A Yarrowia lipolytica genetic engineering strain for increasing the functional fatty acid production of Yarrowia lipolytica, method and application thereof

Abstract

The invention belongs to the field of biotechnology, and discloses a Yarrowia lipolytica genetic engineering strain, method and application thereof for improving the functional fatty acid yield of Yarrowia lipolytica. The strain is obtained by inhibiting the expression of Cdc20 regulatory factor in Yarrowia lipolytica (Po1f) while overexpressing a gene encoding Spt23 protein, and the obtained strain is named Po1f-20-23. Compared with wild-type yeast that has not been modified, Po1f-20-23 has a significantly larger morphology of Yarrowia lipolytica, and the contents of palmitoleic acid and stearic acid are both increased, with palmitoleic acid (C16:1) having a growth rate of more than 100%, and stearic acid (C18:2) having the largest output, with a content accounting for 22.841% of the total fatty acid content, and a growth rate of 23.14%.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular to a Yarrowia lipolytica genetic engineering strain for improving the functional fatty acid yield of Yarrowia lipolytica, a method and an application thereof. Background Art

[0002] Yarrowia lipolytica is a non-pathogenic, dimorphic, Ascomycete yeast. Its entire genome has been sequenced and a large amount of physiological and genetic information has been accumulated.

[0003] Yarrowia lipolytica can not only utilize hexoses such as glucose, fructose and mannose, but also use a variety of hydrophobic substrates as carbon sources to produce single-cell proteins, single-cell lipids and a variety of organic acids. Due to the characteristics of lipid synthesis accumulation and high molecular weight protein secretion, Yarrowia lipolytica can be used to produce high value-added chemicals through genetic engineering and protein engineering, combined with its different metabolic pathways. In recent years, with the development of synthetic biology tools, the application scope of Yarrowia lipolytica has been expanded. It is not only a chassis cell for producing lipid compounds, but also a cell factory for producing value-added lipid derivatives and various special chemicals. After rational design and targeted transformation, Yarrowia lipolytica uses renewable resources to produce lipid compounds, terpenes and other substances. Therefore, Yarrowia lipolytica has always been our key research strain.

[0004] Microbial oils refer to triglycerides, free fatty acids and other lipids that are synthesized and accumulated in large quantities in the cells of oil-producing microorganisms such as molds, yeasts, bacteria and algae under certain culture conditions using carbon sources, nitrogen sources and the like. The screened and cultivated microorganisms can produce hexadecanoic acids such as palmitic acid, palmitic acid (C16:0), palmitoleic acid, and palmitic acid (C16:1); octadecanoic acids such as stearic acid (C18:0), oleic acid, and oleic acid (C18:1); octadecenoic acids such as linoleic acid (C18:2); and eicosanoic acids such as linolenic acid (C18:3). The production of oils by microorganisms not only has the advantages of high oil content, short production cycle, no seasonal influence, and no occupation of arable land, but also can use strategies such as cell mutagenesis, laboratory domestication, and genetic engineering to make microorganisms produce high-value compounds or certain specific fatty acids to form oils, such as EPA and DHA. Therefore, the advantages of using microorganisms to produce functional oils are becoming increasingly obvious.

[0005] The cell cycle refers to the whole process that cells go through from the completion of one division to the end of the next division. It is divided into two stages: interphase (G1 / S / G2 phase) and division phase (M phase). The cell cycle of yeast cells consists of four phases: G1 / S / G2 / M. The cell morphology of each cell cycle can be observed under an inverted microscope, and lipid storage is mainly in the G2 / M phase. Lipid accumulation was inhibited in S phase cells synchronized by hydroxyurea and increased in G2 / M phase cells blocked by noconadazole. In addition, the enrichment of G1 phase cells after rapamycin treatment induced a large amount of lipid accumulation. Based on these results, it was concluded that L. starkeyi cells began to store lipids from the G2 / M phase.

[0006] Cell division cycle 20 homolog (Cdc20) is a G2 / M phase regulator required for the completion of mitosis. Cdc20 can mediate the degradation of phosphatidylcholine-specific phospholipase C (PC-PLC) through the ubiquitin proteasome pathway (UPP). The Wnt / β-catenin signaling pathway (classical Wnt signaling pathway) regulates cell proliferation by regulating the cell cycle. The level of transduction proteins peaks in the G2 / M phase, and knocking out Cdc20 blocks signal transduction through the transduction protein Wnt / β-catenin. Therefore, in order to achieve maximum activity in the G2 / M phase, we decided to inhibit the expression of Cdc20-regulated genes.

[0007] Spt protein is a large class of regulatory factors involved in the yeast transcription process, which is mainly involved in the regulation of unsaturated fatty acid synthesis.

