fermentable monosaccharides. In lignocellulose hydrolysates, glucose and xylose are the main sugar components, with xylose accounting for more than 30% of the hemicellulose hydrolysates. However, because *Saccharomyces cerevisiae* naturally lacks a xylose metabolic pathway, its xylose utilization efficiency is extremely low, resulting in a large amount of xylose resources not being effectively converted into ethanol, becoming a significant bottleneck restricting the development of the second-generation fuel ethanol industry.

[0004] To improve xylose utilization, existing research has explored various technical approaches, such as introducing heterologous xylose metabolism genes to endow yeast with xylose metabolism capabilities; improving xylose transmembrane uptake efficiency by modifying or overexpressing xylose transporters; optimizing the allocation of carbon to ethanol synthesis by regulating key metabolic pathways such as the pentose phosphate pathway and glycolysis; and weakening or deleting bypass genes that lead to byproduct formation to reduce the loss of xylose to non-ethanol products such as xylitol. Despite these advancements, problems such as insufficient xylose conversion efficiency, low ethanol yield, and excessive byproduct accumulation remain unresolved. Summary of the Invention

[0005] The purpose of this invention is to provide a MET2 gene knockout Saccharomyces cerevisiae strain, its construction method, and its application to solve the problem of low ethanol yield in existing strains.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In one aspect, this application provides a MET2 gene knockout Saccharomyces cerevisiae strain, which is obtained by replacing the MET2 gene in Saccharomyces cerevisiae strain BSPC040 with a hygromycin B resistance gene and then transforming it into the pJFE3-XI plasmid.

[0008] Secondly, this application provides a method for constructing a MET2 gene knockout Saccharomyces cerevisiae strain, the method comprising:

[0009] DNA solution was extracted from Saccharomyces cerevisiae strain BSPC040;

[0010] Using the DNA solution as a template, PCR amplification was performed using different primers to obtain the upstream homologous arm sequence MET2-UP and the downstream homologous arm sequence MET2-DOWN of MET2.

[0011] The hygromycin B resistance gene was obtained by PCR amplification using the YG-HYGB plasmid carrying the hygromycin B resistance gene as a template.

[0012] The upstream homologous arm sequence MET2-UP, the hygromycin B resistance gene, and the downstream homologous arm sequence MET2-DOWN of MET2 were ligated using DNA ligase to obtain the ligation product.

[0013] The ligation product was used as a template for PCR amplification and purification to obtain a purified MET2-UP+DOWN+hphB fragment.

[0014] LiAC solution, salmon sperm DNA, and the purified MET2-UP+DOWN+hphB fragment were added to a PEG solution containing Saccharomyces cerevisiae strain BSPC040. The mixture was incubated in a water bath at 30°C for 30 min, followed by heat shock at 42°C for 25 min. After centrifugation, incubation, recentrifugation, and resuspending, the resuspended cells were obtained.

[0015] The resuspended bacterial cells were spread onto YPD solid plates containing hygromycin B and cultured to select positive clones;

[0016] The recombinant plasmid pJFE3-XI carrying the xylose isomerase gene was introduced into the positive clone, and positive transformants were screened using a culture medium containing the hygromycin B antibiotic to obtain the MET2 gene knockout Saccharomyces cerevisiae strain.

[0017] Thirdly, this application provides an application of a MET2 gene knockout Saccharomyces cerevisiae strain, namely, for the preparation of ethanol using xylose as a fermentation carbon source.

[0018] The present invention has the following beneficial effects:

[0019] (1) In this application, the MET2 gene in the Saccharomyces cerevisiae strain BSPC040 is replaced with the hygromycin B resistance gene, thereby obtaining a resistance selection marker while knocking out the MET2 gene. After being replaced with the hygromycin B resistance gene, it is transformed into the pJFE3-XI plasmid to obtain a MET2 gene knockout Saccharomyces cerevisiae strain.

[0020] (2) The MET2 gene knockout Saccharomyces cerevisiae strain can significantly improve the sugar alcohol conversion rate of Saccharomyces cerevisiae BSPC040 strain carrying the xylose metabolism pathway.

[0021] (3) When consuming the same amount of xylose, the ethanol yield of the MET2 gene knockout Saccharomyces cerevisiae strain was increased by 23.1% compared with the Saccharomyces cerevisiae strain without MET2 gene knockout.

[0022] (4) The MET2 gene knockout Saccharomyces cerevisiae strain in this application provides a new theoretical basis and gene editing strategy for strain optimization in the industrial production of lignocellulose ethanol. Attached Figure Description

[0023] Figure 1 This is the recombination process of the MET2-UP+DOWN+hphB fragment, where MET2-UP and MET2-DOWN are the homologous arms flanking the hygromycin B resistance gene, respectively.

