Application of knockout Fum gene in improving yield of erythritol of strain

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of a knockout Fum gene in increasing the yield of erythritol of a strain. The invention provides application of knockout of a Fum gene in a strain in increasing the erythritol yield of the strain, the amino acid sequence of protein coded by the Fum gene is shown as SEQ ID NO: 1, and the strain comprises yarrowia lipolytica. A Fum gene for coding fumarase in a yarrowia lipolytica genome is knocked out by utilizing a CRISPR (clustered regularly interspaced short palindromic repeats) technology, a recombinant yarrowia lipolytica CICC1457-delta Fum with obviously improved erythritol yield is obtained, the erythritol yield is improved by 75.7% and reaches 4.71 g / L compared with that of the original yarrowia lipolytica CICC1457, and in the fermentation culture process, the yield is increased by 75.7% and reaches 4.71 g / L; the thallus growth speed and thallus biomass are not obviously different from those of an original strain.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to knockout Fum Application of genes in increasing erythritol production in bacterial strains. Background Art

[0002] Erythritol, chemically known as 1,2,3,4-butanetetrol, is the smallest sugar alcohol found in nature. It is white, odorless, and heat-stable crystals. It is a functional sugar that tastes cool, is low in calories, and is non-caries-causing. It is currently used in a variety of foods and has a strong market demand. Therefore, breeding high-yield, low-cost, and genetically stable production strains has become one of the key goals of the industry.

[0003] As a powerful gene editing tool, the CRISPR-Cas9 system has great potential in gene knockout. The system can be easily inserted into specific sites of DNA without homologous recombination. Plasmid elimination can eliminate non-specific expression bodies, 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. Microbial fermentation has the advantages of low raw material cost and simple reaction conditions. It is a production method that can be used for large-scale industrial production.

[0004] At present, erythritol is mainly produced by microbial fermentation in industry. In recent years, several strains of Yarrowia lipolytica with high erythritol production have been obtained by gene knockout. Chinese patents CN117187092A and CN116179382A report the production of erythritol by fermentation of recombinant Yarrowia lipolytica. However, the production efficiency of these recombinant Yarrowia lipolytica engineered bacteria is low, and the erythritol production needs to be further improved. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a knockout Fum The present invention finds that by knocking out the Yarrowia lipolytica genome Fum The recombinant strain constructed by the gene can increase the erythritol production, and during the fermentation culture process, the bacterial growth rate and bacterial biomass have no significant difference with the original strain.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a knockout strain Fum The application of gene in increasing the erythritol production of bacteria, Fum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and the bacterial species includes Yarrowia lipolytica ( Yarrowia lipolytic ).

[0007] Preferably, the Fum The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0008] The present invention provides a method for constructing a recombinant strain with improved erythritol production, comprising the following steps: Knockout strains Fum Gene, to obtain a recombinant strain with improved erythritol production; Fum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and the strain includes Yarrowia lipolytica.

[0009] Preferably, the knockout strain Fum The gene reagent 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 targeting gene nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO:3.

[0010] Preferably, the basic vector comprises a pDuRCC-K vector.

[0011] Preferably, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO:7.

[0012] Preferably, the nucleic acid molecules inserted into the basic vector further include a nucleic acid molecule encoding a kanamycin resistance gene and a nucleic acid molecule encoding a green fluorescent protein.

[0013] 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.

[0014] Preferably, the nucleotide sequence of the nucleic acid molecule inserted into the basic vector is as shown in SEQ ID NO:4.

[0015] The present invention provides the use of a recombinant strain constructed by the construction method described in the above technical scheme in the preparation of erythritol.