[0008] Chinese patent CN112391402A co-expresses Kar2 (an endoplasmic reticulum-resident protein) and auxiliary protein Sls1 (a cofactor of Kar2) related to protein synthesis and secretion pathway to increase the expression level of the target protein, Rhizomucor miehei lipase, in Yarrowia lipolytica (Po1f). The enzyme activity of the engineered strain (Po1f-3rol-kar2) reached 327 U / mL, an increase of 82% compared to the initial strain FY5-4rml. This indicates that co-expression of Kar2 and Sls1 can promote the expression of Rhizomucor miehei lipase in Yarrowia lipolytica (Po1f). Chinese patent CN118497020A established a synergistic relationship between the removal of corn slurry vomitoxin and the production of single-cell protein (vomitoxin removal enzyme) by expressing the vomitoxin degrading enzyme gene TRI101. The protein content of Yarrowia lipolytica E150 (CLIB122) after overexpressing TRI101 was 60%~70%, the dry weight was 20 g / L~100g / L, and the degradation rate of vomitoxin in corn slurry was more than 95%. The fermentation liquid was dried and crushed to be an excellent feed, which significantly improved the economic benefits. Chinese patent CN116555362A heterologously expressed lipid droplet-associated proteins (LDAP) in Yarrowia lipolytica (Po1f). The results showed that when LDAP was overexpressed, the lipid droplets increased, more triglycerides were produced, and the total production of fatty acids increased by 85.7%.

[0009] At present, the metabolic engineering means of Yarrowia lipolytica to synthesize fatty acids mainly revolves around the introduction of exogenous thioesterase and the transformation of KS pathway to regulate the fatty acid chain length. However, studies have shown that the accumulation of medium-chain fatty acids will affect the synthesis of essential fatty acids and affect cell growth. The current existing technology is basically a unilateral use of protein engineering or genetic engineering, and there is no report on the production of palmitoleic acid and stearic acid in Yarrowia lipolytica. The present invention simultaneously utilizes genetic engineering and protein engineering to produce functional fatty acids-palmitoleic acid and stearic acid in Yarrowia lipolytica, and the output of palmitoleic acid and stearic acid is improved. Summary of the invention

[0010] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a Yarrowia lipolytica genetic engineering strain, method and application thereof for increasing the functional fatty acid yield of Yarrowia lipolytica.

[0011] The technical solution adopted by the present invention to solve its technical problem is:

[0012] A Yarrowia lipolytica genetic engineering strain for increasing the production of functional fatty acids in Yarrowia lipolytica, wherein the strain is obtained by inhibiting the expression of a Cdc20 regulatory factor in Po1f of Yarrowia lipolytica and overexpressing a gene encoding a Spt23 protein;

[0013] Among them, inhibiting the expression of Cdc20 regulatory factors is achieved by finding the sequence and PAM site of Cdc20 regulatory factors and setting the sgRNA sequence to guide Cas9 to locate the PAM site sequence;

[0014] The gene sequence encoding Cdc20 is shown in SEQ ID NO.1, the gene sequence encoding Spt23 protein is shown in SEQ ID NO.2, and the sgRNA sequence is shown in SEQ ID NO.6.

[0015] Furthermore, the strain increases the content of functional fatty acids by regulating the cell cycle of Yarrowia lipolytica and overexpressing protein genes; wherein regulating the cell cycle of Yarrowia lipolytica Po1f refers to inhibiting the expression of the regulatory factor Cdc20 in Yarrowia lipolytica Po1f; overexpressing protein genes refers to overexpressing the gene encoding Spt23 protein in the genome of Yarrowia lipolytica Po1f; the two methods jointly regulate the cell cycle and protein content of Yarrowia lipolytica Po1f, thereby increasing the production of palmitoleic acid and stearic acid.

[0016] The use of the genetically engineered strain of Yarrowia lipolytica as described above in increasing the production of palmitoleic acid and stearic acid.

[0017] A method for constructing the genetically engineered strain of Yarrowia lipolytica as described above comprises the following steps:

[0018] By inhibiting the expression of the regulatory factor Cdc20, the cell cycle of Yarrowia lipolytica Po1f was regulated, and a positive transformant was obtained, named Po1f-20;

[0019] The gene encoding Spt23 protein was overexpressed in Po1f-20 to regulate the fatty acid production of Yarrowia lipolytica, and the overexpression plasmid DT23 was obtained;

[0020] The DT23 plasmid was transferred into Yarrowia lipolytica Po1f-20 using the lithium acetate method; screening was carried out in yeast SC-URA medium and cultured in a 30°C incubator for 48-96 h; after verification, a positive transformant was obtained and named Po1f-20-23, thereby obtaining a genetically engineered strain of Yarrowia lipolytica that improves the production of functional fatty acids in Yarrowia lipolytica.

[0021] Furthermore, the specific steps are as follows:

[0022] Construction of positive transformant Po1f-20:

[0023] (1) Constructing plasmid C20i that inhibits the expression of regulatory factor Cdc20;

[0024] (2) After consulting the literature, we discovered a regulatory factor in the G2 / M phase: the cell division cycle 20 homolog Cdc20;

[0025] (3) Obtain the gene sequence of the regulatory factor Cdc20 in Po1f of Yarrowia lipolytica from the NCBI website;

[0026] (4) Analyze the 20 nucleotide sequences upstream of the PAM region on the gene sequence of Cdc20 to obtain the gRNA target sequence; name the target sequence Cdc20-N20, and locate it on the Po1f genome map of Yarrowia lipolytica, set the primers for amplifying gRNA, and connect it to the pCRISPR-Cas9 plasmid as a backbone vector, and set the sgRNA sequence to guide Cas9 to locate to the PAM site sequence and prompt Cas9 to cut the DNA double strand at this position; wherein the sgRNA sequence is shown in SEQ ID NO.6;

[0027] (5) Select the pCRISPR-Cas9 plasmid as the backbone and AvrⅡ restriction site, prepare the restriction system, and then use the recovery kit to recover the digested backbone;

[0028] (6) Use 37°C ligase to connect the amplified target fragment and the digested backbone in a 20 μL PCR tube at 37°C for 20 min.

