A recombinant Yarrowia lipolytica and its application in producing erythritol

Abstract

The present invention discloses a recombinant Yarrowia lipolytica and its application in the production of erythritol, belonging to the field of bioengineering technology. The Ku70 gene has been deleted in the recombinant Yarrowia lipolytica genome of the present invention, and the GUT1, GUT2, TKL1 and TAL1 genes are overexpressed using the plasmid pINA1269 combination. The present invention provides a recombinant Yarrowia lipolytica Y-11 / pINA1269-GUT1-TKL1-GUT2-TAL1 that can produce high erythritol. The recombinant Yarrowia lipolytica is inoculated into a 5L fermenter and fermented for 120 hours using crude glycerol as a carbon source. The concentration of erythritol in the fermentation broth can be as high as 110±1.1g / L, and the conversion rate can be as high as 61%.

Description

Technical Field

[0001] The invention relates to a recombinant Yarrowia lipolytica and application thereof in producing erythritol, belonging to the technical field of bioengineering. Background Art

[0002] Erythritol, also known as 1,2,3,4-butanetetrol, is a sweet four-carbon sugar alcohol that is naturally found in seaweed, fungi, fruits and fermented foods. Clinical studies and animal toxicology tests have found that erythritol is not metabolized in the human body, and 90% of the erythritol ingested is excreted through the kidneys. Due to its special nutritional properties, erythritol has been widely used in food and medicine in recent years. Erythritol has the following main functions: (1) Increase the sweetness of food. Erythritol is a sweetener with a sweetness of 60%-70% of sucrose. It has a refreshing sweet taste and is generally added to cakes, candies, beverages and other foods. (2) Helps lose weight. Although erythritol is sweet, it has no calories and is a natural zero-calorie sweetener. For cake products, adding erythritol can reduce calories by at least 30% and will not bring negative effects after use. It can help lose weight after consumption and is very suitable for people who want to lose weight as a sugar substitute. (3) Protect the oral cavity. Many people worry about tooth decay when eating sugar, but erythritol, a sugar substitute, will not be used by oral bacteria, so it will not cause tooth damage. It can also reduce the growth of oral bacteria and effectively protect oral health.

[0003] Currently, erythritol is produced primarily through microbial fermentation and chemical synthesis. Chemical synthesis involves treating starch or cellulose oxidized with periodate with an acid or alkali solution, followed by hydrogenation under high temperature and high pressure. However, this process produces a high number of byproducts, resulting in low yields and difficulty separating the product, making industrialization difficult. Compared to chemical synthesis, microbial fermentation offers advantages in production: a gentle, easily controllable process, minimal environmental pollution, and product safety. Therefore, microbial fermentation is currently the most commonly used method for producing erythritol.

[0004] Huiling et al. overexpressed and isolated two novel erythrose reductase (ER) genes g141.t1 (YALI0D07634g) and g3023.t1 (YALI0C13508g), resulting in an average increase of 14.7% in erythritol production and 31.2% in productivity. By overexpressing the genes ZWF1 and GND1 encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, to balance NADPH cofactor metabolism, erythritol production was increased by 5% and productivity was increased by 50% (for details, see reference: Huiling, C., Siqi, W., Muhammad, B., Xuemei, G., Can, Z., Patrick, F., Hairong, C., 2018. Identification, characterization of two NADPH-dependent erythrose reductases in the yeast Yarrowia lipolytica and improvement of erythritol productivity using metabolic engineering. Microb Cell Fact. 17, 133.). However, currently only a few use glycerol as a carbon source to produce erythritol, especially crude glycerol. Summary of the Invention

[0005] In order to improve the yield and productivity of erythritol production strains in the prior art and to realize the reuse of crude glycerol, a waste of biodiesel, the present invention provides a recombinant Yarrowia lipolytica.

[0006] The main components of the crude glycerol in the waste of biodiesel are (w / w): 80-85% of glycerol, 2.0% of sodium salt, 10-15% of methanol, 2.5% of other organic matter and 2% of water.

[0007] The present invention provides a recombinant Yarrowia lipolytica, wherein the recombinant Yarrowia lipolytica is obtained by knocking out the Ku70 gene of the starting strain Yarrowia lipolytica, and simultaneously overexpressing the glycerol kinase gene GUT1, 3-glyceraldehyde phosphate dehydrogenase gene GUT2, transketolase gene TKL1 and transaldolase gene TAL1 derived from Yarrowia lipolytica.

[0008] In one embodiment of the present invention, the nucleotide sequence of the Ku70 gene is a protein shown in SEQ ID NO.1, and the NCBI Genebank accession number of the Ku70 gene is: 2910012.

[0009] In one embodiment of the present invention, the nucleotide sequence of the glycerol kinase gene GUT1 is a protein as shown in SEQ ID NO. 2, and the NCBI Genebank accession number of the glycerol kinase gene GUT1 is: 2908247.

