Beta-glucan genetically engineered bacterium and construction method thereof

Abstract

The invention discloses a beta-glucan genetically engineered bacterium and a construction method thereof, and belongs to the technical field of genetic engineering, and the beta-glucan genetically engineered bacterium is constructed by replacing a promoter of a PGM2 gene and / or a UGP1 gene in saccharomyces cerevisiae with a strong promoter and knocking out an ALG5 gene by using a gene editing technology. The beta-glucan yield of the genetically engineered bacterium reaches 2.83 g / 100g, which is nearly twice that of a control group. The method is used for efficiently synthesizing the beta-glucan.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a beta-glucan genetically engineered bacterium and a construction method thereof. BACKGROUND

[0002] Beta-glucan is a natural polysaccharide connected by beta-glycoside bonds, and is widely distributed in the cell wall structure of yeast and fungi. Beta-glucan has the functions of immunomodulation, antioxidant, anti-inflammatory and tissue repair, and has a broad application prospect in the fields of food, medicine and cosmetics. However, the traditional source of beta-glucan still has the problems of low yield, unstable structure and single function, which is difficult to meet the actual demand. Natural beta-glucan is usually deposited in the cell wall and is difficult to be secreted outside the cell, which also brings certain difficulties to the downstream separation and purification.

[0003] Compared with the traditional extraction method from the bran of oat, barley and the like, the fermentation production by using genetically edited engineering bacteria is a new strategy for obtaining beta-glucan, but still faces a series of challenges. The core difficulty lies in the complexity of the beta-glucan synthesis pathway, the precise metabolic regulation network inside the cell and the stability of the engineered strain. For example, the biosynthesis of beta-glucan is not controlled by a single gene, but involves a complex pathway in which multiple enzymes such as GSC, FKS and the like catalyze the subunits and regulatory subunits synergistically. At present, the key rate-limiting step in the synthesis pathway is still unclear. Blind overexpression of a single or multiple genes may lead to metabolic imbalance and accumulation of intermediate metabolites, rather than effective improvement of the yield of the end product. SUMMARY

[0004] The purpose of the present application is to provide a beta-glucan genetically engineered bacterium and a construction method thereof. The CRISPR-Cas9 gene editing technology is used to solve the above problems by overexpression and knockout of specific genes, and a genetically engineered bacterium with high yield of beta-glucan is obtained. In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: A construction method of a beta-glucan genetically engineered bacterium, which is to replace the PGM2 gene and / or UGP1 the promoter of the gene with a strong promoter in Saccharomyces cerevisiae.

[0005] As a limitation of the present application, the PGM2 gene and UGP1 the promoter of the gene are replaced with a strong promoter, and the ALG5 gene is knocked out.

[0006] As a further limitation of the present application, the promoter replacement and knockout are realized by CRISPR-Cas9 gene editing technology.

[0007] As a further limitation of the present application, when the promoter is replaced, PGM2 The sgRNA primer of the gene is sg- PGM2 -F and sg- PGM2 -R as shown in SEQ ID NO. 2. UGP1 The sgRNA primer of the gene is sg- UGP1 -F and sg- UGP1 -R as shown in SEQ ID NO. 4.

[0008] As a further limitation of the present application, when the promoter is replaced, PGM2 The sgRNA primer of the gene is sg- PGM2 -F and sg- PGM2 -R as shown in SEQ ID NO. 2. UGP1 The sgRNA primer of the gene is sg- UGP1 -F and sg- UGP1 -R as shown in SEQ ID NO. 4.

[0009] As a further limitation of the present application, the promoter is replaced by preparing a donor DNA, which is Donor DNA-OE- PGM2 or Donor DNA-OE- UGP1 ; The primer nucleotide sequence of the Donor DNA-OE- PGM2 is shown as SEQ ID NO. 5 and SEQ ID NO. 6. The primer nucleotide sequence of the Donor DNA-OE- UGP1 is shown as SEQ ID NO. 7 and SEQ ID NO. 8.