[0029] (7) Add the liquid in the PCR tube in step (6) to the DH5α E. coli competent cells;

[0030] (8) Place the competent cells in step (7) on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min.

[0031] (9) Spread the plate on an LB medium plate containing 0.1% by mass of Amp resistance to screen for positive transformants;

[0032] (10) Use a plasmid extraction kit to extract the constructed plasmid C20i;

[0033] (11) The C20i plasmid was transformed into Yarrowia lipolytica using the lithium acetate method;

[0034] (12) Screening was performed in yeast SC-URA medium to obtain a positive transformant, which was named Po1f-20;

[0035] Construction of the gene overexpression plasmid DT23 encoding Spt23 protein and the strain Po1f-20-23:

[0036] (1) Review the literature and discover the Spt23 protein;

[0037] (2) Obtain the gene sequence encoding the Spt23 protein and locate it on the Po1f genome of Yarrowia lipolytica;

[0038] (3) Design primers for amplification of the gene sequence encoding the Spt23 protein;

[0039] (4) amplifying the sequence encoding the Spt23 protein gene from the Po1f genome of Yarrowia lipolytica and then recovering it using a recovery kit;

[0040] (5) Select the pYLXP' linear vector as the backbone and the restriction site, i.e., the EcoR Ⅰ site, and prepare the restriction system, and then use the recovery kit to recover the digested backbone;

[0041] (6) Use 37°C ligase to connect the amplified target fragment and the digested backbone in a 20 μL PCR tube at 37°C for 20 min.

[0042] (7) Add the liquid in the PCR tube in step (6) to the DH5α E. coli competent cells;

[0043] (8) Place the competent cells in step (7) on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min.

[0044] (9) Spread the plate on an LB medium plate containing 0.1% by mass of Amp resistance to screen for positive transformants;

[0045] (10) Use a plasmid extraction kit to extract the overexpression plasmid DT23;

[0046] (11) The DT23 plasmid was transformed into Yarrowia lipolytica Po1f-20 cells using the lithium acetate method;

[0047] (12) Screening was performed in yeast SC-URA medium, and positive transformants were obtained after verification;

[0048] (13) The positive transformants were fermented in a fermentation medium for 120 h at 28 °C and 220 rpm to obtain a genetically engineered strain of Yarrowia lipolytica Po1f-20-23 that increased the functional fatty acid production of Yarrowia lipolytica.

[0049] Furthermore, the formula of the yeast SC-URA medium is: glucose: 20 g / L; yeast basic nitrogen source: 1.7 g / L; ammonium sulfate: 5 g / L; agar: 3% (mass percentage), and the solvent is water;

[0050] The formula of the fermentation medium is: glucose: 60 g / L; yeast extract: 2.5 g / L; yeast basic nitrogen source: 1.7 g / L, and the solvent is water.

[0051] The fermentation method for increasing the yield of palmitoleic acid and stearic acid by using the genetically engineered strain of Yarrowia lipolytica as described above comprises the following steps:

[0052] The genetically engineered strain of Yarrowia lipolytica was fermented in a fermentation medium for 120 h at a fermentation temperature of 28 °C and a fermentation speed of 220 rpm to obtain palmitoleic acid and stearic acid.

[0053] Furthermore, the formula of the fermentation medium is: glucose: 60 g / L; yeast extract: 2.5 g / L; yeast basic nitrogen source: 1.7 g / L, and the solvent is water.

[0054] The advantages and positive effects achieved by the present invention are:

[0055] 1. The present invention overexpresses the gene encoding Spt23 protein in the genome of Yarrowia lipolytica (Po1f), which can increase the yield of functional fatty acids and participate in regulating the transcription of ole1 gene, which is a key gene for synthesizing palmitoleic acid and stearic acid. Compared with the wild-type yeast that has not been modified, the palmitoleic acid and stearic acid contents of Yarrowia lipolytica in the experimental group are increased, among which palmitoleic acid (C16:1) has a growth rate of more than 100%, and stearic acid (C18:2) has the largest output, accounting for 22.841% of the total fatty acid content, and the growth rate is 23.14%.

[0056] 2. The present invention proposes to inhibit the expression level of Cdc20 regulatory factors in the genome of Yarrowia lipolytica (Po1f), which can regulate the cell cycle of Yarrowia lipolytica, so that most of the cells remain in the G2 / M phase. Compared with the wild-type yeast that has not been modified, the number of cells of Yarrowia lipolytica remaining in the G2 / M phase in the experimental group is significantly increased, and compared with the wild type, the cell morphology in the G2 / M phase, both daughter cells and mother cells are significantly enlarged, and the mother cells are "pea-shaped".

