Application of Fads gene in regulation and control of yield of erythritol produced by fermentation of yarrowia lipolytica

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to application of a Fads gene in regulating and controlling the yield of erythritol produced by fermenting yarrowia lipolytica. The amino acid sequence of the protein coded by the Fads gene is as shown in SEQ ID NO.1, and the yield of the erythritol in the yeast can be remarkably increased by knocking out or knocking down the Fads gene. The result of the embodiment shows that compared with the original strain CICC1457, the growth speed of the Yarrowia lipolytica engineering strain 1k-CICC1457 with the Fads gene knocked out, which is constructed by the invention, is not changed; in addition, the total erythritol yield of the Yarrowia lipolytica engineering strain 1k-CICC1457 strain is obviously increased, and is increased by 1.67 times compared with that of the original strain.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and specifically relates to Fads Application of genes in regulating the yield of erythritol produced by Yarrowia lipolytica fermentation. Background Art

[0002] Erythritol is a polyol widely used in the food industry. It serves as a sweetener for diabetics and those who prefer sugar. It is also used in the processing of chocolate, chewing gum, fudge, and sponge cakes. In addition to its application in the food industry, erythritol is also widely used in organic synthesis as a raw material for alkyd resins, polyesters, and polyethers, and in the production of nitroerythritol, a drug for cardiovascular and cerebrovascular diseases and asthma.

[0003] Currently, the industrial production of erythritol relies primarily on microbial fermentation, with yeast being one of the most commonly used fermentation strains. However, traditional yeast fermentation for erythritol production suffers from relatively low yields and high production costs, limiting its large-scale production and wider application. In yeast cells, multiple genes are involved in regulating the complex metabolic network of erythritol, and the expression and function of these genes directly or indirectly influence the efficiency of erythritol synthesis.

[0004] Fatty acid desaturase (Fads) can change the saturation of fatty acids in yeast cell membranes, thereby regulating the fluidity and permeability of the cell membrane. Currently, there are no reports on the relationship between fatty acid desaturase (Fads) and erythritol production in existing technologies. Summary of the Invention

[0005] The object of the present invention is to provide Fads Application of genes in regulating the production of erythritol by Yarrowia lipolytica, knockout or knockdown Fads Gene, which can significantly increase yeast erythritol production.

[0006] The present invention provides Fads Application of a gene in regulating the yield of erythritol produced by fermentation of Yarrowia lipolytica, the Fads The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1.

[0007] As a preferred solution, the Fads The gene comprises the nucleotide sequence shown in SEQ ID NO.2 or the nucleotide sequence is shown in SEQ ID NO.2.

[0008] As a preferred embodiment, the application includes knocking out or knocking down FadsThe application of gene expression level in improving the yield of erythritol produced by fermentation of Yarrowia lipolytica and / or cultivating Yarrowia lipolytica with high erythritol production.

[0009] The present invention also provides a method for knocking out or knocking down Fads The invention relates to an application of a reagent for regulating gene expression in increasing the yield of erythritol produced by fermentation of Yarrowia lipolytica and / or cultivating Yarrowia lipolytica with high erythritol production.

[0010] As a preferred embodiment, the Yarrowia lipolytica includes Yarrowia lipolytica CICC1457.

[0011] The present invention also provides a targeted knockout Fads The sgRNA of the gene, the nucleotide sequence of the sgRNA is shown as SEQ ID NO.4.

[0012] The present invention also provides a targeted knockout Fads A gene editing vector, wherein the editing vector includes the sgRNA described in the above scheme.

[0013] The present invention also provides a high-yield erythritol yeast engineering bacterium, wherein the yeast engineering bacterium comprises the editing vector described in the above scheme.

[0014] The present invention also provides the use of the sgRNA described in the above scheme, the editing vector described in the above scheme, or the yeast engineering bacteria described in the above scheme in increasing the yield of erythritol produced by Yarrowia lipolytica fermentation and / or cultivating Yarrowia lipolytica with high erythritol production.

