Application of Fum gene knockout in improving the erythritol yield of strains

Abstract

The present invention relates to the technical field of genetic engineering, and particularly to the application of knocking out Fum genes in improving the erythritol yield of strains. The present invention provides the application of knocking out Fum genes in improving the erythritol yield of strains, and the amino acid sequence of the protein encoded by the Fum gene is as shown in SEQ ID NO:1. The strains include Yarrowia lipolytica. The present invention uses CRISPR technology to knock out the Fum gene encoding fumarase in the genome of Yarrowia lipolytica, and obtains a recombinant Yarrowia lipolytica CICC1457-Δ Fum with significantly increased erythritol yield. Compared with the original Yarrowia lipolytica CICC1457, the erythritol yield is increased by 75.7% and reaches 4.71 g / L. During the fermentation culture process, the growth rate and biomass of the cells have no obvious difference from those of the original strain.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to the application of knocking out Fum genes in improving the erythritol yield of strains. Background Art

[0002] Erythritol, chemically named 1,2,3,4-butanetetraol, is the sugar alcohol with the smallest molecular mass found in nature. It is a white, odorless, heat-stable crystal. It is a functional sugar with a cool taste, low calories, and non-cariogenic properties when ingested, and is currently used in a variety of foods, with strong market demand. Therefore, breeding high-yield, low-cost, and genetically stable production strains has become one of the key goals in this industry.

[0003] As a powerful gene editing tool, the CRISPR-Cas9 system has great potential in gene knockout. Using this system, DNA can be easily inserted into specific loci without homologous recombination. Plasmid curing can eliminate non-specific expression vectors, reduce background interference, and effectively improve the credibility of research results. Yarrowia lipolytica is the preferred organism for microbial technology research and application in academia and industry due to its unique metabolic, genetic, and physiological characteristics. The microbial fermentation method has the advantages of low raw material cost and simple reaction conditions, and is a production method that can be used for large-scale industrial production.

[0004] Currently, erythritol is mainly produced by the microbial fermentation method in industry. In recent years, several high-yield erythritol-producing Yarrowia lipolytica strains have been obtained by gene knockout methods. Chinese patents CN117187092A and CN116179382A reported the production of erythritol by fermenting recombinant Yarrowia lipolytica. However, the production efficiency of these recombinant Yarrowia lipolytica engineering strains is relatively low, and the erythritol yield needs to be further improved. Summary of the Invention

[0005] To solve the above problems, the present invention provides the application of knocking out Fum genes in improving the erythritol yield of strains. The present invention discovers that by knocking out the Fum genes in the genome of Yarrowia lipolytica, the recombinant strain constructed can improve the erythritol yield, and during the fermentation culture process, the growth rate and biomass of the cells have no obvious difference from those of the original strain.

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

[0007] The present invention provides the application of knocking out the Fum genes in a strain in improving the erythritol yield of the strain, wherein the amino acid sequence of the protein encoded by the Fum genes is as shown in SEQ ID NO:1, and the strain includes Yarrowia lipolytica (Yarrowia lipolytica ).

[0008] Preferably, the Fum nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0009] The present invention provides a method for constructing a recombinant strain with increased erythritol production, comprising the following steps:

[0010] Knock out the Fum gene in the strain to obtain a recombinant strain with increased erythritol production; the Fum amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1, and the strain includes Yarrowia lipolytica.

[0011] Preferably, the reagent for knocking out the Fum gene in the strain includes a knockout vector; the knockout vector includes a basic vector and a nucleic acid molecule inserted into the basic vector; the nucleic acid molecule includes sgRNA, and the sgRNA includes a nucleic acid molecule targeting the gene with a nucleotide sequence shown in SEQ ID NO:3.

[0012] Preferably, the basic vector includes the pDuRCC-K vector.

[0013] Preferably, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:7.

[0014] Preferably, the nucleic acid molecule inserted into the basic vector further includes a nucleic acid molecule encoding a kanamycin resistance gene and a nucleic acid molecule encoding a green fluorescent protein.

[0015] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the kanamycin resistance gene is shown in SEQ ID NO:5, and the nucleotide sequence of the nucleic acid molecule encoding the green fluorescent protein is shown in SEQ ID NO:8.