[0057] 3. Oil synthesis is mainly in the G2 / M phase, and cell division cycle 20 homolog (Cdc20) is a G2 / M phase regulator necessary for completing mitosis. Cdc20 can mediate the degradation of phosphatidylcholine-specific phospholipase C (PC-PLC) through the ubiquitin proteasome pathway (UPP). The Wnt / β-catenin signaling pathway regulates cell proliferation by regulating the cell cycle, and the level of the transduction protein reaches a peak in the G2 / M phase. Spt protein is a large class of regulatory factors involved in the yeast transcription process. It is mainly involved in regulating the transcription of the ole1 gene, and ole1 is a key gene for the synthesis of palmitoleic acid and stearic acid. Therefore, the present invention inhibits the expression of the Cdc20 regulatory factor in Yarrowia lipolytica (Po1f) and overexpresses the gene encoding the Spt23 protein to increase the yield of palmitoleic acid and stearic acid in Yarrowia lipolytica (Po1f).

[0058] 4. The present invention is a method for increasing the yield of fatty acid fermentation in Yarrowia lipolytica (Po1f), which specifically involves inhibiting the expression of cell division cycle 20 homolog (Cdc20) to regulate the cell cycle of Yarrowia lipolytica (Po1f). At the same time, the gene encoding Spt23 protein is overexpressed in the genome to regulate the transcription of ole1 (Δ 9-Fatty aciddesaturase, Δ 9 desaturase) gene, ole1 is a key gene for synthesizing palmitoleic acid and stearic acid. The two methods are combined to increase the yield of palmitoleic acid and stearic acid in Yarrowia lipolytica (Po1f).

[0059] 5. The present invention utilizes both genetic engineering and protein engineering to transform Yarrowia lipolytica (Po1f) to produce palmitoleic acid and stearic acid in Yarrowia lipolytica. The production of palmitoleic acid and stearic acid in Yarrowia lipolytica has not been reported in previous articles, and the yields of palmitoleic acid and stearic acid in the present invention are improved compared to the wild type (Po1f).

[0060] 6. The modified strains do not require additional exogenous additives and regulatory factors during the cultivation process, and only basic YPD culture medium is needed to achieve the effect of increasing yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the C20i plasmid map of the present invention;

[0062] Figure 2 This is the DT23 plasmid map of the present invention;

[0063] Figure 3 This is a colony PCR verification result diagram of Cdc20-N20 in the present invention;

[0064] Figure 4This is a sequence comparison diagram of Cdc20-N20 in the present invention after being sent for testing in Snap Gene;

[0065] Figure 5 The figure is a comparison diagram of the cell morphology of the original strain Po1f and the strain Po1f-20 in the present invention; wherein the left side is the cell morphology diagram of the original strain Po1f, and the right side is the cell morphology diagram of the strain Po1f-20;

[0066] Figure 6 This is a colony PCR verification result diagram of the overexpression of the spt23 gene in the present invention;

[0067] Figure 7 This is a sequence comparison diagram of the spt23 gene overexpressed in the present invention after being sent for testing in Snap Gene;

[0068] Figure 8 This is a schematic diagram comparing the biomass of the modified strain Po1f-20-23 in the present invention and the biomass of the original strain Po1f. DETAILED DESCRIPTION

[0069] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0070] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0071] A Yarrowia lipolytica genetic engineering strain for increasing the production of functional fatty acids in Yarrowia lipolytica, wherein the strain is obtained by inhibiting the expression of a Cdc20 regulatory factor in Po1f of Yarrowia lipolytica and overexpressing a gene encoding a Spt23 protein;

[0072] Among them, inhibiting the expression of Cdc20 regulatory factors is achieved by finding the sequence and PAM site of Cdc20 regulatory factors and setting the sgRNA sequence to guide Cas9 to locate the PAM site sequence;

[0073] The gene sequence encoding Cdc20 is shown in SEQ ID NO.1, the gene sequence encoding Spt23 protein is shown in SEQ ID NO.2, and the sgRNA sequence is shown in SEQ ID NO.6.

[0074] Preferably, the strain increases the content of functional fatty acids by regulating the cell cycle of Yarrowia lipolytica and overexpressing protein genes; wherein regulating the cell cycle of Yarrowia lipolytica Po1f refers to inhibiting the expression of the regulatory factor Cdc20 in Yarrowia lipolytica Po1f; overexpressing protein genes refers to overexpressing the gene encoding Spt23 protein in the genome of Yarrowia lipolytica Po1f; the two methods jointly regulate the cell cycle and protein content of Yarrowia lipolytica Po1f, thereby increasing the production of palmitoleic acid and stearic acid.

[0075] The use of the genetically engineered strain of Yarrowia lipolytica as described above in increasing the production of palmitoleic acid and stearic acid.

[0076] A method for constructing the genetically engineered strain of Yarrowia lipolytica as described above comprises the following steps:

[0077] By inhibiting the expression of the regulatory factor Cdc20, the cell cycle of Yarrowia lipolytica Po1f was regulated, and a positive transformant was obtained, named Po1f-20;

[0078] The gene encoding Spt23 protein was overexpressed in Po1f-20 to regulate the fatty acid production of Yarrowia lipolytica, and the overexpression plasmid DT23 was obtained;

[0079] The DT23 plasmid was transferred into Yarrowia lipolytica Po1f-20 using the lithium acetate method; screening was carried out in yeast SC-URA medium and cultured in a 30°C incubator for 48-96 h; after verification, a positive transformant was obtained and named Po1f-20-23, thereby obtaining a genetically engineered strain of Yarrowia lipolytica that improves the production of functional fatty acids in Yarrowia lipolytica.