[0015] The present invention also provides a method for increasing the yield of erythritol produced by fermentation of Yarrowia lipolytica, comprising the following steps: inoculating the engineered yeast described in the above scheme into a fermentation medium for fermentation.

[0016] Beneficial effect: The present invention provides Fads Application of a gene in regulating the yield of erythritol produced by fermentation of Yarrowia lipolytica, the Fads The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 1. Fads The results of the embodiment show that the knockout gene of the present invention can significantly increase the erythritol production of yeast. Fads The growth rate of the genetically constructed Yarrowia lipolytica engineered strain 1k-CICC1457 did not change compared with the original strain CICC1457; moreover, the total erythritol production of the genetically constructed Yarrowia lipolytica engineered strain 1k-CICC1457 was significantly increased, which was 1.67 times higher than that of 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 for use in the embodiments are briefly introduced below.

[0018] Figure 1 This is a graph showing the erythritol production of the Yarrowia lipolytica production strain and the selected strain in Example 1; Figure 2 The knockout vector pDuRCC- K - sgRNA-eGFP Plasmid construction diagram; Figure 3 The figure is an agarose gel electrophoresis diagram of the plasmid vector pDuRCC-K digested with enzymes in Example 2; Lane M is a marker; Lanes 1 and 2 are samples of PCR amplification products of the digested pDuRCC-K; Figure 4 The agarose gel electrophoresis diagram of the PCR amplification products in Example 2; Lane M is the marker; Lanes 1 to 4 are [[ID= - ​ PCR amplification product samples; ​ This is the agarose gel electrophoresis diagram of the PCR-amplified Kan expression cassette in Example 2; Lane M is the marker; Lanes 1 and 2 are samples of the PCR-amplified products of kan; ​ This is a comparison of the fluorescence intensities of the control strain CICC1457 and the engineered strain 1k-CICC1457 fermented at different times in Example 3; ​ This is a graph showing the yield of erythritol produced by fermentation of the control strain CICC1457 and the engineered strain 1k-CICC1457 in Example 4. DETAILED DESCRIPTION

[0019] The present invention provides a ​ Genes in the regulation of Yarrowia lipolytica ( ​ ) in the production of erythritol by fermentation, ​The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1: MVKNVDQVDLSQVDTIASGRDVNYKVKYTSGVKMSQGAYDDKGRHISEQPFTWANWHQHINWLNFILVIALPLSSFAAAPFVSFNWKTAAFAVGYYMCTGLGITAGYHRMWAHRAYKAALPVRIILALFGGGAVEGSIRWWASSHRVHHRWTDSNKDPYDARKGFWFSHFGWMLLVPNPKNKGRTDISDLNNDWVVRLQHKYYVYVLVFMAIVLPTLVCGFGWGDWKGGLVYAGIMRY In the art, Yarrowia lipolytica, also known as Yarrowia lipolytica, has rich metabolic pathways, good physiological properties, and is easy to genetically engineer. It can provide sufficient precursors for erythritol synthesis and efficiently secrete the product. It is also highly safe, has mature applications, and can adapt to a variety of conditions in industrial production, effectively reducing costs and improving production efficiency.

[0020] As a preferred embodiment, the ​

[0021] As a preferred embodiment, the application includes knocking out or knocking down ​ The application of gene expression in increasing the yield of erythritol produced by fermentation of Yarrowia lipolytica and / or cultivating Yarrowia lipolytica with high erythritol production. ​ After the gene is introduced, the growth rate of Yarrowia lipolytica is not affected, and the yield of erythritol can be significantly increased.

[0022] The present invention also provides a method for knocking out or knocking down ​ The use of a reagent for increasing gene expression in increasing the yield of erythritol produced by Yarrowia lipolytica fermentation and / or cultivating Yarrowia lipolytica with high erythritol production. As an embodiment, the Yarrowia lipolytica includes Yarrowia lipolytica ( ​ )CICC1457.