[0016] Preferably, the nucleotide sequence of the nucleic acid molecule inserted into the basic vector is shown in SEQ ID NO:4.

[0017] The present invention provides the application of the recombinant strain constructed by the construction method described in the above technical solution in the preparation of erythritol.

[0018] Beneficial effects:

[0019] The present invention provides the application of knocking out the Fum gene in the strain in increasing the erythritol production of the strain, and the FumThe amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and the bacterial strain includes Yarrowia lipolytica. The present invention uses CRISPR technology to knockout the Fum gene encoding fumarase in the genome of Yarrowia lipolytica, and obtains a recombinant Yarrowia lipolytica CICC1457-Δ Fum with significantly increased erythritol production. Compared with the original Yarrowia lipolytica CICC1457, the erythritol production is increased by 75.7% and reaches 4.71 g / L. During the fermentation culture process, the growth rate and biomass of the bacterial cells have no obvious difference from those of the original strain. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0021] Figure 1 It is the map of plasmid pDuRCC-K- sgRNA-eGFP in Example 1;

[0022] Figure 2 It is the electrophoresis result of double enzyme digestion of plasmid pDuRCC-K in Example 1;

[0023] Figure 3 It is the PCR amplification result of the kan-sgRNA-eGFP fragment in Example 2;

[0024] Figure 4 It is the agarose gel electrophoresis result of the Kan fragment in Example 3;

[0025] Figure 5 It is the gene sequencing result of Yarrowia lipolytica CICC1457 and CICC1457-Δ Fum in Example 4;

[0026] Figure 6 It is the fluorescence intensity of Yarrowia lipolytica CICC1457 and CICC1457-Δ Fum in Example 5;

[0027] Figure 7 It is the erythritol production of Yarrowia lipolytica CICC1457 and CICC1457-Δ Fum in Example 6;

[0028] Figure 8 It is the fermentation OD Fum value of Yarrowia lipolytica CICC1457 and CICC1457-Δ 600 in Example 6. Detailed Embodiments

[0029] The present invention provides the application of knocking out the Fum gene in improving the erythritol yield of a strain, wherein the strain includes Yarrowia lipolytica, and the Fum amino acid sequence of the protein encoded by the gene is as shown in SEQ ID NO:1, specifically as follows:

[0030] MLRTVRLAKVSRPLTLRTFSTTPAFLGKRVEKDAFGDIDVDDSHYWGAQTQRSLQNFDIGGEKAKMPEPIVKAFGILKKAAATVNMKYGLDPKVGEAIQKAAQEVIDGKLTKDFPLVVFQTGSGTQSNMNSNEVISNRAIEMLGGKLGSKSPVHPNDHVNMSQSSNDTFPTVMHIAAVMEITKNLIPQLQLLEESLAKKSAEFDKIIKIGRTHLQDATPLTLGQEFSGYVTQVKYGIERVKDVLPRLRHLAQGGTAVGTGLNTKKGFDTAVAAEVSKITGEEFSTAPNKFEALAAHDAIVEASGALNTVAVSLFKIANDIRYLGSGPRCGYGELALPENEPGSSIMPGKVNPTQNEALTMVCCQVFGNNSTITYAGASGQFELNVFKPVMIANLLESIRLIADGSRSFRIHCVDGIVANEKRISQLMNESLMLVTALNPKIGYDMASKVAKNAHKKGITLKESALELGALTSEEFDQWVRPEKMIGPSD.

[0031] As an embodiment, the Fum nucleotide sequence of the gene is as shown in SEQ ID NO:2, specifically as follows:

[0032]

[0033] As an implementation mode, the Yarrowia lipolytica is Yarrowia lipolytica CICC1457, which is purchased from China Center for Industrial Culture Collection of Microorganisms.

[0034] The present invention discovers that by knocking out the Fum gene in the genome of Yarrowia lipolytica, the recombinant strain constructed can improve the production of erythritol, and during the fermentation culture process, the growth rate and biomass of the bacterial cells have no obvious difference from those of the original strain, and it can be used for the industrial fermentation production of erythritol.