[0010] Wherein, by designing a donor DNA template Donor DNA-EO containing an enhanced promoter. By using the homologous recombination repair mechanism, the Donor DNA-EO and the reconstructed plasmid are introduced into the cells with DNA double-strand break, to promote the gene repair process of the cells to achieve the overexpression of the target gene.

[0011] To construct a complete expression vector containing both the sgRNA expression structure and the Cas9 gene, the original plasmid was first subjected to double restriction enzyme digestion using Bcl-I and Swa-I to obtain the vector fragment. Bcl-I digestion produces sticky ends, cleaving the four bases of "GATC"; while Swa-I digestion produces blunt ends. Therefore, in primer design, the nucleotide hybridization sequence was arranged with four bases of "GATC" from 5' to 3', and a 20bp guide RNA was designed based on the target gene. scaffold The first half of the RNA (5'- scaffold RNA (this fragment contains an sgRNA structure). Finally, the vector fragment is ligated with the designed primer nucleotide hybridization sequence using T4 ligase to form a complete expression vector.

[0012] As a limitation of this invention, knockout ALG5 When processing genes, the primer nucleotide sequences of DonorDNA-KO are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0013] As a further limitation of the present invention, the strong promoter is *Saccharomyces cerevisiae*. TDH3 The promoter of the gene, the brewer's yeast is Saccharomyces cerevisiae BY4741.

[0014] The present invention also provides a genetically engineered bacterium, which is constructed by the above-described method for constructing a β-glucan genetically engineered bacterium.

[0015] The present invention also provides a plasmid, which is prepared in accordance with the above-described method for constructing a β-glucan-engineered bacterium.

[0016] The principle of this invention is to use CRISPR-Cas9 technology to... PGM2 , UGP1 The gene promoter was replaced with the strong promoter pTDH3, and single and double overexpression strains were constructed, respectively. The double overexpression strain was knocked out. ALG5 Genes were used to block UDP-glucan shunting, resulting in a high-β-glucan-producing strain, BY4741-pTDH3- PGM2 - UGP1 - ΔALG5 .

[0017] By adopting the above technical solution, the technical progress achieved by this invention compared with the prior art is as follows: This invention unexpectedly discovered during the construction of β-glucan-engineered bacteria that single overexpression... PGM2 Genes and UGP1 In genetically engineered bacteria with β-glucan genes, β-glucan synthesis increased slightly but not significantly, while whenPGM2 Genes and UGP1 When genes are overexpressed synchronously, there is a synergistic effect between the two. Compared with overexpression alone, gene expression is significantly promoted, which in turn enhances the synthesis process of cell wall β-glucan and promotes the operation of the β-glucan synthesis pathway in the cell wall synthesis pathway.

[0018] The β-glucan-engineered bacteria BY4741- prepared by the construction method of the present invention pPGM2 - pUGP1 - ΔALG5 RT-qPCR results showed that PGM2 , UGP1 Expression levels increased by 4.27-fold and 3.85-fold respectively compared to the control group, downstream FKS1 Gene expression was increased by 5.69 times, which is 1.61 times that of double overexpression, while ALG5 expression was almost zero, thus which is more conducive to the efficient synthesis of β-glucan. HPLC detection further confirmed that the β-glucan content of this strain reached 2.83g / 100g, while the β-glucan content of the control group was 1.53g / 100g, which is 1.85 times that of the control group. This confirms that gene editing significantly enhances the ability of β-glucan synthesis by synergistically regulating precursor supply and metabolic diversion.