[0023] The present invention also provides a targeted knockout ​ The sgRNA of the gene, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.4: 5′-TGTAGTTGACATCTCGGCCG-3′.

[0024] The present invention also provides a targeted knockout ​ The editing vector of the gene includes the sgRNA described in the above scheme. As an embodiment, the editing vector also includes a Kan expression cassette and an eGFP expression cassette. In a specific embodiment of the present invention, the editing vector is inserted with a Kan expression cassette, an sgRNA expression cassette and an eGFP expression cassette; the Kan expression cassette is driven by a TEF promoter, ​ The three components of the gene and TEF terminator are connected in sequence. The nucleotide sequence of the Kan expression cassette is shown in SEQ ID NO.3, which can be used for screening positive transformants; the sgRNA expression cassette is composed of the Candida promoter, the gRNA scaffold on the vector pDuRCC-K, the sgRNA (the target gene sequence is shown in SEQ ID NO.4) and the SUP4 terminator, which are connected in sequence. The nucleotide sequence of the sgRN expression cassette is shown in SEQ ID NO.5; the eGFP expression cassette is composed of the hp4d promoter, the green fluorescent protein ​ The three components of the gene and the XPR2 terminator are connected in sequence. The nucleotide sequence of the eGFP expression cassette is shown in SEQ ID NO.6, which can be used to express fluorescent protein for reverse screening and complete plasmid elimination.

[0025] The present invention also provides a yeast engineering bacterium with high erythritol production, wherein the yeast engineering bacterium comprises the editing vector described in the above scheme. The yeast engineering bacterium of the present invention utilizes the editing vector to knock out the ​ The gene is added to the yeast, and the growth rate of the resulting engineered yeast is not affected, and the erythritol production can be increased. As an embodiment, the yeast includes Yarrowia lipolytica. In a specific embodiment of the present invention, the yeast includes Yarrowia lipolytica CICC1457.

[0026] The present invention also provides the use of the sgRNA described in the above scheme, the editing vector described in the above scheme, or the yeast engineering bacteria described in the above scheme in increasing the yield of erythritol produced by Yarrowia lipolytica fermentation and / or cultivating Yarrowia lipolytica with high erythritol production.

[0027] The present invention also provides a method for increasing the yield of erythritol produced by Yarrowia lipolytica fermentation, comprising the following steps: inoculating the engineered yeast described in the above scheme into a fermentation medium for fermentation. In a preferred embodiment, the fermentation temperature is 28-30°C; the fermentation time is 72-96 hours; and the fermentation medium comprises, per liter, 18-22 g / L of glucose, 18-22 g / L of peptone, 8-12 g / L of yeast extract, and the remainder is water. In a specific embodiment of the present invention, the fermentation medium comprises, per liter, 20 g / L of glucose, 20 g / L of peptone, and 10 g / L of yeast extract.

[0028] In order to further illustrate the present invention, the following is a diagram of the present invention with reference to the accompanying drawings and embodiments. ​ The application of the gene in regulating the yield of erythritol produced by Yarrowia lipolytica fermentation is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0029] Example 1 ​ Gene discovery (1) The production strain Yarrowia lipolytica 5-14 (published in the literature: Cui Q, Liu H, GuoC. et.al. Enhancing the erythritol production of Yarrowia lipolytica by high-throughput screening based on highly sensitive artificial sensor and anchor protein cwp2. J Ind Microbiol Biotechnol. 2024 Jan 9;51:kuae045.) is a strain that can efficiently produce erythritol for industrial production after a series of modifications; after breeding the production strain Y.lipolytica 5-14, the bred strain DXMC004-tb was obtained.

[0030] (2) The production strain Y.lipolytica 5-14 and the selected strain DXMC004-tb were inoculated into YPD liquid culture medium respectively, and cultured in a shaking incubator at 30°C and 200 rpm for 24 h to obtain seed solution.