[0035] Based on the above advantages, the present invention provides a method for constructing a recombinant strain with improved erythritol production, comprising the following steps:

[0036] Knock out the Fum gene in the strain to obtain a recombinant strain with improved erythritol production; the Fum gene encodes a protein with the amino acid sequence shown in SEQ ID NO:1, and the strain includes Yarrowia lipolytica.

[0037] As an implementation mode, the Yarrowia lipolytica is Yarrowia lipolytica CICC1457, which is purchased from China Center for Industrial Culture Collection of Microorganisms.

[0038] As an implementation mode, the reagent for knocking out the Fum gene in the strain includes a knockout vector; the knockout vector includes a basic vector and a nucleic acid molecule inserted into the basic vector; the nucleic acid molecule includes sgRNA, and the sgRNA includes a nucleic acid molecule targeting the gene with the nucleotide sequence shown in SEQ ID NO:3. The knockout vector provided by the present invention can guide the Cas9 protein to perform directional cleavage at the gene editing site by selecting a suitable sgRNA Fum gene, thereby knocking out the Fum gene in the genome of Yarrowia lipolytica and improving the erythritol production of the recombinant strain.

[0039] As an implementation mode, the sgRNA is composed of four components: a Candida promoter, a nucleic acid molecule targeting the gene, a gRNA scaffold of vector pDuRCC-K, and a SUP4 terminator, and the nucleotide sequence is shown in SEQ ID NO:7.

[0040] As an implementation mode, the basic vector includes the pDuRCC-K vector.

[0041] As an implementation mode, the nucleic acid molecule inserted into the basic vector further includes a nucleic acid molecule encoding a kanamycin resistance gene and a nucleic acid molecule encoding a green fluorescent protein.

[0042] As an embodiment, the nucleic acid molecule encoding the kanamycin resistance gene consists of three components: the TEF promoter of pDuRCC-K, the kan resistance fragment, and the TEF terminator, and its nucleotide sequence is as shown in SEQ ID NO:5; the nucleic acid molecule encoding the green fluorescent protein consists of three components: the hp4d promoter, the green fluorescent protein eGFP gene, and the XPR2 terminator, and its nucleotide sequence is as shown in SEQ ID NO:8.

[0043] As an embodiment, the nucleotide sequence of the nucleic acid molecule inserted into the basic vector is as shown in SEQ ID NO:4.

[0044] Based on the above advantages, the present invention provides the use of the recombinant strain constructed by the construction method described in the above technical solution in the preparation of erythritol.

[0045] To further illustrate the present invention, the following describes in detail the application of the knockout Fum gene in improving the erythritol yield of the strain with reference to the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1 Construction of the knockout vector pDuRCC-K- sgRNA-eGFP Construction

[0047] (i) Select three components of the TEF promoter, kan resistance fragment, and TEF terminator of the vector pDuRCC-K and connect them in sequence to form the kan fragment; (ii) Connect four components of the Candida promoter, the target gene sequence (5'-GTCCAACATGAACTCCAACG-3', SEQ ID NO:3), the gRNA scaffold of the vector pDuRCC-K, and the SUP4 terminator in sequence to form the sgRNA fragment; (iii) Connect three components of the hp4d promoter, the green fluorescent protein eGFP gene, and the XPR2 terminator in sequence to form the eGFP fragment;

[0048] (iv) Connect the kan fragment, sgRNA fragment, and eGFP fragment obtained in the above steps (i), (ii), and (iii) in sequence, and commission a gene synthesis company to synthesize a fragment with the nucleotide sequence as shown in SEQ ID NO:4, specifically as follows: kan-sgRNA-eGFP Fragment, as follows:

[0049]

[0050] Among them, 1 - 1192 bp is the kan fragment (SEQ ID NO: 5), 1193 - 1269 bp is the linker fragment (SEQ ID NO: 6), 1270 - 1641 bp is the sgRNA fragment (SEQ ID NO: 7), and 1642 - 3261 bp is the eGFP fragment (SEQ ID NO: 8).

[0051] (v) Use Nde I. Pst I to perform double digestion on plasmid pDuRCC-K to obtain a linear pDuRCC-K plasmid. The electrophoresis result of the double digestion of plasmid pDuRCC-K is shown in Figure 2 , where the M lane is Marker, the 1st lane is pDuRCC-K, 10562 bp, and the 2nd lane is the digested plasmid pDuRCC-K. The electrophoresis result indicates that plasmid pDuRCC-K has been cut open.

