Construction of a recombinant yarrowia lipolytica strain with a deletion of a cell wall formation-related gene, method and application

Abstract

The present application belongs to the field of genetic engineering and microbial fermentation technology, and discloses a kind of construction cell wall formation related gene deletion Yarrowia lipolytica Recombinant strain, the recombinant strain is constructed by genetic engineering means to knock out its cell wall formation related gene.The cell wall structure of the recombinant strain of the present application changes, resulting in reduced cell wall stability, and does not affect intracellular synthesis and accumulation of oil.The fermentation broth of the modified strain is treated by conventional cell wall breaking method, and the cell wall breaking rate is obviously improved, which shows that this technical route can provide a better potential choice for improving the cell wall breaking rate of Yarrowia lipolytica.The construction method of the recombinant strain provided by the present application is simple and easy to use, and can be used as an upstream technical means for biological production of Yarrowia lipolytica.Only one modification can obtain sustained technical effect, to fundamentally solve the technical problems of thick cell wall and difficult to break, low cost, high benefit, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial fermentation technology, and in particular to a method for constructing a recombinant strain of Yersinia lipophila with a deletion of cell wall formation-related genes, and its application. Background Technology

[0002] Yarrowia lipolytica, a generally recognized safe (GRAS) yeast, is a model oil-producing yeast. It is rich in acetyl-CoA and malonyl-CoA (precursors to lipid synthesis), with wild-type strains accumulating up to 70% of their cell dry weight in lipids. In recent years, with the development of synthetic biology tools, the application scope of Yarrowia lipolytica has expanded. It is not only a cell factory for producing lipids but also for producing value-added lipid derivatives and various specialty chemicals. Through rational design and targeted modification, Yarrowia lipolytica can utilize renewable resources to produce lipids, oil chemicals, carotenoids, terpenes, and other substances, demonstrating significant application value in renewable energy, chemical, pharmaceutical, and health product fields.

[0003] However, in industrial production, the cell wall of *Yersinia lipolytica* exhibits a dense, three-layered structure of mannan, protein, and glucan, and as a dimorphic species, it can undergo a yeast-to-hyphae transformation when exposed to various extracellular environmental stresses (such as temperature, pH, osmotic pressure, and insufficient carbon and nitrogen sources). This morphological transformation directly affects its cell size and volume, further influencing its specific surface area (the ratio of cell surface area to volume). This directly impacts the opportunity for lipids / fatty acids to adhere to the cell surface, thereby affecting substrate utilization and biotransformation processes. These two influencing factors significantly hinder the industrial extraction of intracellular target products from *Yersinia lipolytica*. Currently, there are few research reports on using genetic engineering to modify cell wall-related genes in *Yersinia lipolytica* to improve cell wall disruption rates, making it difficult to meet the needs of actual production. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and to provide a method for constructing a recombinant strain of Yersinia lipophila with the deletion of cell wall formation-related genes, as well as its application.

[0005] The technical solution adopted by this invention to solve the technical problem is:

[0006] A recombinant strain of *Yersinia lipolytica* with a gene missing from cell wall formation is disclosed. The recombinant strain is constructed by genetically modifying *Yersinia lipolytica* to knock out the gene related to cell wall formation.

[0007] Furthermore, the cell wall formation-related gene is BEM1, which encodes actin cytoskeleton-related protein, and its amino acid sequence is shown in SEQ ID NO.1;

[0008] Alternatively, the cell wall formation-related gene is a C2H2 type zinc finger protease MHY1 that is related to cell morphology transformation, and its amino acid sequence is shown in SEQ ID NO.2;

[0009] Alternatively, the cell wall formation-related gene is a positive gene effector CLA4 that encodes hyphae formation and influences cell size, and its amino acid sequence is shown in SEQ ID NO.3.

[0010] Furthermore, the genetic modification method is homologous recombination or CRISPR-Cas9 gene editing.

[0011] Furthermore, the starting strain of the *Yersinia lipolytica* strain is *Yersinia lipolytica* po1f, which has abundant intracellular lipid accumulation and is often used in the industrial production of bio-oils.

[0012] The method for constructing the recombinant *Yarrowia lipophila* strain as described above includes the following steps:

[0013] Gene knockout was performed using Yersinia lipophila po1f as the starting strain. Its genome is characterized by an artificially modified URA uracil defect, which is beneficial for cyclic gene editing using the uracil selection marker URA.