[0010] In one embodiment of the present invention, the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene GUT2 is a protein as shown in SEQ ID NO. 3, and the NCBI Genebank accession number of the glyceraldehyde-3-phosphate dehydrogenase gene GUT2 is: 2907009.

[0011] In one embodiment of the present invention, the nucleotide sequence of the transketolase gene TKL1 is a protein shown in SEQ ID NO. 4, and the NCBI Genebank accession number of the transketolase gene TKL1 is: 2912397.

[0012] In one embodiment of the present invention, the nucleotide sequence of the transaldolase gene TAL1 is a protein as shown in SEQ ID NO. 5, and the NCBI Genebank accession number of the transaldolase gene TAL1 is: 2909043.

[0013] In one embodiment of the present invention, the recombinant Yarrowia lipolytica uses pCAS1yl plasmid or pINA1269 plasmid as an expression vector.

[0014] In one embodiment of the present invention, the pCAS1yl plasmid expression vector was purchased from Addgene.

[0015] The pINA1269 plasmid expression vector was donated by French professor Catherine Madzak. The preparation method of this expression vector is disclosed in the paper "Madzak C, Gaillardin C, Beckerich J M. Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review [J]. Journal of Biotechnology, 2004, 109 (1-2): 63-81." It can also be purchased from: BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Collection Center, NTCC Type Culture Collection Center.

[0016] In one embodiment of the present invention, the recombinant Yarrowia lipolytica is based on Yarrowia lipolytica PO1f as a starting strain.

[0017] The present invention also provides a method for constructing the above-mentioned recombinant Yarrowia lipolytica, which comprises the following steps:

[0018] (1) Using pCAS1yl, the Ku70 gene was first knocked out in the starting strain Yarrowia lipolytica to obtain the strain Y. lipolytica PO1f-ΔKu70;

[0019] (2) The pINA1269 plasmid was used to overexpress the glycerol kinase gene GUT1, the glyceraldehyde-3-phosphate dehydrogenase gene GUT2, the transketolase gene TKL1, and the transaldolase gene TAL1 to prepare the recombinant plasmid pINA1269-GUT1-TKL1-GUT2-TAL1;

[0020] (3) The prepared recombinant plasmid pINA1269-GUT1-TKL1-GUT2-TAL1 was introduced into the strain obtained in step (1) to prepare recombinant Yarrowia lipolytica / pINA1269-GUT1-TKL1-GUT2-TAL1.

[0021] The present invention also provides a method for producing erythritol, which comprises adding the above-mentioned recombinant Yarrowia lipolytica to a reaction system using biodiesel waste as the sole carbon source to ferment and prepare erythritol.

[0022] In one embodiment of the present invention, the recombinant Yarrowia lipolytica seed liquid is added to a reaction system containing biodiesel waste as the sole carbon source for fermentation to prepare a fermentation broth, and erythritol is separated from the fermentation broth. The biodiesel waste contains crude glycerol; the components of the crude glycerol are as follows by mass fraction: 80-85% glycerol, 2.0% sodium salt, 10-15% methanol, 2.5% other organic matter and 2% water.

[0023] In one embodiment of the present invention, the recombinant Yarrowia lipolytica is inoculated into a seed culture medium for culture to obtain a primary seed liquid; the primary seed liquid is transferred to a seed culture medium at an inoculation rate of 10% and cultured to obtain a secondary seed liquid; the prepared secondary seed liquid is inoculated into a fermentation medium containing crude glycerol at an inoculation rate of 20% for fermentation to prepare erythritol.

[0024] The fermentation medium comprises: 250 g / L crude glycerol, 1.0 g / L yeast extract, 5.0 g / L NH4Cl, 0.25 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 mol / L NaCl, and a pH of 3.0.

[0025] The seed culture medium comprises: 50 g / L crude glycerol, 10 g / L yeast extract, 0.5 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, and pH 3.0.

[0026] In one embodiment of the present invention, the recombinant Yarrowia lipolytica is inoculated into a seed culture medium under the following culture conditions: 30° C., 220 rpm, the pH of the seed culture medium is controlled at 3.0, and the culture time is 20 h.

[0027] In one embodiment of the present invention, the method is to prepare erythritol by fermentation in a 5L fermentation tank, and the method is: inoculating recombinant Yarrowia lipolytica into a seed culture medium for culturing to obtain a primary seed liquid; transferring the primary seed liquid to a seed culture medium at an inoculation rate of 10% (v / v) and culturing to obtain a secondary seed liquid; and inoculating the prepared secondary seed liquid into a fermentation culture medium at an inoculation rate of 20% (v / v) for fermentation to prepare erythritol.

[0028] In one embodiment of the present invention, the seed liquid is inoculated into the fermentation medium, and the fermentation conditions are: 30° C., 800 rpm, 20-30% dissolved oxygen, and the culture time is: 120 h.

[0029] In one embodiment of the present invention, the fermentation medium is: crude glycerol 250 g / L, yeast extract 1.0 g / L, NH4Cl 5.0 g / L, KH2PO4 0.25 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 mol / L, pH 3.0.