[0052] (vi) Use T4 DNA ligase to ligate the kan-sgRNA-eGFP fragment and the linearized pDuRCC-K plasmid to obtain plasmid pDuRCC-K- sgRNA-eGFP (The map is shown in Figure 1 ).

[0053] Example 2 Transformation and verification of the knockout vector pDuRCC-K- sgRNA-eGFP

[0054] Transform the recombinant plasmid pDuRCC-K- sgRNA-eGFP constructed in Example 1 into E. coli BL21 competent cells and perform the transformation according to the instructions of the kit. After single colonies grow on the LB resistant plate containing 100 μg / mL kanamycin, pick a single colony with a sterile inoculation loop and inoculate it into the LB liquid medium containing 100 μg / mL kanamycin, and culture it with shaking at 37°C and 200 r / min for 12 - 16 h. Use the FastPure Plasmid Mini Kit to extract the recombinant plasmid pDuRCC-K- sgRNA-eGFP . Verify the transformation of the recombinant plasmid by PCR.

[0055] The reaction system of the PCR is as follows: 2 μL of the recombinant plasmid pDuRCC-K- sgRNA-eGFP , 2 μL of kan-F, 2 μL of eGFP-R, 25 μL of 2× Phanta Max Master Mix polymerase, and 19 μL of ddH 2 O.

[0056] Kan-F: 5'-ATGGGTAAGGAAAAGACTCA-3' (SEQ ID NO:9);

[0057] eGFP-R: 5'-CGTCCATTCCGAGAGTGATGC-3' (SEQ ID NO:10);

[0058] The amplification program of the PCR is as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, extension at 72 °C for 2 min, for 32 cycles; extension at 72 °C for 10 min, and preservation at 4 °C.

[0059] The PCR products were examined by agarose gel electrophoresis, and the results are shown in Figure 3 , where the M lane is Marker and the 1st lane is the verified Kan-sgRNA-eGFP fragment. It can be seen from the results that using the primers Kan-F and eGFP-R can amplify a specific gene band with a size of about 3900 bp, which is close to the theoretical value of 3953 bp sequence, indicating that the amplification is correct.

[0060] The positive single colonies determined by PCR were inoculated into LB liquid medium containing 100 μg / mL kanamycin, and placed in a shaking incubator at 37 °C and 200 r / min for 12 - 16 h, cultured until OD 600 ≈1.0, 1 mL of 50% glycerol and 1 mL of the bacterial solution were successively added to a sterile glycerol tube and stored in a low-temperature refrigerator at -20 °C.

[0061] Example 3 Construction of the strain Yarrowia lipolytica CICC1457 / pDuRCC-K- sgRNA-eGFP -

[0062] (i) Take 10 μL of plasmid pDuRCC-K- sgRNA - eGFP with a concentration greater than 1000 ng / μL and add it to a 1.5 mL sterile centrifuge tube containing 100 μL of competent cells of Yarrowia lipolytica CICC1457, ice-bath for 5 min, and at the same time place a 2 mm electroporation cuvette on ice for pre-cooling.

[0063] (ii) Aspirate and transfer the competent bacterial solution mixed with the plasmid to the electroporation cuvette, and set the electroporation conditions as: 2.5 kv electric shock for 5 ms.

[0064] (iii) Immediately after the electric shock, add 900 μL of fresh sterile YPD liquid medium to the electroporation cuvette, aspirate and transfer the bacterial solution in the electroporation cuvette to a 1.5 mL sterile centrifuge tube, and place it in a shaking incubator at 30 °C and 200 r / min for 1 h for recovery.

[0065] (iv) Centrifuge at 3000 r / min for 3 min at room temperature, discard the supernatant, and wash 2 - 3 times with 1 mL of sterile water.

[0066] (v) After resuspending the cell pellet with 100 μL of sterile water, spread it onto a YPD resistance screening plate supplemented with 1000 μg / mL G418 (Geneticin), and place the plate in a 30 °C constant temperature incubator for static culture for 2 - 4 days until transformants grow.