[0014] The gene knockout process includes: first constructing a CRISPR-Cas9 gene knockout recombinant vector pCRISPRyl-ura1-BEM1 for the BEM1, MHY1, or CLA4 gene. — or pCRISPRyl-ura1-MHY1 — or pCRISPRyl-ura1-CLA4 — The recombinant strains were then transferred into competent cells of *Yersinia lipophila* po1f. The uracil selection marker URA in the recombinant vector pCRISPRyl-ura1 replaced the BEM1, MHY1, or CLA4 gene in *Yersinia lipophila* po1f. Positive recombinant strains were then screened using YNB solid medium.

[0015] The nucleotide sequence of the uracil selection marker URA is shown in SEQ ID NO.4.

[0016] A method for improving the cell wall disruption rate of *Yarrowia lipolyticis* using the recombinant strain of *Yarrowia lipolyticis* as described above includes the following steps:

[0017] The method is a biological enzymatic method, in which the fermentation broth of *Yarrowia lipolyticis* is centrifuged at 10,000 rpm to precipitate the bacterial cells, and then subjected to the following processing steps:

[0018] Adjust the bacterial cell concentration to 10-14% by volume with water, and then allow it to autolyze at room temperature and natural pH for 1.5-2 hours.

[0019] Adjust the pH value to 5.5-6.5 (this can be achieved by lowering the pH using various acids, such as a 0.1 mol / L hydrochloric acid solution), adjust the temperature to 54-56℃, add a wall-lysing enzyme to a final volume concentration of 0.5%, stir and hydrolyze for 18-24 hours, then raise the temperature to 85-95℃ to inactivate the enzyme for 10-15 minutes. Finally, centrifuge at 10,000 rpm to collect the precipitate, which is the enzymatically treated bacterial cells, with the top layer being the obtained oil.

[0020] Furthermore, the method for preparing the fermentation broth of the Yeast lipolyticis includes the following steps:

[0021] A recombinant strain was constructed by knocking out the cell wall formation-related gene of the *Yersinia lipolytica* strain through genetic modification. The recombinant strain was then inoculated into a fermentation medium for fermentation to obtain the fermentation broth of *Yersinia lipolytica*.

[0022] Furthermore, the fermentation conditions include: adding fermentation medium to the fermenter, adding 3L of fermentation medium to every 5L of fermenter, inoculating 10% (volume percentage), temperature 28-30℃, rotation speed 300-1000rpm, time 80-168h, and feeding in real time throughout the fermentation process.

[0023] The fermentation medium contains: 0.1 g / L peptone, 1-1.3 g / L ammonium sulfate, 3-3.4 g / L YNB (amino-free yeast nitrogen source), and 5 g / L yeast extract;

[0024] The real-time feeding method is as follows: pH adjuster, glucose solution, and defoamer are prepared. The fermenter program is set to maintain the pH in the range of 5.4-5.6. Glucose solution is fed from 24 hours later until the glucose in the fermentation medium is maintained at 20 ml / L. Defoamer is automatically added every time the bubble level reaches the 4L line.

[0025] The pH adjuster consists of 52.6 g / L ammonium sulfate feed, 200 g / L sodium hydroxide, and 20 g / L glucose solution, while the defoamer is Hengxin brand high-efficiency fermentation-specific defoamer.

[0026] The application of the recombinant strain of Yersinia lipophila as described above in the production of bio-oils.

[0027] The application of the methods described above in the production of bio-oils.

[0028] The beneficial effects achieved by this invention are:

[0029] 1. The recombinant strain of this invention is not affected by cell wall modification and can normally complete the intracellular synthesis and accumulation of lipids in microbial cells. Furthermore, its cell wall structure changes, leading to reduced cell wall stability. After treating the fermentation broth of this modified strain with conventional cell wall disruption methods, a significant increase in cell wall disruption rate was observed. This indicates that this technical route can provide a better potential option for improving the cell wall disruption rate of *Yarrowia lipophila*. The recombinant strain construction method provided by this invention is simple, easy to use, and has good application prospects.

[0030] 2. To overcome the problems of tough cells, difficult cell wall disruption, and low target product yield in existing technologies, this invention provides a method for constructing a recombinant strain with low cell wall structural stability. This recombinant strain lacks expression of the BEM1 gene (an actin-cytoskeleton-associated protease playing a prominent role in the spatial backbone of the cell wall structure), the MHY1 gene (a C2H2-type zinc finger protease related to cell morphology transformation), or the CLA4 gene (a positive effector gene related to hyphal formation and cell size). This alters the cell wall structure, leading to reduced cell wall stability and providing a better potential option for improving the cell wall disruption rate of *Yersinia lipolytica*.