[0024] Figure 2 The xylose consumption rate and ethanol production rate of the MET2 gene knockout Saccharomyces cerevisiae strain (i.e., the control strain) in a medium with xylose as the sole carbon source are shown. The solid line represents the xylose consumption rate, and the dashed line represents the ethanol production rate. Detailed Implementation

[0025] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0026] The culture media used in the embodiments of this application include YPD medium, Sc-Ura+X medium with xylose as carbon source, and Sc-Ura+G medium with glucose as carbon source. YPD medium is used for the transformation, activation, culture and preservation of Saccharomyces cerevisiae BSPC040; Sc-Ura+X medium is used for xylose fermentation performance testing experiments; and Sc-Ura+G medium is mainly used for the activation of recombinant strains before fermentation.

[0027] The compositions of the above culture media, according to their concentrations, are as follows:

[0028] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L sterile glucose solution and 20 g / L agar powder, sterilized at 121℃ for 15 min.

[0029] Sc-Ura+X medium: 1.7 g / L Yeast Nitrogen Base (YNB), 5 g / L ammonium sulfate, 0.77 g / L CSM-Ura, 20 g / L xylose and 20 g / L agar powder, sterilized at 121℃ for 15 min.

[0030] Sc-Ura+G medium: 1.7 g / L Yeast Nitrogen Base, 5 g / L ammonium sulfate, 0.77 g / L CSM-Ura, 20 g / L glucose and 20 g / L agar powder, sterilized at 121℃ for 15 min.

[0031] Example 1

[0032] This application includes embodiments of a MET2 gene knockout Saccharomyces cerevisiae strain and its construction method, wherein the construction method includes:

[0033] 1. Extraction of DNA solution from Saccharomyces cerevisiae strain BSPC040

[0034] DNA Extraction Solution I Preparation: Mix 100 mM NaCl, 2% Triton X-100, 1 mM EDTA, 1% SDS and 10 mM Tris-Cl, and adjust the pH to 8.0 to obtain DNA Extraction Solution I.

[0035] Preparation of DNA Extraction Solution II: Phenol, chloroform and isoamyl alcohol are mixed in a volume ratio of 25:24:1 to obtain DNA Extraction Solution II.

[0036] Saccharomyces cerevisiae BSPC040 strain: Saccharomyces cerevisiae BSPC040 strain constructed in application application number 202310974394.X and application title: Application of H3K23A histone point mutation in improving acetic acid tolerance and xylose fermentation performance of Saccharomyces cerevisiae.

[0037] Pick a colony of *Saccharomyces cerevisiae* strain BSPC040 and suspend it in DNA extraction buffer I. Add 0.6 g of acid-washed glass beads to the suspension, then add 200 μL of DNA extraction buffer II. Vortex for 5 min, then centrifuge at 12000 rpm for 5 min. After centrifugation, let it stand for 10 min. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 1 mL of anhydrous ethanol, gently invert to mix, and let stand for 15 min, then centrifuge at 12000 rpm for 10 min. Remove the supernatant and dry thoroughly at room temperature. Add 35 μL of deionized water to the dried DNA to prepare the *Saccharomyces cerevisiae* strain BSPC040 DNA solution.

[0038] 2. PCR amplification of the upstream and downstream homologous arm sequences of the MET2 gene and the hygromycin B resistance gene.

[0039] Using DNA solution from *Saccharomyces cerevisiae* strain BSPC040 as a template, PCR amplification was performed using primers MET2-A and MET2-B, as shown in SEQ ID No. 6 and SEQ ID No. 7, to obtain the upstream homologous arm sequence MET2-UP, as shown in SEQ ID No. 4. Using DNA solution from *Saccharomyces cerevisiae* strain BSPC040 as a template, PCR amplification was performed using primers MET2-C and MET2-D, as shown in SEQ ID No. 8 and SEQ ID No. 9, to obtain the downstream homologous arm sequence MET2-DOWN, as shown in SEQ ID No. 5. Using the YG-HYGB plasmid carrying the hygromycin B resistance gene as a template, PCR amplification was performed using primers HYG-A and HYG-B, as shown in SEQ ID No. 10 and SEQ ID No. 11, to obtain the hygromycin B resistance gene, as shown in SEQ ID No. 2. All PCR amplification products were purified using an agarose gel extraction kit. The primer sequences, PCR amplification system, and PCR amplification procedure used in the embodiments of this application are shown in Tables 1-3.

[0040] Table 1: Primers and sequences used

[0041]

[0042] Table 2: PCR amplification system

[0043]

[0044] Table 3: PCR amplification program

[0045]

[0046] 3. Preparation of MET2-UP+DOWN+hphB fragment

[0047] As attached Figure 1As shown in Table 4, the upstream homologous arm sequence MET2-UP, the hygromycin B resistance gene, and the downstream homologous arm sequence MET2-DOWN of MET2 were ligated using DNA ligase at 50°C for 20 min to obtain the ligation product. After ligation, using the ligation product as a template, PCR amplification was performed using primers MET2-A and MET2-D according to the PCR amplification system shown in Table 2 and the PCR amplification program shown in Table 3. The DNA band size was detected by agarose gel electrophoresis, and the fragment was purified using an agarose gel extraction kit to obtain the MET2-UP+DOWN+hphB fragment shown in SEQ ID No. 3. This fragment includes the hygromycin B resistance gene and the MET2-UP and MET2-DOWN homologous arm sequences located on both sides of the gene, respectively.