[0031] (3) The seed solution from step (2) was transferred to the fermentation tank at an inoculum rate of 10% (v / v), and the culture was continued for 3 days by controlling the rotation speed and dissolved oxygen. The erythritol content in the fermentation broth was detected by HPLC. The test results are as follows: ​ As shown, HPLC detection showed that the erythritol-producing strain Y.lipolytica 5-14 produced 161 g / L of erythritol after fermentation for 72 hours, and the selected strain DXMC004-tb produced 178 g / L of erythritol after fermentation for 72 hours, with a yield increase of 10.6%.

[0032] The whole genome sequencing of the production strain Y.lipolytica 5-14 and the selected strain DXMC004-tb showed that the selected strain DXMC004-tb had a higher ​ The gene mutates.

[0033] Example 2 ​ Gene function verification Construction of knockout vector pDuRCC-K- ​ , plasmid vector construction diagram as shown ​ shown.

[0034] (1) Double enzyme digestion reaction of plasmid vector pDuRCC-K use ​ I and​ I. Double enzyme digestion of plasmid vector pDuRCC-K, total enzyme digestion system 20µL: plasmid vector pDuRCC-K 2.0µL, ​ I 1.0µL, ​ I 1.0µL, 10× Fast Digest buffer 2.0µL, double-distilled water 14.0µL. Enzyme digestion conditions are as follows: 37°C for 2 hours.

[0035] After the enzyme digestion is completed, agarose gel electrophoresis is used to detect whether the enzyme digestion is successful. The identification results are as follows: ​ As shown, the target band appeared and was single, indicating that the plasmid vector was successfully digested.

[0036] (2) Constructing knockout components The knockout component consists of a Kan expression cassette, an sgRNA expression cassette, and an eGFP expression cassette. The Kan expression cassette is used for screening positive transformants, and the sgRNA expression cassette mainly guides the Cas9 protein to the target sequence and cuts it by complementary pairing with the Fads gene target sequence to perform gene knockout. The eGFP expression cassette is mainly used to express fluorescent protein for reverse screening to complete plasmid elimination.

[0037] ① Using pDuRCC-K plasmid as template, select TEF promoter, ​​ Gene( ​ resistance fragment), 1155~1159bp is the linker, and 1160~1357bp is the TEF terminator.

[0038] ②According to ​ The gene (SEQ ID NO. 2) sequence was used to design the sgRNA, whose sequence is shown in SEQ ID NO. 4: 5′-TGTAGTTGACATCTCGGCCG-3′. The Candida promoter, the gRNA scaffold on the vector pDuRCC-K, the targeting gene sequence (sgRNA), and the SUP4 terminator were sequentially connected to form the sgRNA expression cassette. The nucleotide sequence of the sgRNA expression cassette is shown in SEQ ID NO. Shown in NO.5: 5′-AGACATAAAAAACAAAAAAAGCACCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTGTAGTTGACATCTCGGCCGCAAATTAAAAATAGTTTACGCAAGTCTCGAACTTCCACCACAATTTAATTGTAAGAACGCTGCG AAAACATATTCTCCAATCCTATTGGGCCGAAGTTTCAGCTATGCCAAAGGCATCAAATTTTTTTTTATAAAAATTTTCCACTTTTCCATACCAGTGAAATTATCACGTGATGGAGTGAGTATCTATAATATTTGATTGTTTCTTTGATGAGGCAATACTCGAGTATAGTTATTGACCAATGACTTTTAGC-3′. Among them, 1~20bp is the SUP4 terminator, 21~24bp is the linker, 25~99bp is the gRNA scaffold, 100~119bp is the target gene sequence, and 120~372bp is the promoter.

[0039]

[0040] We commissioned Nanjing GenScript Biotechnology Co., Ltd. to synthesize the knockout component and connect the above three components in sequence to form ​ - ​

[0041] (3) Connect the linearized vector and the knockout component The linearized vector from step (1) and the knockout component from step (2) were ligated using T4 DNA ligase. The total ligation reaction volume was 10 µL: 1.0 µL of linearized plasmid, 1.0 µL of knockout component, 1.0 µL of T4 DNA Ligase, 1.0 µL of 10× Ligase Buffer, and 6.0 µL of double-distilled water. The reaction was carried out at 16°C for 5 h to obtain the recombinant plasmid pDuRCC- K - ​ .