[0080] Preferably, the specific steps are as follows:

[0081] Construction of positive transformant Po1f-20:

[0082] (1) Constructing plasmid C20i that inhibits the expression of regulatory factor Cdc20;

[0083] (2) After consulting the literature, we discovered a regulatory factor in the G2 / M phase: the cell division cycle 20 homolog Cdc20;

[0084] (3) Obtain the gene sequence of the regulatory factor Cdc20 in Po1f of Yarrowia lipolytica from the NCBI website;

[0085] (4) Analyze the 20 nucleotide sequences upstream of the PAM region on the gene sequence of Cdc20 to obtain the gRNA target sequence; name the target sequence Cdc20-N20, and locate it on the Po1f genome map of Yarrowia lipolytica, set the primers for amplifying gRNA, and connect it to the pCRISPR-Cas9 plasmid as a backbone vector, and set the sgRNA sequence to guide Cas9 to locate to the PAM site sequence and prompt Cas9 to cut the DNA double strand at this position; wherein the sgRNA sequence is shown in SEQ ID NO.6;

[0086] (5) Select the pCRISPR-Cas9 plasmid as the backbone and AvrⅡ restriction site, prepare the restriction system, and then use the recovery kit to recover the digested backbone;

[0087] (6) Use 37°C ligase to connect the amplified target fragment and the digested backbone in a 20 μL PCR tube at 37°C for 20 min.

[0088] (7) Add the liquid in the PCR tube in step (6) to the DH5α E. coli competent cells;

[0089] (8) Place the competent cells in step (7) on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min.

[0090] (9) Spread the plate on an LB medium plate containing 0.1% by mass of Amp resistance to screen for positive transformants;

[0091] (10) Use a plasmid extraction kit to extract the constructed plasmid C20i;

[0092] (11) The C20i plasmid was transformed into Yarrowia lipolytica using the lithium acetate method;

[0093] (12) Screening was performed in yeast SC-URA medium to obtain a positive transformant, which was named Po1f-20;

[0094] Construction of the gene overexpression plasmid DT23 encoding Spt23 protein and the strain Po1f-20-23:

[0095] (1) Review the literature and discover the Spt23 protein;

[0096] (2) Obtain the gene sequence encoding the Spt23 protein and locate it on the Po1f genome of Yarrowia lipolytica;

[0097] (3) Design primers for amplification of the gene sequence encoding the Spt23 protein;

[0098] (4) amplifying the sequence encoding the Spt23 protein gene from the Po1f genome of Yarrowia lipolytica and then recovering it using a recovery kit;

[0099] (5) Select the pYLXP' linear vector as the backbone and the restriction site, i.e., the EcoR Ⅰ site, and prepare the restriction system, and then use the recovery kit to recover the digested backbone;

[0100] (6) Use 37°C ligase to connect the amplified target fragment and the digested backbone in a 20 μL PCR tube at 37°C for 20 min.

[0101] (7) Add the liquid in the PCR tube (6) to the DH5α E. coli competent cells;

[0102] (8) Place the competent cells in (7) on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min.

[0103] (9) Spread the plate on an LB medium plate containing 0.1% by mass of Amp resistance to screen for positive transformants;

[0104] (10) Use a plasmid extraction kit to extract the overexpression plasmid DT23;

[0105] (11) The DT23 plasmid was transformed into Yarrowia lipolytica Po1f-20 cells using the lithium acetate method;

[0106] (12) Screening was performed in yeast SC-URA medium, and positive transformants were obtained after verification;

[0107] (13) The positive transformants were fermented in a fermentation medium for 120 h at 28 °C and 220 rpm to obtain a genetically engineered strain of Yarrowia lipolytica Po1f-20-23 that increased the functional fatty acid production of Yarrowia lipolytica.

[0108] Preferably, the formula of the yeast SC-URA medium is: glucose: 20 g / L; yeast basic nitrogen source: 1.7 g / L; ammonium sulfate: 5 g / L; agar: 3% (mass percentage), and the solvent is water;

[0109] The formula of the fermentation medium is: glucose: 60 g / L; yeast extract: 2.5 g / L; yeast basic nitrogen source: 1.7 g / L, and the solvent is water.

[0110] The fermentation method for increasing the yield of palmitoleic acid and stearic acid by using the genetically engineered strain of Yarrowia lipolytica as described above comprises the following steps:

[0111] The genetically engineered strain of Yarrowia lipolytica was fermented in a fermentation medium for 120 h at a fermentation temperature of 28 °C and a fermentation speed of 220 rpm to obtain palmitoleic acid and stearic acid.

[0112] Preferably, the formula of the fermentation medium is: glucose: 60 g / L; yeast extract: 2.5 g / L; yeast basic nitrogen source: 1.7 g / L, and the solvent is water.

[0113] Specifically, the relevant preparation and detection are as follows:

[0114] The competent cells used in the present invention are DH5α Escherichia coli competent cells (Qingke Bio-Trelief ® 5αChemically Competent Cell, catalog number TSC-C01-100).