[0048] Table 4: DNA Ligation System

[0049]

[0050] 4. Transformation of PCR products

[0051] Saccharomyces cerevisiae BSPC040 was added to YPD liquid medium and cultured at 30°C until the logarithmic growth phase. The culture was centrifuged at 5000 rpm for 5 min, and the Saccharomyces cerevisiae BSPC040 culture was collected. After removing the supernatant, the cells were resuspended in sterile water and centrifuged again at 5000 rpm for 5 min to collect the cells. The cells were then resuspended in 0.1 M LiAC solution and centrifuged at 5000 rpm for 1 min, and the supernatant was removed. 240 μL of 50% PEG solution was added to the centrifuge tube containing the Saccharomyces cerevisiae BSPC040 cells, and after thorough mixing, 36 μL of 1 M LiAC solution, 10 μL of 10 mg / mL salmon sperm DNA, and 30 μL of purified MET2-UP+DOWN+hphB fragment were added. The solution was thoroughly mixed, incubated in a 30°C water bath for 30 min, and then heat-shocked at 42°C for 25 min. After heat shock, centrifuge at 5000 rpm for 5 min, remove the supernatant, add 1 mL of YPD medium, and incubate at 30℃ for 2 h. After incubation, centrifuge at 5000 rpm for 1 min, remove the supernatant, and resuspend the bacterial cells in 400 μL of sterile water to obtain resuspended bacterial cells.

[0052] 70 μL of bacterial culture was spread onto YPD agar plates containing 300 mg / L hygromycin B antibiotic and incubated upside down at 30°C for 3 days. After incubation, single colonies were selected from the plates, DNA was extracted, and transformants were identified by PCR using primers MET2-A and MET2-D. The recombinant plasmid pJFE3-XI carrying the xylose isomerase gene was transformed into positive clones identified by PCR, and the clones were cultured on SC-Ura agar plates containing 300 mg / L hygromycin B antibiotic to screen for positive transformants, thus obtaining the MET2 gene knockout Saccharomyces cerevisiae strain.

[0053] Comparative Example 1

[0054] The recombinant plasmid pJFE3-XI was transformed into the Saccharomyces cerevisiae BSPC040 strain using the method described in "4. Transformation of PCR products" above, and positive transformants were screened on SC-Ura solid plates to obtain the control strain.

[0055] In this embodiment, the xylose fermentation performance of the experimental strain and the control strain were tested. The specific testing process and results are as follows:

[0056] The MET2 gene knockout Saccharomyces cerevisiae strain and the control strain were placed in Sc-Ura+G liquid medium and cultured at 30℃ and 200 rpm until OD. 600 The concentration should be 0.5-1.0. After incubation, centrifuge at 5000 rpm for 1 min and remove the supernatant. Resuspend the bacterial culture in sterile water and collect the cells again. Then, resuspend the cells in Sc-Ura+X liquid medium with xylose as the carbon source and dilute the culture to OD. 600 The concentration was set at 0.2, and the culture was continued in a shaker at 200 rpm and 30℃. During the culture, samples were taken every 24 hours, with 1 mL of fermentation broth taken each time. The samples were centrifuged at 12000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane and injected into sample vials. The xylose and ethanol contents of the sample supernatant were determined using a high-performance liquid chromatography (HPLC) system and an Aminex HPX-87H ion exchange column. The column temperature of the ion exchange column was controlled at 45℃, 5 mM H2SO4 was used as the mobile phase, and the flow rate was set at 0.6 mL / min. Parameters were measured using a differential refractometer. The following is the formula for calculating the sample consumption or generation rate.

[0057]

[0058] Where r is the utilization or generation rate of the detected object during the time interval 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.

[0059] From the appendix Figure 2 It is evident that the control strain consumed all xylose within 120 hours, while the MET2 gene knockout *Saccharomyces cerevisiae* strain consumed it within 192 hours. This indicates that the MET2 gene knockout *Saccharomyces cerevisiae* strain consumed xylose at a lower rate than the control strain. Regarding ethanol production, when all xylose in the culture medium was consumed, the ethanol yield of the control strain was 0.307 ± 0.001 gg. -1 The ethanol yield of the MET2 gene knockout Saccharomyces cerevisiae strain was 0.378 ± 0.002 gg. -1 The yield was 23.1% higher than that of the control strain. This indicates that MET2 gene knockout can significantly improve the sugar alcohol conversion rate of BSPC040 strain, thereby increasing the ethanol yield.

[0060] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of MET2 gene knockout Saccharomyces cerevisiae strain in the preparation of ethanol using xylose as a fermentation carbon source. The MET2 gene knockout Saccharomyces cerevisiae strain was obtained by replacing the MET2 gene in Saccharomyces cerevisiae strain BSPC040 with the hygromycin B resistance gene and then transforming it into the pJFE3-XI plasmid.

2. The application according to claim 1, characterized in that, The sequence of the MET2 gene is shown in SEQ ID No. 1, and the sequence of the hygromycin B resistance gene is shown in SEQ ID No.

2.

3. The application according to claim 1, characterized in that, The ethanol yield increased by 23.1%.

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