[0030] The crude glycerol comprises (w / w): 80-85% glycerol, 2.0% sodium salt, 10-15% methanol, 2.5% other organic matter and 2% water.

[0031] In one embodiment of the present invention, the seed culture medium comprises: 50 g / L crude glycerol, 10 g / L yeast extract, 0.5 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, and pH 3.0.

[0032] In one embodiment of the present invention, during the fermentation process in a 5 L fermenter, the stirring speed is set to 800 rpm, the temperature is set to 30° C., the dissolved oxygen is controlled at 20%-30%, and the fermentation time is 120 h.

[0033] The present invention also provides the use of the recombinant Yarrowia lipolytica or the method in preparing products containing erythritol.

[0034] Beneficial effects

[0035] (1) The present invention uses crude glycerol, a waste product of biodiesel, as a raw material for the production of erythritol, and provides a recombinant Yarrowia lipolytica that can utilize crude glycerol to produce high levels of erythritol. The recombinant Yarrowia lipolytica is inoculated into a 5-L fermenter and fermented for 120 h. The erythritol concentration is as high as 110±1.1 g / L, and the conversion rate is as high as 61%, which is greatly improved compared to the currently reported glycerol conversion rate.

[0036] (2) The present invention greatly improves the integration efficiency by knocking out the Ku70 gene, and enhances the utilization of glycerol and the supply of precursors by combining the overexpression of the glycerol kinase gene GUT1, the 3-glyceraldehyde phosphate dehydrogenase gene GUT2, the transketolase gene TKL1 and the transaldolase gene TAL1, thereby further achieving high production of erythritol.

[0037] (3) The recombinant Yarrowia lipolytica obtained by the present invention can achieve high production of erythritol under high osmotic pressure and low pH conditions, which on the one hand protects the bacteria from contamination by other bacteria, and on the other hand effectively inhibits the synthesis of by-products mannitol and arabitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Schematic diagram of the construction of the knockout plasmid pCAS2yl-ΔKu70.

[0039] Figure 2 : Schematic diagram of the construction of overexpression plasmid.

[0040] Figure 3 : Fermentation process curves of Y-01 and Y-11. DETAILED DESCRIPTION

[0041] The present invention discloses a recombinant Yarrowia lipolytica and its use in erythritol production in a preferred embodiment. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] In order to further understand the present invention, the present invention is described in detail below with reference to specific examples. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products and can be purchased through commercial channels.

[0043] The Y. lipolytica PO1f involved in the following examples was purchased from: BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Collection Center, NTCC Type Culture Collection Center.

[0044] The culture medium involved in the following examples is as follows:

[0045] YPD liquid medium (1 L): 20 g glucose, 20 g peptone, 10 g yeast extract.

[0046] YPD solid medium (1 L): Add 2% agar powder to YPD liquid medium.

[0047] SC liquid medium (1 L): 20 g glucose, 1.7 g YNB, 5 g ammonium sulfate.

[0048] SC solid medium (1 L): Add 2% agar powder to SC liquid medium.

[0049] LB solid medium (1 L): 10 g NaCl, 10 g tryptone, 5 g yeast extract, 20 g agar powder.

[0050] The detection methods involved in the following embodiments are as follows:

[0051] The cell density OD600 was measured using an ultraviolet spectrophotometer;

[0052] Detection of erythritol and glycerol content:

[0053] High-performance liquid chromatography (Agilent 1200 series; Agilent Technologies) was used for detection.

[0054] The sample volume is 10ul and the amino column is NH2 (250×4.6 mm; 5 μm), 80% acetonitrile as the mobile phase, flow rate of 1.0 mL / min. Detector: RID detector, detector temperature 35°C, column temperature 40°C.

[0055] The conversion rate is calculated as follows:

[0056]

[0057] Where: η: conversion rate; C 赤藓糖醇 : erythritol concentration in the fermentation broth after fermentation, g / L; C0: initial crude glycerol concentration in the fermentation broth, g / L; C 粗甘油 : crude glycerol concentration in the fermentation broth after the fermentation is completed, g / L; V1: volume of crude glycerol consumed, L; V: volume of the fermentation broth after the fermentation is completed, L.

[0058] Example 1: Preparation of recombinant Yarrowia lipolytica PO1fΔKu70 strain

[0059] (1) Construction of knockout plasmid pCAS2yl-ΔKu70

[0060] The Ku70 gene sequence from the Y. lipolytica CLIB122 genome (NCBI Genebank Accession No. 2910012) was used to determine the location of the Ku70 gene and the 1758-bp deletion. Approximately 1000-bp fragments before and after the Ku70 gene were selected as upstream and downstream homology arms, as shown in SEQ ID NOs. 6 and 7. To knock out the Ku70 gene, two primer pairs (Ku70-sg-1 and Ku70-sg-2) were designed to construct the plasmid pCAS1yl-ΔKu70. Hygromycin B (SEQ ID NO. 8) was used as a selection marker to knock out the Ku70 gene.