[0042] (4) Recombinant plasmid pDuRCC- K - ​ Conversion ① Add 10µL of recombinant plasmid pDuRCC- ​ Add to 100 µL of E. coli BL21 competent cells, mix gently, and place on ice for 30 minutes; heat shock at 42°C for 45 seconds, then quickly cool in an ice bath for 2 minutes; ② Add 900µL of LB medium without resistance to the mixture in step ① and resuscitate at 37℃ for 1 hour.

[0043] ③ Centrifuge the bacterial solution from step ② at 2500×g for 3 min, aspirate 900µL of the supernatant, and resuspend the solution to obtain a resuspended bacterial solution.

[0044] ④ Spread the resuspended bacteria in step ③ onto LB solid medium containing 100µg / mL kanamycin resistance and culture overnight until normal colonies of about 2mm appear. At this time, the strain is E. coli BL21 (pDuRCC- K - ​ ).

[0045] (5) Identification of positive clones: ① Colony PCR identification The single colony cultured in step (4) was picked and added to 1 mL of LB medium containing kanamycin, and cultured at 37°C and 200 rpm for 6-8 h. 2 μL of bacterial solution was aspirated and colony PCR identification was performed according to the 50 μL PCR reaction system.

[0046] The recombinant plasmid pDuRCC-K- ​ PCR amplification was performed using the template, and the sequences of the PCR amplification primers are as follows: pDuRCC-F (SEQ ID NO.8): 5′-ATGGGTAAGGAAAAGACTCA-3′; pDuRCC-R (SEQ ID NO. 9): 5′-CGTCCATTCCGAGAGTGATGC-3′.

[0047] The total PCR amplification system (50 µL) consisted of 2.0 µL of 100 µM upstream primer, 2.0 µL of 100 µM downstream primer, 2.0 µL of template, 25 µL of 5 U / µL phantalide enzyme, and 19 µL of ddH2O. The PCR amplification conditions were: 95°C pre-denaturation for 3 min; 30 cycles of 95°C denaturation for 15 s, 55°C annealing for 15 s, and 72°C extension for 30 s; and 72°C extension for 5 min. The PCR results were as follows: ​ As shown in the figure, the colony with a single target band is a positive clone.

[0048] ② Bacteria sample sequencing The positive clones after identification were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to further prove the correctness of the constructed positive clones.

[0049] Example 3 Recombinant plasmid pDuRCC- K - ​ Transformation into Yarrowia lipolytica CICC1457 (1) Preparation of competent yeast ① Inoculate a frozen glycerol tube of preserved Yarrowia lipolytica CICC 1457 (purchased from the China Industrial Microbiological Culture Collection) into 10 mL of YPD liquid medium and culture overnight in a shaking incubator at 30°C and 200 rpm to obtain an activated CICC1457 culture. This strain is the original strain and has not undergone any modification.

[0050] ② Transfer the activated CICC1457 bacterial solution in step ① into 50 mL of 2×YPD liquid medium, and the initial OD 600 The cells were cultured in a shaking incubator at 30°C and 200 rpm until the OD 600 It is 0.8~1.0.

[0051] ③Pipette 10 mL of the bacterial solution from step ② into a 10 mL centrifuge tube. Place the bacterial solution on ice for 15 minutes and then centrifuge at 4°C, 5000 rpm for 5 minutes. Discard the supernatant to obtain the bacterial cells. All subsequent steps are performed on ice.

[0052] ④ Take 5 mL of sterile 1 mol / L sorbitol solution to resuspend the cells obtained in step ③, centrifuge at 5000 rpm for 5 min at 4°C, and remove the supernatant.

[0053] ⑤ Take 5 mL of sterile 10% glycerol solution to resuspend the cells prepared in step ④, centrifuge at 5000 rpm for 5 min at 4°C, and remove the supernatant.