[0115] LB-Amp R Solid culture medium: Tryptone 10 g / L; Yeast extract 5 g / L; Sodium chloride (NaCl) 10 g / L; Ampicillin: 0.1% (mass percentage); Agar: 2% (mass percentage), the solvent is water;

[0116] LB-Amp R Liquid culture medium: Tryptone 10 g / L; Yeast extract 5 g / L; Sodium chloride (NaCl) 10 g / L; Ampicillin: 0.1% (mass percentage), the solvent is water;

[0117] Yeast SC-URA medium: Dextrose: 20 g / L; Yeast Nitrogenbase (YNB): 1.7 g / L; Ammonium sulfate (NH 4 ) 2 SO 4 ): 5 g / L; agar: 3% (mass percentage), the solvent is water. Compared with SC medium, SC-URA medium lacks uracil, so it can be used to identify the URA3 auxotrophic type of Saccharomyces cerevisiae. Since the present invention uses the HisG-URA3-HisG method to discard URA3 in the original genome, SC-URA medium can be used to identify whether the strain is successfully edited.

[0118] Seed culture medium: Yeast Extract: 10 g / L; Peptone: 20 g / L; Dextrose: 20 g / L, the solvent is water;

[0119] Fermentation medium (oil-producing medium): Glucose (Dextrose): 60 g / L; Yeast Extract (Yeast Extract): 2.5 g / L; Yeast Nitrogen base (YNB): 1.7 g / L, and the solvent is water.

[0120] Example 1 A method for regulating the cell cycle of Yarrowia lipolytica (Po1f) by inhibiting the expression of the regulatory factor Cdc20 comprises the following steps:

[0121] Construction of plasmid C20i that inhibits Cdc20 expression:

[0122] 1. After consulting the literature, we found the regulatory factor of G2 / M phase: cell division cycle 20 homolog (Cdc20);

[0123] 2. Obtain the gene sequence of the regulatory factor Cdc20 from the NCBI website (as shown in SEQ ID NO.1);

[0124] 3. Use the CRISPRdirect website to analyze the 20 nucleotide sequences upstream of the PAM region on the gene sequence of Cdc20, and perform nucleotide BLAST on the NCBI website to select 20 nucleotides with high specificity, and finally obtain the gRNA target sequence. The target sequence is named Cdc20-N20, and its sequence is shown in SEQ ID NO.3. It is located on the (Po1f) genome map, and the primers Cdc20-gRNA-F / R are set to amplify gRNA and connect it to the pCRISPR-Cas9 backbone vector. Its sequence is shown in SEQ ID NO.4 and SEQ ID NO.5, and the sgRNA sequence (its sequence is shown in SEQ ID NO.6) is set to guide Cas9 to locate to the PAM site sequence and prompt Cas9 to cut the DNA double strand at this position. The above-mentioned related sequences are detailed in Table 1.

[0125]

[0126] 4. Select pCRISPR-Cas9 plasmid (published in authorized patent CN201710264591.7) as the backbone vector and Avr Ⅱ restriction site, prepare the restriction system (as shown in Table 2), and then use a recovery kit (such as UE-GX-250 DNA gel recovery kit) to recover the enzyme-cut backbone.

[0127]

[0128] 5. Use 37 ℃ ligase to connect the amplified Cdc20-N20 target fragment and the digested backbone in a 20 μL PCR tube at 37 ℃ for 20 min.

[0129] 6. Add the liquid in the PCR tube in the previous step to the DH5α E. coli competent cells;

[0130] 7. Place the competent cells on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min.

[0131] 8. Coating on LB-Amp R On solid culture plates, positive transformants were screened using Taq enzyme (such as Vazyme 2 × Rapid TaqMaster Mix). Figure 3 and Figure 4 As shown, it can be seen that the Cdc20-N20 target fragment has been successfully connected to the pCRISPR-Cas9 plasmid backbone vector without deletions and errors.

[0132] 9. Inoculate the positive transformants into LB-Amp R In liquid culture medium, a plasmid extraction kit was used to extract and obtain C20i plasmid;

[0133] 10. The C20i plasmid was transferred into Yarrowia lipolytica (Po1f) using the lithium acetate method;

[0134] Lithium Acetate Method:

[0135] (1) Inoculate a single transformed yeast colony into YPD medium;

[0136] (2) Incubate overnight at 28°C in a shaking incubator until OD 600 In the range of 0.8-1.0;

[0137] (3) Transfer 1 mL of bacterial solution into a sterile 1.5 mL centrifuge tube and centrifuge at 3000 rpm for 4 min.

[0138] (4) Take 5 μL of salmon sperm (ssDNA) and heat it at 98 °C for 3 min;

[0139] (5) Mix 5 μL of lithium acetate, 5 μL of DTT solution, and 80 μL of PEG4000 solution to obtain a mixed solution;

[0140] (6) Pour off the supernatant from step (3), leaving the bacterial cells, and add 500 ng of the corresponding C20i plasmid into the supernatant;

[0141] (7) Add the heated ssDNA in (4) to the mixed solution in (5), mix well, and then add to the centrifuge tube in (6). Pipet and mix the bacteria, and vortex for 10 seconds.

[0142] (8) Place the centrifuge tube mixed in (7) in a 37°C water bath, vortex for 10 s every 10 min, and spread on a yeast SC-URA medium plate 1 h later.

[0143] 11. Screening was performed in yeast SC-URA medium to obtain a positive transformant, which was named Po1f-20;

[0144] 12. The obtained positive transformants were inoculated into seed culture medium and cultured at 28°C and 220 rpm;

[0145] 13. Take 10 μL of bacterial solution and place it on a glass slide. Observe the cell morphology under an inverted microscope with a 100× oil lens. Figure 5 As shown, it can be seen that by comparing the cell morphology of the original strain Po1f in the same cell cycle and the strain Po1f-20 after inhibiting the expression level of the Cdc20 regulatory factor, it can be seen that the yeast cell mother morphology of the latter strain Po1f-20 has become significantly larger, with a trend of more than 200% enlargement, and appears "pea-shaped".