[0061] The upstream and downstream homologous arm fragments of the Ku70 gene were amplified from the Y.lipolytica PO1f genome using primers Ku70-UP-F / R and Ku70-DOWN-F / R. The PCR program was: 95℃, 10 min; 95℃, 30 s; 58℃, 30 s; 72℃, 1 min; 72℃, 10 min, and 30 cycles.

[0062] Novazons (Suzhou, China) synthesized the hygB gene (SEQ ID NO. 8) from Coccidioides posadasii in pUC-GW to generate the vector pUC-GW-hygB, which was then codon-optimized. Using the hygB-F / R primer pair, the hygB gene was cloned into KpnI-linearized pINA1269 to generate the plasmid pINA1269-hygB. The pINA1269-hygB fragment was then amplified using the hp4d-hygB-XPR2t-F / R primers to obtain the hp4d-hygB-XPR2t fragment.

[0063] The primer sequences involved are as follows:

[0064] Ku70-sg-1F: 5'-gggtcggcgcaggttgacgttgatagagtgctgaaaaggcgttttagagctagaaatagc-3';

[0065] Ku70-sg-1R:5’-gctatttctagctctaaaacgccttttcagcactctatcaacgtcaacctgcgccgaccc-3’;

[0066] Ku70-sg-2F:5’-gggtcggcgcaggttgacgtgtcgaacgtcttgtcttccggttttagagctagaaatagc-3’;

[0067] Ku70-sg-2R:5’-gctatttctagctctaaaaccggaagacaagacgttcgacacgtcaacctgcgccgaccc-3’;

[0068] Ku70-UP F:5’-catgattacgccaagcttgtttcactacactacataacttgtaccattctaccc-3’;

[0069] Ku70-UP R:5’-tttcaaaaagcggcggttcgtg-3’;

[0070] Ku70-DOWN F:5’-ctagggaggcacatctaaacgaataacg-3’;

[0071] Ku70-DOWN R:5’-aacccggtctctgtttaaacagtgaacgaccaagactaaagggtg-3’;

[0072] hygB F:5’-gaacccgaaactaaggatccatgcctgaactcaccgcg-3’;

[0073] hygB R:5’-ctcgtccgagggcaaaggaatagggtacctccatggcctgtcc-3’;

[0074] hp4d-hygB-XPR2 F:5’-cacgaaccgccgctttttgaaagtagtaggttgaggccgttgagc-3’;

[0075] hp4d-hygB-XPR2 R:5’-cgttattcgtttagatgtgcctccctagacacgggcatctcacttgc-3’。

[0076] After purification, the three fragments obtained above were amplified by overlap extension PCR using the primer set Ku70-UP-F / Ku70-DOWN-R to obtain the fusion fragment Ku70 UP-hp4d-hygB-XPR2t-Ku70 DOWN. After purification, the fragment was ligated with the PmeI-linearized plasmid pCAS1yl-ΔKu70 using the homologous recombinase ClonExpress II One Step Cloning Kit (Norwegian) and transformed into E. coli JM109 competent cells to obtain transformants.

[0077] After the transformant colonies were picked for PCR verification of the correct band size, the plasmid was extracted and sent to Novozymes for sequencing. If the sequencing was correct, the knockout plasmid pCAS2yl-ΔKu70 was successfully constructed (e.g. Figure 1 shown).

[0078] (2) Preparation of Yarrowia lipolytica competent cells

[0079] Y. lipolytica PO1f was picked from a -80°C glycerol tube using an inoculating loop, streaked three times onto YPD solid medium, and incubated at 30°C in an incubator for 48 hours. A single colony was picked from the plate and inoculated into a 50mL vial containing 10mL of YPD liquid medium and incubated at 30°C at 220 rpm for 20 hours. The cells were collected in a clean hood into a 50mL centrifuge tube and centrifuged at 6000 rpm for 5 minutes at 4°C, discarding the supernatant. The cells were resuspended in 20mL of sterile water and washed, centrifuged at 6000 rpm for 5 minutes at 4°C, discarding the supernatant, and repeated the wash twice. The cells were resuspended in 1mL of 0.1mol / L lithium acetate (pH 6.0) and incubated at room temperature for 10 minutes. 100μL of the competent cells were aliquoted into a sterile 2mL centrifuge tube for transformation or glycerol was added to a final concentration of 25% (v / v) and stored at -80°C to prepare Y. lipolytica PO1f competent cells.

[0080] (3) Preparation of Y. lipolytica PO1fΔKu70

[0081] 10 μL of plasmid pCAS2yl-ΔKu70 and 10 μL of salmon sperm DNA (10 mg / mL) were added to Y. lipolytica PO1f competent cells, mixed by pipetting, and incubated at 30°C for 15 min. 600 μL of 50% PEG4000 was added to the above mixture, mixed, and incubated at 30°C and 220 rpm for 1 h.