[0016] Beneficial effects: The present invention provides a knockout strain Fum The application of gene in increasing the erythritol production of bacteria, Fum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and the bacterial species includes Yarrowia lipolytica. The present invention uses CRISPR technology to knock out the gene encoding fumarase in the Yarrowia lipolytica genome. FumGene, a recombinant Yarrowia lipolytica CICC1457-Δ with significantly improved erythritol production was obtained Fum Compared with the original Yarrowia lipolytica CICC1457, the erythritol production increased by 75.7% to 4.71 g / L, and during the fermentation process, there was no significant difference in the bacterial growth rate and biomass compared with the original strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 The plasmid pDuRCC-K- sgRNA-eGFP Spectrum of Figure 2 The electrophoresis results of double restriction enzyme digestion of plasmid pDuRCC-K in Example 1; Figure 3 For Example 2 can-sgRNA-eGFP PCR amplification results of the fragments; Figure 4 For Example 3 Can Agarose gel electrophoresis results of the fragments; Figure 5 Yarrowia lipolytica CICC1457 and CICC1457-Δ Fum Gene sequencing results; Figure 6 Yarrowia lipolytica CICC1457 and CICC1457-Δ Fum Fluorescence intensity; Figure 7 Yarrowia lipolytica CICC1457 and CICC1457-Δ Fum Erythritol production; Figure 8 Yarrowia lipolytica CICC1457 and CICC1457-Δ Fum Fermentation OD 600 value. DETAILED DESCRIPTION

[0019] The present invention provides a knockout strain Fum Application of a gene in increasing the erythritol production of a strain, wherein the strain includes Yarrowia lipolytica, Fum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and is as follows: MLRTVRLAKVSRPLTLRTFSTTPAFLGKRVEKDAFGDIDVDDSHYWGAQTQRSLQNFDIGGEKAKMPEPIVKAFGILKKAAATVNMKYGLDPKVGEAIQKAAQEVIDGKLTKDFPLVVFQTG SGTQSNMNSNEVISNRAIEMLGGKLGSKSPVHPNDHVNMSQSSNDTFPTVMHIAAVMEITKNLIPQLQLLEESLAKKSAEFDKIIKIGRTHLQDATPLTLGQEFSGYVTQVKYGIERVKDVLP RLRHLAQGGTAVGTGLNTKKGFDTAVAAEVSKITGEEFSTAPNKFEALAAHDAIVEASGALNTVAVSLFKIANDIRYLGSGPRCGYGELALPENEPGSSIMPGKVNPTQNEALTMVCCQVFG NNSTITYAGASGQFELNVFKPVMIANLLESIRLIADGSRSFRIHCVDGIVANEKRISQLMNESLMLVTALNPKIGYDMASKVAKNAHKKGITLKESALELGALTSEEFDQWVRPEKMIGPSD.

[0020] As an implementation mode, the Fum The nucleotide sequence of the gene is shown in SEQ ID NO: 2, and is as follows:

[0021] As an embodiment, the Yarrowia lipolytica is Yarrowia lipolytica CICC1457, which is purchased from China Industrial Microbiological Culture Collection Center.

[0022] The present invention found that by knocking out the Yarrowia lipolytica genome Fum The recombinant strain constructed by the gene can increase the erythritol production, and during the fermentation culture process, the bacterial growth rate and bacterial biomass have no obvious differences with the original strain, and can be used for industrial fermentation production of erythritol.

[0023] Based on the above advantages, the present invention provides a method for constructing a recombinant strain with improved erythritol production, comprising the following steps: Knockout strains Fum Gene, to obtain a recombinant strain with improved erythritol production; Fum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and the strain includes Yarrowia lipolytica.

[0024] As an embodiment, the Yarrowia lipolytica is Yarrowia lipolytica CICC1457, which is purchased from China Industrial Microbiological Culture Collection Center.

[0025] As an embodiment, the knockout strain Fum The gene knockout reagent 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 an sgRNA, and the sgRNA includes a targeting gene nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO: 3. The knockout vector provided by the present invention can guide the Cas9 protein to perform directional cutting at the gene editing site by selecting a suitable sgRNA. Fum knocking out the gene in the Yarrowia lipolytica genome Fum Gene, improve the erythritol production of the recombinant strain.

[0026] As an embodiment, the sgRNA consists of four components: a Candida promoter, a targeting gene nucleic acid molecule, a gRNA scaffold of a vector pDuRCC-K, and a SUP4 terminator, and the nucleotide sequence is shown in SEQ ID NO:7.