[0031] 3. Compared with the original strain, the recombinant strain of the present invention has different cell wall morphology and stability. The constructed recombinant strain of Yersinia lipolytica with missing cell wall formation-related genes can improve its cell wall disruption rate.

[0032] 4. This invention uses the Yeast lipolyticis strain po1f as the starting strain and employs homologous recombination or CRISPR-Cas9 gene editing to knock out three genes related to the formation of the Yeast lipolyticis cell wall. Compared with the starting strain, the recombinant strain with reduced cell wall stability can normally complete the intracellular synthesis and accumulation of lipids in microbial cells, and its cell wall structure and morphology change. Attached Figure Description

[0033] Figure 1 The images are electrophoresis diagrams of the BEM1 gene YAIL0_E16060g, MHY1 gene YALI1_B28150g, and CLA4 gene YALI_1C31453g verified in Examples 1, 2, and 3 of this invention.

[0034] Figure 2 The recombinant Yersinia lipophila strain po1f-BEM1 in Examples 1, 2, and 3 of this invention. — po1f-MHY1 — po1f-CLA4 — TEM electron microscope images of Comparative Example 1;

[0035] Figure 3 The recombinant Yersinia lipophila strain po1f-BEM1 in Examples 1, 2, and 3 of this invention. — po1f-MHY1 — po1f-CLA4 — The total bio-oil content of Comparative Example 1 is shown in the figure.

[0036] Figure 4 The recombinant Yersinia lipophila strain po1f-BEM1 in Examples 1, 2, and 3 of this invention. — po1f-MHY1 — po1f-CLA4 — The cell wall breakage rate diagram of Comparative Example 1. Detailed Implementation

[0037] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments. However, the scope of protection of the present invention is not limited to the scope represented by the embodiments.

[0038] Unless otherwise specified, all raw materials used in this invention are conventional commercially available products. Unless otherwise specified, all methods used in this invention are conventional methods in the field. All substances used in this invention are of conventional usage quality.

[0039] A recombinant strain of *Yersinia lipolytica* with a gene missing from cell wall formation is disclosed. The recombinant strain is constructed by genetically modifying *Yersinia lipolytica* to knock out the gene related to cell wall formation.

[0040] Preferably, the cell wall formation-related gene is BEM1, which encodes actin cytoskeleton-related protein, and its amino acid sequence is shown in SEQ ID NO.1;

[0041] Alternatively, the cell wall formation-related gene is a C2H2 type zinc finger protease MHY1 that is related to cell morphology transformation, and its amino acid sequence is shown in SEQ ID NO.2;

[0042] Alternatively, the cell wall formation-related gene is a positive gene effector CLA4 that encodes hyphae formation and influences cell size, and its amino acid sequence is shown in SEQ ID NO.3.

[0043] Preferably, the genetic modification method is homologous recombination or CRISPR-Cas9 gene editing.

[0044] Preferably, the starting strain of the said Yersinia lipophila strain is Yersinia lipophila po1f, which has abundant intracellular lipid accumulation and is often used in the industrial production of bio-oils.

[0045] The method for constructing the recombinant *Yarrowia lipophila* strain as described above includes the following steps:

[0046] Gene knockout was performed using Yersinia lipophila po1f as the starting strain. Its genome is characterized by an artificially modified URA uracil defect, which is beneficial for cyclic gene editing using the uracil selection marker URA.

[0047] The gene knockout process includes: first constructing a CRISPR-Cas9 gene knockout recombinant vector pCRISPRyl-ura1-BEM1 for the BEM1, MHY1, or CLA4 gene. — or pCRISPRyl-ura1-MHY1 — or pCRISPRyl-ura1-CLA4 — The recombinant strains were then transferred into competent cells of *Yersinia lipophila* po1f. The uracil selection marker URA in the recombinant vector pCRISPRyl-ura1 replaced the BEM1, MHY1, or CLA4 gene in *Yersinia lipophila* po1f. Positive recombinant strains were then screened using YNB solid medium.

[0048] The nucleotide sequence of the uracil selection marker URA is shown in SEQ ID NO.4.