[0054] ⑥ Take 100µL of sterile 1 mol / L sorbitol solution to resuspend the cells from step ⑤, aliquot 100µL of competent medium into 1.5mL sterile centrifuge tubes and store at -80℃.

[0055] (2) Transformation of recombinant plasmid ① Take 10µL of plasmid pDuRCC- ​ Add to a 1.5 mL sterile centrifuge tube containing 100 µL of CICC1457 competent cells and place on ice for 5 minutes. Meanwhile, place a 2 mm electroporation cuvette on ice to pre-cool.

[0056] ② Transfer the competent bacterial solution mixed with the plasmid to an electroporation cup, and set the electroporation conditions to: 2.5kV electric shock for 5ms.

[0057] ③ Immediately after the electroporation, add 900 µL of fresh sterile YPD liquid medium to the electroporation cup, transfer the bacterial liquid in the electroporation cup to a 1.5 mL sterile centrifuge tube, and place it in a shaking incubator at 30°C and 200 rpm to recover for 1 hour.

[0058] ④ Centrifuge at 3000 rpm for 3 min at room temperature, discard the supernatant, and wash 2 to 3 times with 1 mL of sterile water.

[0059] ⑤ Resuspend the cells in 100 µL of sterile water and spread them onto YPD resistance selection plates supplemented with 1000 µg / mL Geneticin (G418). Place the plates in a 30°C incubator and incubate for 2–4 days until transformants emerge.

[0060] (3) Identification of positive clones ① Colony PCR identification Add 50 µL of 20 mM NaOH solution to a sterile 1.5 mL centrifuge tube, pick up 20 colonies that have grown successfully on the plate and add them to the tube for resuspending, boil for 10 minutes, and centrifuge at 12,000°C for 1 minute.

[0061] 1 μL of supernatant was taken as a template for PCR amplification. The primer sequences used are as follows: pDuRCC-F (SEQ ID NO.8): 5′-ATGGGTAAGGAAAAGACTCA-3′; Kan-R (SEQ ID NO. 10): 5′-TTAGAAAAACTCATCGAGCA-3′.

[0062] PCR amplification system (10 µL): 100 µM upstream primer 0.5 µL, 100 µM downstream primer 0.5 µL, template 1.0 µL, KODOne TM PCR Master Mix polymerase 5.0µL, ddH2O 3.0µL.

[0063] PCR amplification conditions were as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 56°C for 30 s, extension at 68°C for 10 s, 40 cycles; extension at 68°C for 5 min; and storage at 16°C for 1 min.

[0064] PCR identification results ​ As shown, the results showed that the target band appeared and was single, indicating that the colony was a positive clone.

[0065] ② Bacteria sample sequencing The positive clones after identification were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to further verify the correctness of the constructed positive clones and obtain the positive knockout strain 1k-CICC1457.

[0066] Example 4 Fluorescence Detection of Knockout Strain 1k-CICC1457 (1) Activation of strains: The positive knockout strain 1k-CICC1457 and the control strain CICC1457 in Example 2 were inoculated into YPD liquid culture medium, respectively, and cultured in a shaking incubator at 30°C and 200 rpm for 24 h to obtain the activated strain 1k-CICC1457 and the activated strain CICC1457; (2) Bacterial transfer: Take the activated strain in step (1) and transfer it to a 500 mL conical flask containing 50 mL of liquid fermentation medium at a 10% (v / v) inoculation rate, and culture it in a shaking incubator at 30°C and 200 rpm for 3 days.

[0067] (3) Fluorescence detection: Samples were taken every 12 h, 1 mL of fermentation broth was centrifuged to remove the supernatant, the bacteria were resuspended in 1 mL of sterile water, and the fluorescence intensity of the knockout strain 1k-CICC1457 and the control strain CICC1457 at 484-507 nm was detected using an enzyme-labeled instrument.