[0027] As one embodiment, the basic vector includes a pDuRCC-K vector.

[0028] As an embodiment, 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.

[0029] As an embodiment, the nucleic acid molecule encoding the kanamycin resistance gene consists of three components: the TEF promoter, the kan resistance fragment and the TEF terminator of pDuRCC-K, and the nucleotide sequence is 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 the nucleotide sequence is shown in SEQ ID NO:8.

[0030] As an embodiment, the nucleotide sequence of the nucleic acid molecule inserted into the basic vector is shown as SEQ ID NO:4.

[0031] Based on the above advantages, the present invention provides the use of a recombinant strain obtained by the construction method described in the above technical scheme in the preparation of erythritol.

[0032] In order to further illustrate the present invention, the knockout provided by the present invention is described below in conjunction with the accompanying drawings and embodiments. Fum The application of the gene in increasing the erythritol production of the strain is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0033] Example 1 Knockout vector pDuRCC-K- sgRNA-eGFP Construction (i) The three components of the TEF promoter, kan resistance fragment, and TEF terminator of the vector pDuRCC-K were selected and connected in sequence to form the kan fragment; (ii) the four components of the Candida promoter, the targeting gene sequence (5'-GTCCAACATGAACTCCAACG-3', SEQID NO: 3), the gRNA scaffold of the vector pDuRCC-K, and the SUP4 terminator were connected in sequence to form the sgRNA fragment; (iii) the three components of the hp4d promoter, the green fluorescent protein eGFP gene, and the XPR2 terminator were connected in sequence to form the eGFP fragment; (iv) The kan fragment, sgRNA fragment, and eGFP fragment obtained in steps (i), (ii), and (iii) above are sequentially connected and commissioned to a gene synthesis company to synthesize the nucleotide sequence shown in SEQ ID NO:4 can-sgRNA-eGFP The snippet is as follows: Among them, 1~1192bp is the kan fragment (SEQ ID NO:5), 1193~1269bp is the linker fragment (SEQID NO:6), 1270~1641bp is the sgRNA fragment (SEQ ID NO:7), and 1642~3261bp is the eGFP fragment (SEQ ID NO:8).

[0034] (v) Use Nth I. Psst I double-digested the plasmid pDuRCC-K to obtain a linear pDuRCC-K plasmid. The electrophoresis results of the double-digested plasmid pDuRCC-K are shown in Figure 2 , where lane M is the marker, lane 1 is pDuRCC-K, 10562 bp, and lane 2 is the plasmid pDuRCC-K after enzyme digestion. The electrophoresis results show that the plasmid pDuRCC-K has been cut.

[0035] (vi) Use T4 DNA ligase to ligate the can-sgRNA-eGFP The fragment was ligated with the linearized pDuRCC-K plasmid to obtain the plasmid pDuRCC-K- sgRNA-eGFP (See the diagram Figure 1 ).

[0036] Example 2 Knockout vector pDuRCC-K- sgRNA-eGFP Conversion and verification The recombinant plasmid pDuRCC-K- sgRNA-eGFP Convert to E. coli Transform BL21 competent cells according to the kit instructions. After a single colony grows on the LB resistance plate containing 100 μg / mL kanamycin, pick up a single colony with a sterile inoculation loop and inoculate it into LB liquid medium containing 100 μg / mL kanamycin. Culture at 37°C and 200 r / min for 12-16 hours. Use the FastPure Plasmid Mini Kit to extract the recombinant plasmid pDuRCC-K- sgRNA-eGFP . PCR was used to verify the transformation of the recombinant plasmid.

[0037] The PCR reaction system is: recombinant plasmid pDuRCC-K- sgRNA-eGFP 2 μL, kan-F 2 μL, eGFP-R2 μL, 2× Phanta Max Master Mix polymerase 25 μL and ddH2O 19 μL.