[0049] A method for improving the cell wall disruption rate of *Yarrowia lipolyticis* using the recombinant strain of *Yarrowia lipolyticis* as described above includes the following steps:

[0050] The method is a biological enzymatic method, in which the fermentation broth of *Yarrowia lipolyticis* is centrifuged at 10,000 rpm to precipitate the bacterial cells, and then subjected to the following processing steps:

[0051] Adjust the bacterial cell concentration to 10-14% by volume with water, and then allow it to autolyze at room temperature and natural pH for 1.5-2 hours.

[0052] Adjust the pH value to 5.5-6.5 (this can be achieved by lowering the pH using various acids, such as a 0.1 mol / L hydrochloric acid solution), adjust the temperature to 54-56℃, add a wall-lysing enzyme to a final volume concentration of 0.5%, stir and hydrolyze for 18-24 hours, then raise the temperature to 85-95℃ to inactivate the enzyme for 10-15 minutes. Finally, centrifuge at 10,000 rpm to collect the precipitate, which is the enzymatically treated bacterial cells, with the top layer being the obtained oil.

[0053] Preferably, the method for preparing the fermentation broth of the lipophilic yeast includes the following steps:

[0054] A recombinant strain was constructed by knocking out the cell wall formation-related gene of the *Yersinia lipolytica* strain through genetic modification. The recombinant strain was then inoculated into a fermentation medium for fermentation to obtain the fermentation broth of *Yersinia lipolytica*.

[0055] Preferably, the fermentation conditions include: adding fermentation medium to a fermenter, adding 3L of fermentation medium to every 5L of fermenter, inoculating 10% (volume percentage), temperature 28-30℃, rotation speed 300-1000rpm, time 80-168h, and feeding in real time throughout the fermentation process.

[0056] The fermentation medium contains: 0.1 g / L peptone, 1-1.3 g / L ammonium sulfate, 3-3.4 g / L YNB (amino-free yeast nitrogen source), and 5 g / L yeast extract;

[0057] The real-time feeding method is as follows: pH adjuster, glucose solution, and defoamer are prepared. The fermenter program is set to maintain the pH in the range of 5.4-5.6. Glucose solution is fed from 24 hours later until the glucose in the fermentation medium is maintained at 20 ml / L. Defoamer is automatically added every time the bubble level reaches the 4L line.

[0058] The pH adjuster consists of 52.6 g / L ammonium sulfate feed, 200 g / L sodium hydroxide, and 20 g / L glucose solution, while the defoamer is Hengxin brand high-efficiency fermentation-specific defoamer.

[0059] Specifically, the relevant preparation and testing methods are as follows:

[0060] In the following examples, *Yersinia lipolyticis* po1f is a derivative strain of *Yersinia lipolyticis* ATCC20460, directly provided by Professor Peng Xu of the University of Maryland, and purchased from Yeastern Biotech (Taiwan); it was used as the CRISPR-Cas9 recombinant vector pCRISPRyl-ura1-BEM1. — pCRISPRyl-ura1-MHY1 — pCRISPRyl-ura1-CLA4 — The series of plasmids were constructed in-house; unless otherwise specified, all reagents and culture media used were commercially available, and all methods used were conventional.

[0061] The terms “increase” or “enhancement” used in this invention generally mean an increase of a statistically significant amount. However, to avoid ambiguity, the terms “increase” or “enhancement” mean an increase of at least 10% compared to a reference level (e.g., the level in the starting strain), such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including an increase of 100%, or any amount between 10% and 100% compared to a reference level; or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level, or any amount between 2 times and 10 times, or a greater amount.

[0062] In a first aspect, the present invention provides a recombinant bacterial strain lacking three cell wall formation-related genes. This recombinant strain is obtained through genetic modification of a starting strain, wherein the BEM1 gene, MHY1 gene, or CLA4 gene is knocked out compared to the starting strain. Preferably, the amino acid sequence encoded by the knocked-out BEM1 gene is shown in SEQ ID NO.1. More preferably, the amino acid sequence of the knocked-out MHY1 gene is shown in SEQ ID NO.2. The inventors of the present invention have found that the knockout of the MHY1 gene can more significantly lead to changes in cell wall morphology and uneven structural distribution. Preferably, the amino acid sequence encoded by the knocked-out CLA4 gene is shown in SEQ ID NO.3.

[0063] In this invention, knockout, or gene knockout, refers to a technique that uses CRISPR-Cas9 to precisely integrate a foreign gene into a specific site on the genome of a target cell, thereby achieving the purpose of site-specific modification of a gene on a chromosome. More preferably, these three genes in the recombinant strain are knocked out, thereby replacing the knocked-out BEM1, MHY1, or CLA4 genes in the three recombinant strains with the gene for the uracil selection marker URA.