[0019] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the Donor DNA-OE primer design process in Example 1 of the present invention; Figure 2 This is an electrophoresis diagram of the construction results of the single-gene overexpression strain in Example 1 of the present invention. In the diagram, (a) is BY4741-pTDH3- PGM2 Electrophoresis verification results of the engineered strains: lane M is for Maker, and lane 2 is for strain BY4741. PGM2 Gene PCR amplification results, (b) is BY4741-pTDH3- UGP1 Electrophoresis verification results of engineered strains: lane M is for Maker, and lane 1 is for strain BY4741. UGP1 Gene PCR amplification results, lane 2 is strain BY4741-pTDH3- UGP1 of UGP1 Gene PCR amplification results; Figure 3 In Embodiment 1 of the present invention PGM2 The image shows the sequencing results of the CRISPR expression vector for the target gene. Figure 4 In Embodiment 1 of the present invention UGP1The image shows the sequencing results of the CRISPR expression vector for the target gene. Figure 5 This is an electrophoresis diagram of the construction results of the dual-gene overexpression strain in Example 1 of this invention. Lane M in the diagram is Maker, and lanes 1 and 2 are for strain BY4741, respectively. PGM2 , UGP1 The gene PCR amplification results showed that lanes 3 and 4 contained strain BY4741-pTDH3- PGM2 - UGP1 of PGM2 , UGP1 Gene PCR amplification results; Figure 6 This is a schematic diagram of the Donor DNA-KO primer design process in Example 1 of the present invention; Figure 7 This is an electrophoresis diagram of the gene knockout strain construction results in Example 1 of the present invention. Lane M is the Maker lane, and lane 1 is the gene knockout strain BY4741. ALG5 Gene PCR amplification results, lane 2 is strain BY4741-pTDH3-PGM2-UGP1- Δ After editing ALG5 ALG5 Gene PCR amplification results; Figure 8 The image shows the results of real-time fluorescence quantitative PCR detection of genetically engineered bacteria in Example 1 for efficacy verification. Figure 9 The chromatogram of group BY4741 in Example 2 for efficacy verification; Figure 10 To verify the effect, in Example 2, BY4741- pPGM2 - pUGP1 - ΔALG5 Chromatographic analysis of the group. Detailed Implementation

[0021] The present invention will be further described in detail below through specific embodiments. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.

[0022] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0023] The strains and plasmids used in the examples include: Escherichia coli competent cells DH5α, Escherichia coli competent cells JM110, plasmid pML104, and Saccharomyces cerevisiae strain BY4741 (MATa, His3Δ1, Leu2Δ0, Met15Δ0, Ura3Δ0).

[0024] The molecular biology experiments described in this example, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly performed in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the competent cell method used for transformation and the method for preparing competent cells were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions can be determined through simple experiments if necessary.

[0025] Example 1: A β-glucan-engineered bacterium and its construction method This embodiment describes a β-glucan genetically engineered bacterium, BY4741- pPGM2 - pUGP1 - ΔALG5 Its construction method, using CRISPR-Cas9 gene editing technology to introduce Saccharomyces cerevisiae BY4741 ( Saccharomyces cerevisiae )middle PGM2 Genes and UGP1 The original promoter of the gene was replaced with Saccharomyces cerevisiae. TDH3 Gene promoter, knockout ALG5 Gene generation. This specifically includes the following steps performed sequentially: (a) Overexpression of Saccharomyces cerevisiae PGM2 and UGP1 Gene (1) Obtaining hybridization nucleotide fragment sgRNA: The target gene of Saccharomyces cerevisiae BY4741 was obtained from the NCBI website. PGM2 and UGP1 Based on the DNA sequence, sgRNA primer sequences were designed, as shown in Table 1.

[0026] Table 1 sgRNA primer sequences Anneal the primer fragments to obtain samples containing... PGM2 -guide RNA and UGP1 -The hybrid nucleotide fragment sgRNA of the guide RNA. The primer fragment annealing system consisted of 10 μL containing 3 μL each of guide-F and guide-R, 3 μL of NEBbuffer 2, and 1 μL of water. The primer fragment annealing program was as follows: Step 1: 95ºC, 10 min; Step 2: 85ºC, 5 min 30 s; Step 3: 25ºC, 5 min.

[0027] (2) Construction of reconstructed plasmid pML104- PGM2 and pML104- UGP1The pML104 original plasmid containing the Cas9 gene was double-digested with restriction endonucleases BclⅠ and SwaⅠ to obtain the vector fragment. BclⅠ digestion produced sticky ends, cleaving the four bases "GATC"; while SwaⅠ digestion resulted in blunt ends. Therefore, in primer design, the nucleotide hybridization sequence was arranged from 5' to 3' as four bases "GATC". A 20bp guide RNA was designed based on the target gene. scaffold The first half of the RNA (5'- scaffold RNA (this fragment contains the sgRNA structure). Finally, the vector fragment was ligated with the designed primer nucleotide hybridization sequence using T4 ligase to obtain the complete expression vector pML104- containing the sgRNA expression structure and the Cas9 gene. PGM2 and pML104- UGP1 .