[0146] Example 2 A method for overexpressing a gene encoding Spt23 protein in Po1f-20 to regulate the fatty acid production of Yarrowia lipolytica comprises the following steps:

[0147] Construction of gene overexpression plasmid DT23 encoding Spt23 protein:

[0148] 1. Consult the literature and find the Spt23 protein (sequence 5′-3′, SEQ ID NO.2);

[0149] 2. Obtain the gene sequence encoding Spt23 protein from the NCBI website and locate it on the (Po1f) genome;

[0150] 3. Design amplification primers encoding the Spt23 protein gene sequence, whose sequences are shown in SEQ ID NO.7 and SEQ ID NO.8, see Table 3 for details;

[0151]

[0152] 4. The gene sequence encoding Spt23 protein was amplified from the genome of Yarrowia lipolytica (Po1f), with an unwinding temperature of 98 °C, an annealing temperature of 56 °C, an annealing time of 50 s, 30 cycles, and an extension temperature of 72 °C. The gel was then recovered using a recovery kit (UE-GX-250 DNA gel recovery kit). The amplification system is shown in Table 4.

[0153]

[0154] 5. Select pYLXP' linear vector as the vector and restriction site (EcoR Ⅰ site), prepare 50 μL restriction system, and then use a recovery kit (such as UE-GX-250 DNA gel recovery kit) to recover the digested backbone. The restriction system is shown in Table 5.

[0155]

[0156] 6. Use 37 ℃ ligase to connect the target fragment of the gene sequence encoding spt23 protein and the digested backbone in a 20 μL PCR tube at 37 ℃ for 20 min.

[0157] 7. Add the liquid in the PCR tube in the previous step to the DH5α E. coli competent cells;

[0158] 8. Place the competent cells on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min.

[0159] 9. Coated on LB-Amp containing Amp resistance (mass percentage 0.1%) R On solid medium plates, use Taq enzyme (such as Vazyme 2 × Rapid Taq Master Mix) to screen positive transformants;

[0160] 10. Inoculate the positive transformants into LB-Amp R The overexpression plasmid DT23 was obtained by extracting from the liquid culture medium using a plasmid extraction kit.

[0161] Example 3 Fermentation to verify the role of the gene encoding Spt23 protein in Yarrowia lipolytica

[0162] Using the method of embodiment 1

[0163] 1. The DT23 plasmid was transferred into Yarrowia lipolytica Po1f-20 using the lithium acetate method (the same as the lithium acetate method in step 10 in constructing the plasmid C20i for inhibiting Cdc20 expression in the above example);

[0164] 2. Screen in yeast SC-URA medium and culture in a 30 ℃ incubator for 48-96 hours;

[0165] 3. Design primers for transformant verification, whose sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, as shown in Table 6. After verification, positive transformants were obtained and named Po1f-20-23. Figure 6 , Figure 7 As shown, it can be seen that the gene encoding Spt23 protein has been successfully connected to the pYLXP' linear vector backbone vector without deletions and errors.

[0166]

[0167] 4. The obtained positive transformant Po1f-20-23 was fermented in the fermentation medium (oil-producing medium) for 120 h at a fermentation temperature of 28 °C and a fermentation speed of 220 rpm, and samples were taken every 24 h;

[0168] 5. Centrifuge the bacterial sample at 12000 rpm for 1 min, discard the supernatant, and perform methyl esterification treatment;

[0169] Methylation treatment method:

[0170] (1) Add 500 μL of NaOH-methanol solution to 1 mL of bacterial sample and place it in an oscillator for 4-5 h;

[0171] (2) Add 40 μL of concentrated sulfuric acid to terminate the reaction;

[0172] (3) Add 1 mL of n-hexane solution and shake for about 20 min; centrifuge at 12,000 rpm for 1 min.

[0173] 6. Use a sterilized syringe to suck out the supernatant after centrifugation, filter it with a filter membrane, and then add it to the gas phase vial;

[0174] 7. Use gas chromatograph to detect the content of fatty acids;

[0175] The results are as follows:

[0176]

[0177] As shown in Table 7 and Figure 8As shown in the data, after inhibiting the expression level of Cdc20 regulatory factor and overexpressing the gene encoding Spt23 protein in the genome of Yarrowia lipolytica (Po1f), the fatty acid production of Yarrowia lipolytica can be effectively increased. The contents of fatty acids such as palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), linoleic acid (C18:2), and linolenic acid (C18:3) have all increased, among which the growth of palmitoleic acid (C16:1) is more obvious, with a growth rate of more than 100%, but its share of total lipids is only 4.851%; stearic acid (C18:0) accounts for a large proportion of total lipids, about 22.841%, and the growth rate is 23.14%.