[0064] A second aspect of the present invention provides a method for constructing a recombinant bacterial strain, the method comprising: genetically modifying a starting strain such that the BEM1 gene, MHY1 gene, or CLA4 gene of the starting strain is knocked out.

[0065] According to the present invention, preferably, the starting strain is *Yarrowia lipolytica*. More preferably, the starting strain is *Yarrowia lipolytica* po1f, which has been artificially modified to be uracil-deficient in URA, and whose genome is characterized by being conducive to cyclic gene editing operations using the uracil selection marker URA, the nucleotide sequence of which is shown in SEQ ID NO. 4.

[0066] According to the present invention, preferably, the BEM1 gene, MHY1 gene, or CLA4 gene of the starting strain is knocked out by CRISPR-Cas9 gene knockout.

[0067] In this invention, it is preferred to knock out specific open reading frame sequences or promoter sequences in the genome of Yersinia lipophila by CRISPR-Cas9 gene knockout.

[0068] In this invention, the homologous sequence fragments used for CRISPR-Cas9 gene knockout can be obtained in the following ways: They can be artificially synthesized as homologous arms based on upstream and downstream fragment sequences of target genes (e.g., BEM1 gene YAIL0_E16060g, MHY1 gene YALI1_B28150g, CLA4 gene YALI1_C31453g) in *Yersinia lipolytica* disclosed in databases known in the art (e.g., GenBank database, https: / / www.ncbi.nlm.nih.gov / genbank / ); or they can be amplified from the genome of the starting strain (e.g., *Yersinia lipolytica*) using PCR to obtain the initial homologous sequence fragments of the target gene. However, this invention is not limited to these methods. The initial homologous sequence of the target gene, whether partial or complete, refers to a sequence containing the aforementioned target gene.

[0069] In this invention, a CRISPR-Cas9 gene knockout recombinant vector pCRISPRyl-ura1-BEM1 containing three BEM1, MHY1, or CLA4 genes can be constructed first. — or pCRISPRyl-ura1-MHY1 — or pCRISPRyl-ura1-CLA4 — The recombinant vectors described above can knock out the BEM1, MHY1, or CLA4 genes of the starting strain (such as *Yarrowia lipophila* po1f). Various methods for constructing recombinant vectors to ligate target gene fragments into the vector to prepare gene knockout recombinant vectors are known in the art, such as, but not limited to, the classic enzyme digestion-ligation method, the Gateway cloning system developed by Invitrogen, the Creator cloning system developed by Clontech, the Univector cloning system developed by Stephen Elledge's laboratory, and the GoldenGate cloning method based on type IIs restriction endonucleases.

[0070] For example, the recombinant vector of the present invention can be constructed using the recombinase method: based on the genome of the starting strain (such as Yeast lipophila po1f), the upstream and downstream homologous arm sequences targeting the insertion site are amplified by PCR; the gene sequence to be inserted, the upstream and downstream homologous arm sequences, and the resistance gene expression cassette are tandemly linked to obtain the recombinant vector, but the present invention is not limited thereto.

[0071] Subsequently, the recombinant vector can be introduced into the starting strain (e.g., *Yersinia lipophila*) using conventional methods in the art, such as, but not limited to, microinjection, gene gun, transformation (e.g., electroporation), infection, or transfection. Microinjection, gene gun, transformation, infection, or transfection are all conventional procedures in the art. For example, transformation refers to treating cells using known methods in molecular biology and genetic engineering to make the treated cells competent, thereby allowing them to come into contact with exogenous DNA, which then enters the competent cells. Commonly used transformation methods include protoplast transformation, chemical transformation, and electroporation transformation. Infection refers to using an artificially modified live bacteriophage virus as a vector, recombining the vector with the target DNA sequence, and then packaging the recombinant DNA into a viable bacteriophage or virus in vitro using the capsid protein of the bacteriophage or virus, thereby allowing the recombinant DNA to enter the host cell via infection. Transfection refers to treating cells into competent cells using methods such as CaCl2 or electroporation, and then allowing these competent cells to accept the recombinant bacteriophage DNA.

[0072] After introducing the three recombinant vectors into the starting strain (such as Yersinia lipophila po1f), positive clones can be screened by selection markers (such as resistance genes) and verified by genomic PCR or by genomic DNA sequencing, thereby obtaining the recombinant strains that lack the BEM1 gene, MHY1 gene, or CLA4 gene.