[0028] For the reconstructed plasmid pML104- PGM2 and pML104- UGP1 The ligation was confirmed to be correct after transformation and sequencing analysis.

[0029] (3) Preparation of Donor DNA-OE: 40 bases from the latter half of the target gene promoter, TDH3 The promoter of the gene is sequentially linked to the first 38 bases of the target gene, designed as DonorDNA-OE-. PGM2 DonorDNA-OE- UGP1 ,like Figure 1 DonorDNA-OE- PGM2 DonorDNA-OE- UGP1 The upstream sequence 5' end 59bp is the upstream primer, and the downstream sequence 5' end 59bp is the downstream primer, as shown in Table 2.

[0030] Table 2 Donor DNA-OE PCR Primer Sequences A 50 μL amplification system was used: 1 μL template DNA (yeast cell BY4741), 2 μL each of primers Donor-F and Donor-R, 25 μL of PCR mix, and 20 μL of water. Donor DNA-OE-PGM2 and Donor DNA-OE-UGP1 fragments were obtained according to the program in Table 3. After amplification, the fragments were separated by 1% agarose gel electrophoresis. The approximately 772 bp band was excised from the agarose gel and purified using a TIANGEN gel recovery kit. Two types of donor DNA were obtained: Donor DNA-OE-... PGM2and Donor DNA-OE- UGP1 .

[0031] Table 3 Donor DNA-OE PCR reaction procedure (4) Preparation of a single-gene overexpression strain of Saccharomyces cerevisiae: The constructed recombinant plasmid pML104- PGM2 pML104- UGP1 With the prepared donor DNA (Donor DNA-OE- PGM2 Or DonorDNA-OE- UGP1 The strains were transformed into competent Saccharomyces cerevisiae BY4741 cells, and after PCR verification, sequencing, and identification, the successfully sequenced strains were named BY4741-pTDH3- PGM2 BY4741-pTDH3- UGP1 .

[0032] In the PCR verification, self-designed primers for verifying gene deletion were used to perform PCR amplification with extracted DNA as a template. The amplification products were then subjected to gel electrophoresis, and preliminary verification was performed based on fragment length. PGM2 , UGP1 If overexpression is successful, the PCR product length of the primer should be greater than 500 bp (500 bp plus the length of the replaced promoter). If overexpression fails, the PCR product length of the primer should be 500 bp. The results are as follows: Figure 2 This indicates that the amplification was successful.

[0033] Sequence alignment: The product is sent for sequencing, and the sequencing results are as follows: Figure 3 with PGM2 Sequencing results of CRISPR system expression vector for the target gene and Figure 4 with UGP1 The sequencing results of the CRISPR expression vector of the target gene are compared with those downloaded from the NCBI database. PGM2 , UPG1 Startup subregion sequence and TDH3 The promoter sequences were compared and analyzed. PGM2 A portion of the gene promoter region (bases located at positions 477607 to 477764 on chromosome XIII) was replaced with... TDH3 Promoter (located at positions 883811 to 884477 on chromosome VII); UGP1 A portion of the bases in the gene promoter region (located at positions 369790 to 369890 on chromosome XI) were also replaced with... TDH3The promoter (located at bases 883811 to 884477 on chromosome VII). Through the above comparison and verification, two single-gene overexpression strains were successfully constructed, namely BY4741-pTDH3- PGM2 With BY4741-pTDH3- UGP1 The conclusion is that the structure was successfully constructed.

[0034] The primer sequences for verification are as follows: Val.Primers- PGM2 -F:5'-TGCAGTTCCAGGAACAAACA-3'; Val.Primers- PGM2 -R:5'-ACCATGCTGGCCAATAACTA-3'; Val.Primers- UGP1 -F: 5'-GTTCTCACACAGATCCCTGTA-3'; Val.Primers- UGP1 -R:5'-CCAAATATCTCCTGAAAAGCG-3'.