[0178] The sequences used in the present invention are as follows:

[0179] Gene sequence encoding cell division cycle 20 homolog (Cdc20) (sequence 5′-3′, SEQ ID NO.1):

[0180]

[0181] The genomic sequence encoding the spt23 protein (sequence 5′-3′, SEQ ID NO.2):

[0182]

[0183] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A genetically engineered strain of Yarrowia lipolytica for increasing the yield of functional fatty acids in Yarrowia lipolytica, characterized in that: The strain is obtained by growing Yarrowia lipolytica ( Yarrowia lipolytica ) Po1f inhibition Cdc20 Overexpression of regulatory factors Spt23 The gene of the protein is obtained; Among them, inhibition Cdc20 Regulator expression is achieved by finding Cdc20 The sequence and PAM site of the regulatory factor are determined, and the sgRNA sequence is set to guide Cas9 to locate the PAM site sequence; coding Cdc20 The gene sequence of the regulatory factor is shown in SEQ ID NO.1, encoding Spt23 The gene sequence of the protein is shown in SEQ ID NO.2, and the sgRNA sequence is shown in SEQ ID NO.

6.

2. Use of the genetically engineered strain of Yarrowia lipolytica as claimed in claim 1 in increasing the production of palmitoleic acid and stearic acid.

3. A method for constructing a genetically engineered strain of Yarrowia lipolytica as claimed in claim 1, characterized in that: The specific steps are as follows: (1) Obtaining regulatory factors in Po1f of Yarrowia lipolytica Cdc20 The gene sequence of (2) Yes Cdc20 The 20 nucleotide sequences upstream of the PAM region on the gene sequence of Cdc20 were analyzed to obtain the gRNA target sequence; the target sequence was named Cdc20-N20 and located on the Po1f genome map of Yarrowia lipolytica, the gRNA was amplified and connected to the pCRISPR-Cas9 plasmid as a primer for the backbone vector, and the sgRNA sequence was set to guide Cas9 to locate to the PAM site sequence and cause Cas9 to cut the DNA double strand at this position; (3) Select the pCRISPR-Cas9 plasmid as the backbone and AvrⅡ restriction site, prepare the restriction system, and then use the recovery kit to recover the digested backbone; (4) Use 37°C ligase to connect the amplified target fragment and the digested backbone in a 20 μL PCR tube at 37°C for 20 min. (5) Add the liquid in the PCR tube in step (5) to the DH5α E. coli competent cells; (6) Place the competent cells in step (6) on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min. (7) Spread the plate on an LB medium plate containing 0.1% by mass of Amp resistance to screen for positive transformants; (8) Use a plasmid extraction kit to extract the constructed plasmid C20i ; (9) C20i The plasmids were transformed into Yarrowia lipolytica using the lithium acetate method; (10) Screening was performed in yeast SC-URA medium to obtain a positive transformant, which was named Po1f-20; Build Code Spt23 Protein gene overexpression plasmid DT23 And construct strain Po1f-20-23: 1) Get the code Spt23 The gene sequence of the protein was located on the Po1f genome of Yarrowia lipolytica; 2) Design coding Spt23 Primers for amplification of protein gene sequences; 3) and encode Spt23 The protein gene sequence was amplified from the Yarrowia lipolytica Po1f genome and then recovered using a recovery kit; 4) Select the pYLXP' linear vector as the backbone and the restriction site, i.e., the EcoR Ⅰ site, and prepare the restriction system, and then use the recovery kit to recover the backbone after the restriction digestion; 5) Use 37 ℃ ligase to connect the amplified target fragment and the digested backbone in a 20 μL PCR tube at 37 ℃ for 20 min; 6) Add the liquid in the PCR tube in step 5) to the DH5α E. coli competent cells; 7) Place the competent cells in step 6) on ice for 30 min, heat shock them in a 42°C water bath for 90 s, and then continue to place them on ice for 3 min; 8) Spread on LB medium plates containing 0.1% Amp resistance by mass to screen positive transformants; 9) Use the plasmid extraction kit to extract the overexpression plasmid DT23 ; 10) DT23 The plasmid was transformed into Yarrowia lipolytica Po1f-20 cells using the lithium acetate method; 11) Screening was performed in yeast SC-URA medium, and positive transformants were obtained after verification; 12) The obtained positive transformant was fermented in a fermentation medium for 120 h at 28°C and a rotation speed of 220 rpm to obtain a genetically engineered strain of Yarrowia lipolytica Po1f-20-23 with increased functional fatty acid production of Yarrowia lipolytica.

4. The construction method according to claim 3, characterized in that: The formula of the yeast SC-URA medium is: glucose: 20 g / L; yeast basic nitrogen source: 1.7 g / L; ammonium sulfate: 5 g / L; agar: 3% by mass, and the solvent is water; The formula of the fermentation medium is: glucose: 60 g / L; yeast extract: 2.5 g / L; yeast basic nitrogen source: 1.7 g / L, and the solvent is water.

5. A fermentation method for increasing the yield of palmitoleic acid and stearic acid using the genetically engineered strain of Yarrowia lipolytica as claimed in claim 1, characterized in that: The steps include: The genetically engineered strain of Yarrowia lipolytica was fermented in a fermentation medium for 120 h at a fermentation temperature of 28 °C and a fermentation speed of 220 rpm to obtain palmitoleic acid and stearic acid.

6. The fermentation method according to claim 5, characterized in that: The formula of the fermentation medium is: glucose: 60 g / L; yeast extract: 2.5 g / L; yeast basic nitrogen source: 1.7 g / L, and the solvent is water.

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