[0067] After single colonies grow on the resistance plate, randomly pick 10 single colonies with a sterile inoculation loop into a 1.5 mL sterile centrifuge tube containing 50 μL of sterile water and pipette to mix evenly; take 10 μL of the bacterial solution into a new centrifuge tube, add 20 μL of lysis solution, boil at 100 °C for 12 min, then centrifuge at 12000 r / min at room temperature for 1 min, and take the supernatant as a template for PCR amplification.

[0068] The reaction system of the said PCR amplification is: 1 μL of supernatant, 0.5 μL of Kan-F (SEQ ID NO:9), 0.5 μL of Kan-R, 5 μL of KOD One TM PCR Master Mix polymerase, and 3 μL of ddH 2 O;

[0069] Kan-R: 5'-TTAGAAAAACTCATCGAGCA-3' (SEQ ID NO:11);

[0070] The reaction program of the said PCR amplification is: pre-denaturation at 98 °C for 2 min; denaturation at 98 °C for 10 sec, annealing at 56 °C for 30 sec, extension at 68 °C for 30 sec, 40 cycles; extension at 68 °C for 5 min, and preservation at 16 °C.

[0071] Detect the PCR products by agarose gel electrophoresis, and the results are shown in Figure 4 , the M lane is Marker, and the 1 - 10 lanes are the verified Kan fragments. The results show that using primers Kan-F and Kan-R can amplify a specific gene band with a size of approximately 800 bp. The bands in lanes 1, 3 - 10 are close to the theoretical value of 810 bp, indicating correct amplification.

[0072] Example 4 Sequencing analysis of Yarrowia lipolytica CICC1457 / pDuRCC-K- sgRNA-eGFP

[0073] Randomly pick the correctly amplified recombinant strain CICC1457 / pDuRCC-K- in Example 3 using a sterile inoculation loop sgRNA-eGFP and pipette and mix it evenly into 50 μL of sterile water; take 10 μL of the bacterial solution into a new centrifuge tube and add 20 μL of lysis solution. After boiling at 100 °C for 12 min, centrifuge at 12000 r / min at room temperature for 1 min, and use the supernatant as a template for PCR amplification.

[0074] The reaction system for the PCR amplification is as follows: 1 μL of supernatant, 0.5 μL of Fum-F, 0.5 μL of Fum-R, KODOne TM PCR Master Mix polymerase 5 μL and ddH 2 O 3 μL;

[0075] Fum-F: 5'-ATGCTCCGAACAGTCCGACTAG-3' (SEQ ID NO:12);

[0076] Fum-R: 5'-GTCAGAGGGGCCAATCATCTTC-3' (SEQ ID NO:13);

[0077] The reaction procedure for the PCR amplification is the same as that in Example 3.

[0078] After PCR, use the SanPrep column DNA gel extraction kit for gel extraction, and then perform sequencing analysis on the Fum gene to verify the Fum gene knockout situation. The sequencing results are as Figure 5 shown. It is found that the Fum target gene sequence of the gene in the strain Yarrowia lipolytica CICC1457 / pDuRCC-K-sgRNA-eGFP has mutated. The N20 region has mutated, the base C at 396bp has mutated to A, and the base G at 397bp has mutated to T, resulting in the amino acid at position 132 changing from N to K. It can be seen that the Fum gene has been successfully knocked out, and a recombinant strain with the Fum gene knocked out (Yarrowia lipolytica CICC1457-Δ Fum ) has been successfully constructed.

[0079] Example 5 Fluorescence detection and plasmid elimination of Yarrowia lipolytica CICC1457-Δ Fum

[0080] Fluorescence detection: The Yarrowia lipolytica CICC1457- prepared in Example 4 ΔFumThe control strain Yarrowia lipolytica CICC1457 was inoculated into YPD liquid medium and cultured in a shaking incubator at 30 ℃ and 200 r / min for 24 hours. It was used as a seed liquid and transferred to a 500 mL conical flask containing 50 mL of liquid fermentation medium at a 10% (v / v) inoculation rate. It was cultured in a shaking incubator at 30 ℃ and 200 r / min for 3 days, and samples were taken every 12 hours. 1 mL of the fermentation liquid was centrifuged to remove the supernatant, and the bacteria were resuspended with 1 mL of sterile water, and the Yarrowia lipolytica CICC1457-Δ was detected by an ELISA instrument. Fum The fluorescence intensity of the control strain Yarrowia lipolytica CICC1457 at 484-507 nm is shown in Figure 6 , Yarrowia lipolytica CICC1457-Δ Fum The fluorescence intensity of the knockout vector pDuRCC-K- sgRNA-eGFP Effect of plasmid transformation in Yarrowia lipolytica.