[0068] The stronger the fluorescence, the better the plasmid transformation. The identification results are as follows: ​ As shown, the fluorescent protein in the knockout strain 1k-CICC1457 is continuously expressed and is higher than that in the control strain, indicating that the knockout strain 1k-CICC1457 has good growth. In addition, the OD values ​​of the production strain and the knockout strain were measured during the growth process. 600 The values ​​were not significantly different, indicating that the growth rate and biomass of the knockout strain 1k-CICC1457 were not affected.

[0069] Example 5 Vector Elimination and Shake Flask Fermentation Production of Erythritol in Knockout Strain 1k-CICC1457 (1) Vector elimination: The knockout strain 1k-CICC1457 of Example 2 and the control strain CICC1457 were inoculated into YPD liquid culture medium, respectively, and cultured in a shaking incubator at 30°C and 200 rpm for 24 h. The cells were subcultured three times.

[0070] (2) Shake flask fermentation: The 1k-CICC1457 seed solution after passage in step (1) was transferred to a 500 mL conical flask containing 50 mL of liquid fermentation medium at a 10% (v / v) inoculation rate, and cultured in a shaker at 30°C and 200 rpm for 3 days to obtain a fermentation broth.

[0071] (3) Fermentation broth treatment: Centrifuge at 6000 rpm and 4°C for 5 min. After centrifugation, dilute the supernatant 50 times with distilled water, filter through a 0.22 µm filter membrane, immediately add to a brown liquid vial, and store at -20°C in the dark.

[0072] (4) Detection of the generated product by differential high performance liquid chromatography: equipped with a Hi-Plex H (300×7.7 mm, Agilent Technologies) column, the column was operated at 65°C. The mobile phase was pure water, the flow rate was 0.6 mL / min, and the injection volume was 10 μL. Five different concentrations of erythritol standard solutions were prepared, namely 0.25 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, 4.0 g / L, and 8.0 g / L, and the peak time was determined.

[0073] Identification results such as ​ As shown, compared with the original strain, the total erythritol production of the Yarrowia lipolytica engineered strain 1k-CICC1457 constructed in the present invention was significantly improved, reaching 4.5 g / L, an increase of 1.67 times.

[0074] It can be seen that knockout ​ The gene can significantly increase the production of erythritol; and during the fermentation culture process, the growth rate and biomass of the engineered strain are no significantly different from those of the original strain.

[0075] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Fads Application of a gene in regulating the yield of erythritol produced by fermentation of Yarrowia lipolytica, the Fads The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Said Fads The gene comprises the nucleotide sequence shown in SEQ ID NO.2 or the nucleotide sequence is as shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The applications include knockout or knockdown Fads The application of gene expression level in improving the yield of erythritol produced by fermentation of Yarrowia lipolytica and / or cultivating Yarrowia lipolytica with high erythritol production.

4. Knockout or knockdown Fads The invention discloses an application of a reagent for measuring the gene expression level in improving the yield of erythritol produced by fermentation of Yarrowia lipolytica and / or cultivating Yarrowia lipolytica with high erythritol yield.

5. The use according to any one of claims 1 to 4, characterized in that: The Yarrowia lipolytica includes Yarrowia lipolytica CICC1457.

6. A targeted knockout Fads The sgRNA of a gene is characterized by: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

4.

7. A targeted knockout Fads A gene editing vector, characterized in that The editing vector comprises the sgRNA of claim 6.

8. A yeast engineering bacterium with high erythritol production, characterized in that: The yeast engineering bacteria comprises the editing vector according to claim 7.

9. Use of the sgRNA according to claim 6, the editing vector according to claim 7, or the yeast engineering bacteria according to claim 8 in increasing the yield of erythritol produced by fermentation of Yarrowia lipolytica and / or cultivating Yarrowia lipolytica with high erythritol production.

10. A method for increasing the yield of erythritol produced by fermentation of Yarrowia lipolytica, characterized in that: The method comprises the following steps: inoculating the yeast engineering bacteria according to claim 8 into a fermentation medium for fermentation.

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