[0035] (5) Preparation of a dual-gene overexpression strain of Saccharomyces cerevisiae: The obtained Donor DNA-OE- UGP1 With pML104- UGP1 Transfer to brewer's yeast BY4741-pTDH3- PGM2 In competent cells, the genetically engineered strain BY4741-pTDH3-, exhibiting dual gene overexpression, was obtained through screening on SD-URA selection medium, PCR verification, and sequence alignment. PGM2 - UGP1 .

[0036] Among them, the electrophoresis results obtained from PCR verification are as follows: Figure 5 Compared to the control group, the *Saccharomyces cerevisiae* strain BY4741... PGM2 and UGP1 Total length of the gene fragment, BY4741-pTDH3- PGM2 - UGP1 Double overexpression strains PGM2 The total length of the gene fragments has increased significantly, from the theoretical 511bp to 1047bp; UGP1 The total length of the gene fragments also increased significantly, from the theoretical 502bp to 1017bp.

[0037] Sequence alignment showed that in the dual-gene overexpression strain BY4741-pTDH3- PGM2 - UGP1 middle PGM2Partial bases in the gene promoter region (chromosome XIII 477607 to 477764) and UGP1 A portion of the bases in the gene promoter region (chromosome XI, 369790 to 369890) were replaced with... TDH3 Promoter (chromosome VII, 883811 to 884477).

[0038] (ii) Knockout of brewing yeast ALG5 Gene (1) Obtaining ALG5 -guide RNA hybridization nucleotide fragment sgRNA: target gene from Saccharomyces cerevisiae BY4741 obtained from the NCBI website. ALG5 Based on the DNA sequence, design the sgRNA primer sequences as follows: sg- ALG5 -F:5'-GATCACCAAGGCCGCCATATCCAGGTTTTAGAGCTAG-3' sg- ALG5 -R:5'-CTAGCTCTAAAACCTGGATATGGCGGCCTTGGT-3' Anneal the primer fragments according to the procedure in (I) to obtain the primer fragments containing... ALG5 -guide RNA hybridization nucleotide fragment sgRNA.

[0039] (2) Construction of reconstructed plasmid pML104- ALG5 Purification of enzyme digestion products, pML104 gel recovery of fragments and containing ALG5 -GuideRNA hybridization nucleotide fragment sgRNA was used to perform an enzyme ligation reaction to obtain the reconstructed plasmid pML104- with sgRNA expression structure and Cas9 gene. ALG5 The ligation was confirmed to be correct after transformation and sequencing analysis.

[0040] (3) Preparation of Donor DNA-KO: such as Figure 6 As shown, 45 bases were selected from the 5' and 3' ends of the target gene, and these two base segments were linked together to construct a DonorDNA-KO-N* with a length of approximately 90 bp. The primer sequences were designed and synthesized using a method of overlapping upstream and downstream primers and using each other as templates, as shown in Table 4.

[0041] Table 4. Donor DNA-KO PCR Primer Sequences A 50 μL amplification system was used: primers Donor- ALG5 -F and Donor- ALG52 μL and 25 μL of PCR mix (R) and 21 μL of water were added, and the Donor DNA-KO-1 was amplified according to the procedure in Table 5. ALG5 Excerpt.

[0042] Table 5 Donor DNA-KO- ALG5 Amplification program (4) Preparation of Saccharomyces cerevisiae gene knockout strain: The constructed recombinant plasmid pML104- ALG5 Donor DNA-KO- ALG5 Transformed into competent Saccharomyces cerevisiae BY4741 cells, the strains were verified by PCR and identified by sequencing. Successfully sequenced strains were named BY4741- pPGM2 - pUGP1 -Δ ALG5 .

[0043] Among them, PCR verification showed that, compared to the control group BY4741-pTDH3- PGM2 - UGP1 Double overexpression strains ALG5 The length of the gene fragment, BY4741- pPGM2 - pUGP1 - ΔALG5 of ALG5 The gene length was significantly shortened, from 1475 bp to 554 bp, with clear bands and a significantly shorter fragment length, such as... Figure 7 The results were in line with expectations, providing preliminary evidence that the gene knockout was successful.