[0038] kan-F: 5'-ATGGGTAAGGAAAAGACTCA-3' (SEQ ID NO: 9); eGFP-R: 5'-CGTCCATTCCGAGAGTGATGC-3' (SEQ ID NO: 10); The PCR amplification program is: 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, 32 cycles; extension at 72°C for 10 min, and storage at 4°C.

[0039] The PCR products were tested by agarose gel electrophoresis. Figure 3 , where lane M is the marker and lane 1 is the verification Kan-sgRNA-eGFP The results show that 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, indicating that the amplification is correct.

[0040] The positive single colony determined by PCR was inoculated into LB liquid medium containing 100 μg / mL kanamycin and cultured in a shaking incubator at 37 °C and 200 rpm for 12-16 h until the OD 600 ≈1.0, pipette 1 mL of 50% glycerol and 1 mL of bacterial solution into a sterile glycerol tube, and freeze it in a -20 ℃ low-temperature refrigerator.

[0041] Example 3 Strain Yarrowia lipolytica CICC1457 / pDuRCC-K- sgRNA-eGFP Construction (i) Take 10 μL of plasmid pDuRCC-K- sgRNA - eGFP Add to a 1.5 mL sterile centrifuge tube containing 100 μL of Yarrowia lipolytica CICC1457 competent cells and place on ice for 5 min. Meanwhile, place a 2 mm electroporation cuvette on ice to pre-cool.

[0042] (ii) The competent bacterial solution mixed with the plasmid was transferred to an electroporation cuvette and the electroporation conditions were set as follows: 2.5 kV electric shock for 5 ms.

[0043] (iii) Immediately after the electroporation, add 900 μL of fresh sterile YPD liquid culture medium to the electroporation cup, transfer the bacterial solution in the electroporation cup to a 1.5 mL sterile centrifuge tube, and place the tube in a shaking incubator at 30 °C and 200 rpm for 1 h to recover.

[0044] (iv) Centrifuge at 3000 rpm for 3 min at room temperature, discard the supernatant, and wash 2-3 times with 1 mL of sterile water.

[0045] (v) Resuspend the cells in 100 μL of sterile water and spread them on YPD resistance selection plates containing 1000 μg / mL G418 (Geneticin). Place the plates in a 30°C incubator for 2-4 days until transformants grow.

[0046] After a single colony grows on the resistance plate, use a sterile inoculation loop to randomly pick 10 single colonies and pipette them into a 1.5 mL sterile centrifuge tube with 50 μL of sterile water to mix them evenly; take 10 μL of bacterial solution into a new centrifuge tube and add 20 μL of lysis buffer, boil at 100 ℃ for 12 min, then centrifuge at 12000 r / min at room temperature for 1 min, and use the supernatant as a template for PCR amplification.

[0047] The reaction system of the PCR amplification is: 1 μL of supernatant, 0.5 μL of Kan-F (SEQ ID NO: 9), 0.5 μL of Kan-R, and KOD One TM PCR Master Mix polymerase 5 μL and ddH2O 3 μL; Kan-R: 5'-TTAGAAAAACTCATCGAGCA-3' (SEQ ID NO: 11); The reaction procedure of the 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 storage at 16°C.

[0048] The PCR products were tested by agarose gel electrophoresis. Figure 4 , Lane M is the marker, and lanes 1 to 10 are for verification Can The results showed that primers Kan-F and Kan-R could amplify a specific gene band with a size of about 800 bp. Lanes 1, 3 to 10 were close to the theoretical value of 810 bp, indicating that the amplification was correct.

[0049] Example 4 Yarrowia lipolytica CICC1457 / pDuRCC-K- sgRNA-eGFP Sequencing analysis Use a sterile inoculation loop to randomly pick the recombinant strain CICC1457 / pDuRCC-K- sgRNA-eGFPPipette and mix in 50 μL sterile water; take 10 μL of bacterial solution into a new centrifuge tube and add 20 μL of lysis buffer, boil at 100 ℃ for 12 min, centrifuge at 12000 r / min at room temperature for 1 min, and use the supernatant as a template for PCR amplification.