[0081] Plasmid elimination: Yarrowia lipolytica CICC1457-Δ Fum The cells were placed in a shaking incubator at 30 °C and 200 rpm for 24 h and subcultured three times. When the fluorescence disappeared, the vector pDuRCC-K- sgRNA-eGFP disappear.

[0082] Example 6 Yarrowia lipolytica CICC1457-Δ Fum Shake flask fermentation and yield detection

[0083] Shake flask fermentation: Yarrowia lipolytica CICC1457-Δ prepared in Example 4 Fum The control strain Yarrowia lipolytica CICC1457 was inoculated into YPD liquid medium, and the seed solution was cultured in a shaking incubator at 30 ℃ and 200 r / min. The inoculation amount was transferred to a 500 mL conical flask containing 50 mL liquid fermentation medium at 10% (v / v). The culture was carried out in a shaking incubator at 30 ℃ and 200 r / min for 3 days, and samples were taken every 12 h. Erythritol production and OD were determined. 600 value.

[0084] Detection of erythritol production: The fermentation broth was centrifuged at 6000 r / min for 5 min at 4 °C. After centrifugation, the supernatant was diluted 50-fold with distilled water, filtered through a 0.22 µm filter membrane, immediately added to a brown liquid-phase vial, and stored at -20 °C in the dark. It was detected using a differential high-performance liquid chromatograph equipped with a Hi-Plex H (300×7.7 mm, Agilent Technologies) chromatographic column, and the chromatographic column was operated at 65 °C. The mobile phase was pure water, and the flow rate was 0.6 mL / min. The injection volume was 10 μL. The results are shown in Figure 7 and Figure 8 .

[0085] It can be seen from Figure 7 that after 72 h of fermentation, the content of erythritol in the fermentation broth of Yarrowia lipolytica CICC1457-Δ Fum reached 4.71 g / L, while that of the original Yarrowia lipolytica CICC1457 at 72 h was 2.68 g / L. The erythritol production of Yarrowia lipolytica CICC1457-Δ Fum increased by 75.7%.

[0086] It can be seen from Figure 8 that during the fermentation culture process, the growth rate and biomass of the cells showed no significant difference from those of the original strain. The above data indicate that knocking out Fum gene increased the level of erythritol production by Yarrowia lipolytica.

[0087] In summary, the Yarrowia lipolytica CICC1457-Δ Fum constructed by knocking out Fum gene in the present invention increased the synthetic yield of erythritol, and during the fermentation culture process, the growth rate and biomass of the cells showed no significant difference from those of the original strain.

[0088] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of knocking out the Fum gene in a strain to increase the erythritol production of the strain, characterized in that: The amino acid sequence of the protein encoded by the Fum gene is shown in SEQ ID NO: 1, and the bacterial species is Yarrowia lipolytica.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the Fum gene is shown in SEQ ID NO:

2.

3. A method for constructing a recombinant strain with improved erythritol production, characterized in that: The following steps are involved: The Fum gene in the strain is knocked out to obtain a recombinant strain with improved erythritol production; the amino acid sequence of the protein encoded by the Fum gene is shown in SEQ ID NO:1, and the strain is Yarrowia lipolytica; the reagent for knocking out the Fum gene in the strain includes a knockout vector; the knockout vector includes a basic vector and a nucleic acid molecule inserted into the basic vector; the nucleic acid molecule includes sgRNA, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO:

7.

4. The construction method according to claim 3, characterized in that: The basic vector includes the pDuRCC-K vector.

5. The construction method according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid molecule inserted into the basic vector is shown in SEQ ID NO:

4.

6. Use of the recombinant strain constructed by the construction method according to any one of claims 3 to 5 in the preparation of erythritol.

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