[0082] Place the centrifuge tube containing the above mixture in a 39℃ water bath for 1 hour; add 1ml YPD medium, activate and culture at 30℃ and 220rpm for 2 hours; centrifuge at 9000rpm for 1 minute, remove the supernatant and suspend the bacteria with 1ml sterile water; centrifuge at 9000rpm for 1 minute, collect the bacteria, spread the bacteria on YPD solid culture medium containing 400μg / mL hygromycin B, and culture in a constant temperature incubator at 30℃ for 3-4 days; pick a single colony from the plate, and verify the single colony by colony PCR using Taq DNA polymerase with Ku70-UP-F / Ku70-DOWN-R as primers; the positive single colony with the target band size is inoculated into a vial containing YPD liquid culture medium, and after culture at 30℃ and 220rpm for 24 hours, the genome is extracted and sent to Jinweizhi Company for sequencing using Ku70-UP-F / Ku70-DOWN-R as primers for further verification, thus obtaining Y.lipolytica The strain Y.lipolytica PO1f-ΔKu70, in which the Ku70 gene was successfully knocked out in PO1f, was named Y-01.

[0083] Example 2: Construction of recombinant Yarrowia lipolytica

[0084] Overexpression plasmids were constructed as follows Figure 2 The specific steps are as follows:

[0085] (1) Construction of single gene expression plasmid

[0086] The NCBI Genebank accession number of the glycerol kinase gene GUT1 involved in the following examples is: 2908247, the NCBI Genebank accession number of the glyceraldehyde-3-phosphate dehydrogenase gene GUT2 is: 2907009, the NCBI Genebank accession number of the transketolase gene TKL1 is: 2912397, and the NCBI Genebank accession number of the transaldolase gene TAL1 is: 2909043.

[0087] Using the construction of the plasmid pINA1269-GUT1 as an example, primers GUT1-F and GUT1-R were designed based on the sequence of the glycerol kinase-encoding gene GUT1 published in Genbank (2908247) at NCBI. The 1512-bp GUT1 gene was amplified from the Y. lipolytica PO1f genome. The pINA1269 plasmid was digested with the restriction endonuclease KpnI to obtain a linearized plasmid.

[0088] Enzyme digestion system (50 μL): 3000-5000 ng of plasmid, 2.5 μL of Q.Cut KpnI, 5 μL of 10x Q.Cut Buffer, and 50 μL of ddH2O. The digestion temperature was 37°C and the digestion time was 1 h.

[0089] The primer sequences involved are as follows:

[0090] GUT1-F: 5'-caaccacacacatccacgtgatgtcttcctacgtaggagctctcg-3';

[0091] GUT1-R: 5'-ggacaggccatggaggtaccttactcaagccagccaacagc-3';

[0092] The target gene GUT1 and the linearized vector pINA1269 were purified and recovered using an agarose gel DNA recovery kit (Shanghai Jierui), and then ligated using the homologous recombinase ClonExpress II One Step Cloning Kit (Novozymes). The ligation product was transformed into E. coli BL21 competent cells to obtain the transformation product.

[0093] The transformation product was plated onto LB solid medium (containing 200 mg / L ampicillin) and incubated in an inverted incubator at 37°C for 12 hours to obtain transformants. Transformants were selected from the plate for colony PCR verification, and plasmids were extracted and sent for sequencing. Once verified, the recombinant plasmid pINA1269-GUT1 was obtained.

[0094] Plasmids pINA1269-GUT2, pINA1269-TKL1, and pINA1269-TAL1 were obtained using the above method. The primer sequences involved are as follows:

[0095] GUT2-F: 5'-caaccacacacatccacgtgatgttcagaaccattcgaaaacccg-3';

[0096] GUT2-R: 5'-ggacaggccatggaggtaccttatatttgtccttgggggtaaggccc-3';

[0097] TKL1-F: 5'-caaccacacacatccacgtgatggctccccaattttcaaagactg-3';

[0098] TKL1-R: 5'-ggggacaggccatggaggtaccttagacaccgtggccggg-3';

[0099] TAL1-F: 5'-atacaaccacacacatccacgtgatgtcttccaactctcttgaacagct-3';

[0100] TAL1-R: 5'-ggacaggccatggaggtaccctaagcggagagcttggtctcaat-3'.

[0101] (2) Construction of dual-gene expression plasmid

[0102] Taking the construction of plasmid pINA1269-GUT1-TKL1 as an example, using the pINA1269-TKL1 prepared in step (1) as a template, primers hp4d-TKL1-XPR2t-F / R were designed to amplify the hp4d-TKL1-XPR2t fragment from pINA1269-TKL1. The pINA1269-GUT1 prepared in step (1) was digested with restriction endonuclease KpnI to obtain a linearized plasmid.

[0103] Enzyme digestion system (50 μL): 3000-5000 ng of plasmid, 2.5 μL of Q.Cut KpnI, 5 μL of 10x Q.Cut Buffer, and ddH2O to 50 μL. Digestion temperature: 37°C, digestion time: 1 hour.