[0050] The reaction system of the PCR amplification is: 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 ddH2O 3 μL; Fum-F: 5'-ATGCTCCGAACAGTCCGACTAG-3' (SEQ ID NO: 12); Fum-R: 5'-GTCAGAGGGGCCAATCATCTTC-3' (SEQ ID NO: 13); The reaction procedure of the PCR amplification is the same as that of Example 3.

[0051] After PCR, the DNA was recovered using the SanPrep column-based DNA gel recovery kit. Fum Gene sequencing analysis to verify Fum The sequencing results are as follows: Figure 5 As shown, it was found that the strain Yarrowia lipolytica CICC1457 / pDuRCC-K-sgRNA-eGFP Fum The targeted gene sequence of the gene mutated, the N20 region mutated, the 396bp base C mutated to A, the 397bp base G mutated to T, resulting in the 132nd amino acid mutation from N to K. Fum The gene was successfully knocked out and the knockout was successfully constructed Fum The recombinant strain (Yarrowia lipolytica CICC1457-Δ Fum ).

[0052] Example 5 Yarrowia lipolytica CICC1457-Δ Fum Fluorescence detection and plasmid elimination Fluorescence detection: Yarrowia lipolytica CICC1457- 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.

[0053] 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.

[0054] Example 6 Yarrowia lipolytica CICC1457-Δ Fum Shake flask fermentation and yield detection 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.

[0055] Detection of erythritol production: The fermentation broth was centrifuged at 6000 r / min and 4°C for 5 min. After centrifugation, the supernatant was diluted 50 times 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. Detection was performed using a differential high performance liquid chromatograph equipped with a Hi-Plex H (300×7.7 mm, Agilent Technologies) column, and the column was operated at 65°C. The mobile phase was pure water at a flow rate of 0.6 mL / min. The injection volume was 10 μL. Results are shown in Figure 7 and Figure 8 .

[0056] Depend on Figure 7 It can be seen that after 72h of fermentation, Yarrowia lipolytica CICC1457-Δ Fum The content of erythritol in the fermentation broth reached 4.71 g / L, while the content of erythritol in the fermentation broth of the original Yarrowia lipolytica CICC1457 was 2.68 g / L at 72 h. Fum The erythritol production increased by 75.7%.

[0057] Depend on Figure 8 It can be seen that during the fermentation process, the bacterial growth rate and biomass were not significantly different from those of the original strain. Fum After the gene was introduced, the level of erythritol production in Yarrowia lipolytica was increased.

[0058] In summary, the present invention knocks out Fum Genetic construction of Yarrowia lipolytica CICC1457-Δ Fum The synthetic yield of erythritol was increased, and during the fermentation culture process, the bacterial growth rate and bacterial biomass had no significant difference from the original strain.

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockout strains Fum The application of the gene in increasing the erythritol production of the bacterial strain is characterized in that: Said Fum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and the bacterial species includes Yarrowia lipolytica ( Yarrowia lipolytica ).

2. The use according to claim 1, characterized in that: Said Fum The nucleotide sequence of the 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: Knockout strains Fum Gene, to obtain a recombinant strain with improved erythritol production; Said Fum The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1, and the strain includes Yarrowia lipolytica.

4. The construction method according to claim 3, characterized in that: The knockout strain Fum The gene reagent 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 targeting gene nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO:

3.

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

6. The construction method according to claim 4, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

7.

7. The construction method according to claim 4, characterized in that: The nucleic acid molecules inserted into the basic vector also include a nucleic acid molecule encoding a kanamycin resistance gene and a nucleic acid molecule encoding a green fluorescent protein.

8. The construction method according to claim 7, characterized in that: 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.

9. The construction method according to claim 4 or 7, characterized in that: The nucleotide sequence of the nucleic acid molecule inserted into the basic vector is shown in SEQ ID NO:

4.

10. Use of the recombinant strain constructed by the construction method according to any one of claims 3 to 9 in the preparation of erythritol.

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