[0044] Sequence alignment: for BY4741- pPGM2 - pUGP1 - ΔALG5 In strains ALG5 Gene sequencing revealed a deletion in the gene at positions 19 to 939 (bases 120229 to 121149 on chromosome XVI), indicating the successful construction of a β-glucan-derived engineered bacterium, BY4741-. pPGM2 - pUGP1 - ΔALG5 .

[0045] Example 1: Analysis of Relative Gene Expression Levels Real-time quantitative PCR was performed on the genetically engineered bacteria constructed in Example 1 to detect the activity of each strain. PGM2 , UGP1, FKS1 (lie in UGP1 Downstream) and ALG5 Gene expression levels, results as follows Figure 8 .

[0046] Figure 8 In the text, B represents strain BY4741; BP, BU, and BPU represent the gene overexpression strain BY4741-pTDH3- PGM2 BY4741-pTDH3- UGP1 and BY4741-pTDH3- PGM2 - UGP1 BPUA stands for gene knockout strain BY4741- pPGM2 - pUGP1 - ΔALG5 Different letters on the bars in the graph indicate significant differences. p <0.05). The results showed that, compared with the control group BY4741 strain, single overexpression... PGM2's strain UGP1 Gene expression levels increased slightly but not significantly, with single overexpression. UGP1 strains of genes PGM2 Gene expression levels also increased slightly but not significantly, while the double overexpression strain BY4741-pTDH3- PGM2 - UGP1 middle, PGM2 as well as UGP1 The relative expression levels of these two compounds increased significantly, reaching 4.63-fold and 4.13-fold respectively, both far exceeding their individual expression levels under single overexpression. The results indicate that when... PGM2 Genes and UGP1 When genes are overexpressed synchronously, there is a synergistic effect between the two, which significantly promotes gene expression compared to overexpression of one gene alone.

[0047] PGM2 and UGP1 Compared to the control group, the gene expression levels increased by 4.27-fold and 3.85-fold, respectively. There was no significant increase compared to double overexpression; in fact, the relative expression levels decreased slightly. This is presumably related to a negative feedback mechanism within the cell, but it may not affect downstream genes. FKS1 (β-glucan synthase) expression levels were significantly increased during testing, reaching 5.69 times that of the control group and 1.61 times that of the double-overexpression strain, indicating a greater potential for efficient β-glucan synthesis. ALG5 The relative expression level of [a substance] decreased significantly, almost to zero.

[0048] In summary, real-time quantitative PCR data analysis, BY4741- pPGM2 - pUGP1 - ΔALG5 Key genes in the β-glucan synthesis pathway in strains PGM2 , UGP1 , FKS1 The ALG5 gene showed a significant increase, with a clear fold increase, while the ALG5 gene showed a significant decrease, almost to zero, consistent with gene editing results.

[0049] Example 2: Analysis of Relative Gene Expression Levels High-performance liquid chromatography (HPLC) was used to detect the differences between the control group strain BY4741 and the experimental group strain BY4741- pPGM2 - pUGP1 - ΔALG5 The β-glucan content in the two groups of samples to be tested.

[0050] (1) Sample preparation: Take an appropriate amount of sample into a test tube, add 0.5 mL of concentrated hydrochloric acid, place in a 30℃ water bath for 1 hour, then add 5 mL of water, wash the test tube several times and transfer the washing solution to a hydrolysis tube, hydrolyze at 120℃ for 1 hour. After hydrolysis is complete, remove the sample and let it cool to room temperature. Adjust the pH of the hydrolysate to about 7 with sodium hydroxide solution, then perform volume adjustment and membrane treatment, and use the samples as strain BY4741 and strain BY4741-, respectively. pPGM2 - pUGP1 - ΔALG5 The samples to be tested are used for instrumental analysis.

[0051] (2) Preparation of glucose standard curve: Prepare glucose standard solution and plot the standard curve. The curve shows good linearity in the range of 100-5000 μg / mL, and the linear equation is y=a x +b, where a=404.9253, b=0, and the correlation coefficient is 0.99944.