[0104] The primer sequences involved are as follows:

[0105] hp4d-TKL1-XPR2t-F: 5'-cacgaaccgccgctttttgaaagtagtaggttgaggccgttgagc-3';

[0106] hp4d-TKL1-XPR2t-R: 5'-cgttattcgtttagatgtgcctccctagacacgggcatctcacttgc-3';

[0107] The target fragment hp4d-TKL1-XPR2t and the linearized vector pINA1269-TKL1 were purified and recovered using an agarose gel DNA recovery kit (Shanghai Jierui), and then ligated using the homologous recombinase ClonExpress II One Step Cloning Kit (Novozymes). The ligation product was transformed into E. coli BL21 competent cells to obtain the transformation product.

[0108] The transformation product was plated onto LB solid medium (containing 200 mg / L ampicillin) and incubated upside down in a 37°C incubator for 12 hours to obtain transformants. Transformants were selected from the plate for colony PCR verification, and plasmids were extracted and sent for sequencing. Once verified, the recombinant plasmid pINA1269-GUT1-TKL1 was obtained.

[0109] Plasmids pINA1269-GUT1-GUT2 and pINA1269-GUT1-TAL1 were obtained according to the above method. The primer sequences involved are as follows:

[0110] hp4d-GUT2-XPR2t-F: 5'-cacgaaccgccgctttttgaaagtagtaggttgaggccgttgagc-3';

[0111] hp4d-GUT2-XPR2t-R: 5'-cgttattcgtttagatgtgcctccctagacacgggcatctcacttgc-3';

[0112] hp4d-TAL1-XPR2t-F: 5'-cacgaaccgccgctttttgaaagtagtaggttgaggccgttgagc-3';

[0113] hp4d-TAL1-XPR2t-R:5'-cgttattcgtttagatgtgcctccctagacacgggcatctcacttgc-3'.

[0114] (3) Construction of triple gene expression plasmid

[0115] Using the construction of the plasmid pINA1269-GUT1-TKL1-GUT2 as an example, primers hp4d-GUT2-XPR2t-F / R were designed using pINA1269-GUT2 as a template to amplify the hp4d-GUT2-XPR2t fragment from pINA1269-GUT2. pINA1269-GUT1-TKL1 was then digested with the restriction endonuclease KpnI to obtain a linearized plasmid.

[0116] Enzyme digestion system (50 μL): 3000-5000 ng of plasmid, 2.5 μL of Q.Cut KpnI, 5 μL of 10x Q.Cut Buffer, and ddH2O to 50 μL. Digestion temperature: 37°C, digestion time: 1 hour.

[0117] The target fragment hp4d-GUT2-XPR2t was purified and recovered from the linearized vector pINA1269-GUT1-TKL1 using an agarose gel DNA recovery kit (Shanghai Jierui). The ligated fragments were then ligated using the homologous recombinase ClonExpress II One Step Cloning Kit (Novozymes). The ligated product was transformed into E. coli BL21 competent cells to obtain the transformant. The transformant was plated onto LB solid medium (containing 200 mg / L ampicillin) and incubated inverted at 37°C for 12 hours to obtain transformants. Transformants were selected from the plate for colony PCR verification, and plasmids were extracted and sequenced. Once verified, the recombinant plasmid pINA1269-GUT1-TKL1-GUT2 was obtained.

[0118] According to the above method, plasmid pINA1269-GUT1-TKL1-TAL1 can be prepared.

[0119] (4) Construction of four-gene expression plasmid

[0120] Using pINA1269-TAL1 as a template, primers hp4d-TAL1-XPR2t-F / R were designed to amplify the hp4d-TAL1-XPR2t fragment from pINA1269-TAL1. pINA1269-GUT1-TKL1-GUT2 was digested with the restriction endonuclease KpnI to obtain a linearized plasmid.

[0121] Enzyme digestion system (50 μL): 3000-5000 ng of plasmid, 2.5 μL of Q.Cut KpnI, 5 μL of 10x Q.Cut Buffer, and ddH2O to 50 μL. Digestion temperature: 37°C, digestion time: 1 hour.

[0122] The target fragment hp4d-TAL1-XPR2t was purified and recovered from the linearized vector pINA1269-GUT1-TKL1-GUT2 using an agarose gel DNA recovery kit (Shanghai Jierui). The ligated fragments were then ligated using the homologous recombination enzyme ClonExpress II OneStep Cloning Kit (Novozymes). The ligated product was transformed into E. coli BL21 competent cells to obtain the transformant. The transformant was plated onto LB solid medium (containing 200 mg / L ampicillin) and incubated inverted in a 37°C incubator for 12 hours to obtain transformants. Transformants were selected from the plate for colony PCR verification, and plasmids were extracted and sequenced. Once verified, the recombinant plasmid pINA1269-GUT1-TKL1-GUT2-TAL1 was obtained.