[0052] (3) High performance liquid chromatography analysis: The chromatogram of group BY4741 is shown below. Figure 9 BY4741- pPGM2 - pUGP1 - ΔALG5 The chromatogram of the group is as follows Figure 10 The test results showed that the glucose concentration in group BY4741 was 238.421 μg / mL. pPGM2 - pUGP1 - ΔALG5 The concentration in group B was 386.575 μg / mL, which, converted to β-glucan content using the formula, yielded 1.53 g / 100 g and 2.83 g / 100 g, respectively. Therefore, the experimental group strain BY4741- pPGM2 - pUGP1 - Δ ALG5 The β-glucan content in the medium was 1.85 times that of the control group BY4741.

[0053] The above results indicate that the genetically engineered strain BY4741- pPGM2 - pUGP1 - ΔALG5It has the characteristic of producing high levels of β-glucan.

[0054] Example 2: A plasmid This plasmid is the reconstructed plasmid pML104- PGM2 It was prepared in the method for constructing a β-glucan genetically engineered bacterium in Example 1.

[0055] Example 3 A plasmid This plasmid is the reconstructed plasmid pML104- UGP1 It was prepared in the method for constructing a β-glucan genetically engineered bacterium in Example 1.

[0056] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a β-glucan-engineered bacterium, characterized in that, In brewer's yeast PGM2 Genes and / or UGP1 The gene promoter is replaced with a strong promoter.

2. The method for constructing a β-glucan-engineered bacterium according to claim 1, characterized in that, In brewer's yeast PGM2 Genes and UGP1 The gene promoter is replaced with a strong promoter, or the gene is knocked out. ALG5 Gene.

3. The method for constructing a β-glucan-engineered bacterium according to claim 2, characterized in that, Promoter replacement and knockout ALG5 The gene was edited using CRISPR-Cas9 gene editing technology.

4. The method for constructing a β-glucan-engineered bacterium according to claim 3, characterized in that, When the promoter is replaced, PGM2 The sgRNA primer for the gene is an nucleotide sequence as shown in SEQ ID NO.1, sg- PGM2 -F and sg- as shown in SEQ ID NO.2 PGM2 -R; UGP1 The sgRNA primer for the gene is an sg-nucleotide sequence as shown in SEQ ID NO.

3. UGP1 -F and sg- as shown in SEQ ID NO.4 UGP1 -R.

5. The method for constructing a β-glucan-engineered bacterium according to claim 3, characterized in that, When the promoter is replaced, PGM2 The sgRNA primer for the gene is an nucleotide sequence as shown in SEQ ID NO.1, sg- PGM2 -F and sg- as shown in SEQ ID NO.2 PGM2 -R; UGP1 The sgRNA primer for the gene is an sg-nucleotide sequence as shown in SEQ ID NO.

3. UGP1 -F and sg- as shown in SEQ ID NO.4 UGP1 -R.

6. The method for constructing a β-glucan-engineered bacterium according to claim 5, characterized in that, Promoter replacement was achieved by preparing donor DNA, wherein the donor DNA was Donor DNA-OE- PGM2 Or Donor DNA-OE- UGP1 ; Among them, the Donor DNA-OE- PGM2 The primer nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6; The Donor DNA-OE- UGP1 The primer nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.

8.

7. A method for constructing a β-glucan-engineered bacterium according to any one of claims 2 to 6, characterized in that, Knockout ALG5 When processing genes, the primer nucleotide sequences of DonorDNA-KO are shown in SEQ ID NO.9 and SEQ ID NO.

10.

8. A method for constructing a β-glucan-engineered bacterium according to claim 7, characterized in that, The strong promoter is Saccharomyces cerevisiae. TDH3 The promoter of the gene; the brewer's yeast is Saccharomy cescerevisiae BY4741.

9. A genetically engineered bacterium, characterized in that, It is constructed using the method for constructing β-glucan-engineered bacteria as described in any one of claims 1 to 8.

10. A plasmid, characterized in that, It is prepared in any one of the methods for constructing a β-glucan-engineered bacterium according to claims 1 to 8.

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