[0123] (5) Transformation of expression plasmid

[0124] Prepare Y.lipolytica PO1f-ΔKu70 competent cells and add 5-10 μL of overexpression plasmids pINA1269-GUT1, pINA1269-GUT2, pINA1269-TKL1, pINA1269-TAL1, pINA1269-GUT1-GUT2, pINA1269-GUT1-TKL1, pINA1269-GUT1-TAL1, pINA1269-GUT1-TKL1-GUT2, pINA1269-GUT1-TKL1-TAL1, and pINA1269-GUT1-TKL1-GUT2-TAL1 to Y.lipolytica The transformation was carried out in PO1f-ΔKu70 competent cells, and the bacteria were spread on SC solid medium containing uracil and cultured in a constant temperature incubator at 30°C for 3-4 days; single colonies were picked from the plates for colony PCR verification; positive transformants were inoculated into 10 mL YPD vials and preserved in glycerol tubes to prepare genetically engineered bacteria Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1, Y.lipolyticaPO1f-ΔKu70 / pINA1269-GUT2, Y.lipolytica PO1f-ΔKu70 / pINA1269-TKL1, Y.lipolyticaPO1f-ΔKu70 / pINA1269-TAL1, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-GUT2, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TKL1, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TAL1, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TKL1-GUT2, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TKL1-TAL1, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TKL1-GUT2-TAL1, respectively named: Y-02, Y-03, Y-04, Y-05, Y-06, Y-07, Y-08, Y-09, Y-10, Y-11.

[0125] Example 3: Fermentation of erythritol by genetically engineered bacteria

[0126] 1. Preparation of erythritol by shake flask fermentation

[0127] Fermentation process:

[0128] (1) Using an inoculation loop, the recombinant Yarrowia lipolytica Y-01, Y-02, Y-03, Y-04, Y-05, Y-06, Y-07, Y-08, Y-09, Y-10, and Y-11 prepared in Example 1 and Example 2 were picked from a -80°C glycerol tube, three lines were drawn on YPD solid medium, and the cells were placed in a 30°C incubator for 48 h for activation;

[0129] (2) Preparation of culture medium

[0130] 1) Preparation of crude glycerol (w / w): 80-85% glycerol, 2.0% sodium salt, 10-15% methanol, 2.5% other organic matter and 2% water.

[0131] 2) Preparation of seed culture medium: crude glycerol 50 g / L, yeast extract 10 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 1.0 g / L, pH 3.0.

[0132] 3) Preparation of fermentation medium: crude glycerol 250 g / L, yeast extract 1.0 g / L, NH4Cl 5.0 g / L, KH2PO4 0.25 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 mol / L, pH 3.0.

[0133] (3) Picking activated single colonies, inoculating them into seed culture medium, and culturing them at 30°C and 220 rpm for 20 h to prepare seed solution;

[0134] (4) The seed solution was transferred to a 500 mL baffled shake flask containing 50 mL of fermentation medium at an inoculum rate of 20% (v / v) and fermentation was started at a temperature of 30°C, a rotation speed of 220 rpm, and a fermentation time of 120 h to prepare a fermentation broth.

[0135] According to the above method, recombinant Yarrowia lipolytica Y-01, Y-02, Y-03, Y-04, Y-05, Y-06, Y-07, Y-08, Y-09, Y-10 and Y-11 were fermented in shake flasks to produce erythritol. The results are shown in Table 1.

[0136] Table 1 Erythritol concentration and conversion rate of different strains

[0137]

[0138] The results showed that the starting strain Y-01 had a stronger ability to synthesize erythritol than the strain Y.lipolytica PO1f and could be used for further metabolic modification.

[0139] The Ku70 gene was knocked out in the Y.lipolytica PO1f strain to obtain strain Y-01, which significantly improved the gene integration efficiency.

[0140] GUT1, GUT2, TKL1 and TAL1 were overexpressed in Y-01 to obtain strains Y-02, Y-03, Y-04, Y-05, Y-06, Y-07, Y-08, Y-09, Y-10 and Y-11, respectively, which increased the production of erythritol. The final Y-11 strain Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TKL1-GUT2-TAL1 had an erythritol concentration of up to 59±0.7g / L.

[0141] 2. Fermentation of erythritol in 5L fermenter

[0142] (1) Recombinant Yarrowia lipolytica was picked from the -80℃ glycerol tube using an inoculation loop, streaked onto three zones of YPD plate medium, and incubated in a 30℃ incubator for 48 h.

[0143] (2) Preparation of culture medium

[0144] 1) Preparation of crude glycerol (w / w): 80-85% glycerol, 2.0% sodium salt, 10-15% methanol, 2.5% other organic matter and 2% water.

[0145] 2) Preparation of seed culture medium: crude glycerol 50 g / L, yeast extract 10 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 1.0 g / L, pH 3.0.

[0146] 3) Preparation of fermentation medium: crude glycerol 250 g / L, yeast extract 1.0 g / L, NH4Cl 5.0 g / L, KH2PO4 0.25 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 mol / L, pH 3.0.

[0147] (3) Picking activated single colonies from the plate, inoculating them into seed culture medium, and culturing them at 30°C and 220 rpm for 20 h to prepare the first-level seed solution;

[0148] The first-stage seed solution was transferred to a 1 L baffled shake flask containing 200 mL of the above seed culture medium at a 10% (v / v) inoculum volume and cultured at 30°C and 220 rpm for 20 h to obtain the second-stage seed solution;

[0149] (4) Transferring the inoculum of the secondary seed solution at a volume ratio of 20% (v / v) to a 5-L fermenter containing 1.6 L of fermentation medium for fermentation; the fermentation parameters were controlled as follows: stirring speed of 800 rpm, temperature of 30°C, dissolved oxygen of 20% to 30%, and fermentation time of 120 h;

[0150] Recombinant Yarrowia lipolytica Y-01 and Y-11 were fermented in 5L fermenters to prepare fermentation broths containing erythritol. The erythritol content in the fermentation broths was detected. The results were as follows: Figure 3 As shown in Table 2.

[0151] (5) As a control, the specific implementation method is the same as steps (1) to (4), except that the crude glycerol in the culture medium prepared in step (2) is replaced by pure glycerol, that is, step (2) is:

[0152] 1) Preparation of seed culture medium: 50 g / L pure glycerol, 10 g / L yeast extract, 0.5 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, pH 3.0.

[0153] 2) Preparation of fermentation medium: 250 g / L pure glycerol, 1.0 g / L yeast extract, 5.0 g / L NH4Cl, 0.25 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.5 mol / L NaCl, pH 3.0.

[0154] According to the methods of steps (3) to (4), fermentation broth containing erythritol was prepared, and the content of erythritol in the fermentation broth was detected. The results are shown in Table 2.

[0155] Table 2: Erythritol concentration and conversion rate of different strains

[0156]

[0157] The results showed that the resulting recombinant strain Y-11, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TKL1-GUT2-TAL1, produced 110±1.1 g / L of erythritol after 120 hours of cultivation using crude glycerol as a carbon source, a 340% increase compared to the original strain, and a conversion rate of 61%. Furthermore, the resulting Y-11 strain, Y.lipolytica PO1f-ΔKu70 / pINA1269-GUT1-TKL1-GUT2-TAL1, had a higher capacity for utilizing crude glycerol than pure glycerol, demonstrating that the strain of the present application can produce erythritol from biodiesel waste.

[0158] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Yarrowia lipolytica, characterized in that The recombinant Yarrowia lipolytica is a yeast strain in which the original strain Yarrowia lipolytica is deleted. Yarrowialipolytica PO1f Ku 70 genes, and overexpressed the glycerol kinase gene from Yarrowia lipolytica GUT1 , glyceraldehyde-3-phosphate dehydrogenase gene GUT2 , transketolase gene TKL1 and transaldolase genes TAL1 , described Ku70 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the glycerol kinase gene GUT1 The nucleotide sequence of the protein is as shown in SEQ ID NO.2, the 3-phosphoglyceraldehyde dehydrogenase gene GUT2 The nucleotide sequence of the protein is as shown in SEQ ID NO.3, the transketolase gene TKL1 The nucleotide sequence of the protein is shown in SEQ ID NO.4, the transaldolase gene TAL1 The nucleotide sequence of the protein is shown in SEQ ID NO.

5.

2. The recombinant Yarrowia lipolytica according to claim 1, wherein The recombinant Yarrowia lipolytica uses pCAS1yl plasmid and / or pINA1269 plasmid as expression vector.

3. A method for preparing the recombinant Yarrowia lipolytica according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) Using the pCAS1yl plasmid to knock out the Yarrowia lipolytica genome Ku70 Gene; (2) Overexpression of glycerol kinase gene using pINA1269 plasmid GUT1 , glyceraldehyde-3-phosphate dehydrogenase gene GUT2 , transketolase gene TKL1 and transaldolase genes TAL1 , and the recombinant plasmid pINA1269- GUT1 - TKL1 - GUT2 - TAL1 ; (3) The prepared recombinant plasmid pINA1269- GUT1 - TKL1 - GUT2 - TAL1 The recombinant Yarrowia lipolytica / pINA1269- GUT1 - TKL1 - GUT2 - TAL1 .

4. A method for producing erythritol, characterized in that: The method comprises adding the recombinant Yarrowia lipolytica according to claim 1 or 2 to a reaction system using crude glycerol from biodiesel waste as the sole carbon source to ferment and prepare erythritol.

5. The method according to claim 4, wherein The method comprises inoculating the recombinant Yarrowia lipolytica into a seed culture medium for culturing to obtain a first-level seed solution; Transfer 10% of the first-stage seed solution to the seed culture medium for cultivation to obtain the second-stage seed solution; The prepared secondary seed liquid was inoculated into a fermentation medium containing crude glycerol at an inoculum rate of 20% for fermentation to prepare erythritol.

6. The method according to claim 5, wherein Inoculate the seed liquid into the fermentation medium and control the dissolved oxygen at 20%~30%.

7. Use of the recombinant Yarrowia lipolytica according to claim 1 or 2 in the preparation of a product containing erythritol.

Images