[0073] In this invention, preferably, the gene knockout process includes: first constructing a CRISPR-Cas9 gene knockout recombinant vector pCRISPRyl-ura1-BEM1 containing the BEM1 gene, MHY1 gene, or CLA4 gene. — or pCRISPRyl-ura1-MHY1 — or pCRISPRyl-ura1-CLA4 — Then, through homologous recombination, the uracil selection marker URA in the recombinant vector pCRISPRyl-ura1 was replaced.

[0074] A third aspect of the present invention provides a method for fermenting bio-oils from the three recombinant strains described above. Preferably, the method for fermenting the bio-oils includes: inoculating the recombinant strains as described above into a fermentation medium for fermentation.

[0075] Alternatively, a recombinant strain can be constructed as described above, and the resulting recombinant strain can be inoculated into a fermentation medium for fermentation.

[0076] According to the present invention, preferably, the method for preparing the seed liquid includes: picking a single colony of the recombinant strain and inoculating it into a seed culture medium for seed culture to obtain the seed liquid.

[0077] In this invention, the single colonies of the three recombinant strains can be obtained fresh or have been cryopreserved at low temperatures (e.g., the three recombinant strains are cryopreserved in glycerol cryovials at -80°C).

[0078] In this invention, there are no particular limitations on the seed culture medium, which can be any seed culture medium conventionally used in the art.

[0079] Preferably, the seed culture medium contains a carbon source and a nitrogen source.

[0080] More preferably, the seed culture medium contains: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose.

[0081] In this invention, there are no particular limitations on the conditions for seed culture (including temperature, rotation speed, time, etc.), and conventional culture conditions in the art can be used. Preferably, when the starting strain is *Yarrowia lipophila*, the seed culture conditions include: a temperature of 28-30°C, a rotation speed of 200-250 rpm, preferably 220 rpm, and a time of 40-60 h, preferably 48 h.

[0082] In this invention, there are no particular limitations on the fermentation method. It can be a fermentation method for biological oils that is commonly used in the art, such as inoculating the seed liquid into the fermentation culture medium (e.g., a shake flask or fermenter containing the fermentation culture medium) to carry out fermentation culture to obtain fermentation broth.

[0083] In this invention, in order to investigate whether the bio-oil fermentation of recombinant strains lacking the BEM1 gene, MHY1 gene, or CLA4 gene is affected, and in order to obtain more bacterial cells that can be used for cell wall disruption, preferably, the inoculation amount of the seed liquid is 10 parts by volume relative to 100 parts by volume of the fermentation medium.

[0084] In this invention, in order to investigate whether the bio-oil fermentation of recombinant strains lacking the BEM1 gene, MHY1 gene, or CLA4 gene is affected, and in order to obtain more bacterial cells that can be used for cell wall disruption, the fermentation conditions are further preferably as follows: fermentation in a 5L fermenter, 3L of fermentation medium, inoculum size of 10% by volume, temperature of 28-30℃, rotation speed of 300-1000rpm, time of 80-168h, and real-time feeding throughout the fermentation process.

[0085] Preferably, the fermentation medium contains a carbon source and a nitrogen source.

[0086] Preferably, the fermentation medium contains: 0.1 g / L peptone, 3-1.3 g / L ammonium sulfate, 3-3.4 g / L YNB (amino-free yeast nitrogen source), and 5 g / L yeast extract.

[0087] In this invention, in order to investigate whether the bio-oil fermentation of recombinant strains lacking the BEM1 gene, MHY1 gene, or CLA4 gene is affected, and in order to obtain more cells that can be used for cell wall disruption, preferably, the real-time feeding method is as follows: prepare pH adjuster, glucose solution, and defoamer; set the fermenter program to maintain pH in the range of 5.4-5.6; start feeding glucose solution after 24 hours until the glucose in the fermentation medium is maintained at 20 ml / L; and automatically add defoamer when the foam level reaches the 4L line.

[0088] More preferably, the real-time feeding method is as follows: the pH adjuster is 52.6 g / L ammonium sulfate feeder, 200 g / L sodium hydroxide, and 20 g / L glucose solution; the defoamer is Hengxin brand high-efficiency fermentation-specific defoamer.

[0089] The fourth aspect of this invention provides a method for detecting the biomass content of the three recombinant strains mentioned above.

[0090] Preferably, the method for detecting the amount of bio-oil is as follows: the fermentation broth is subjected to fatty acid methyl esterification treatment, the relative content of each fatty acid is detected by gas chromatography, and then the total oil content is calculated.

[0091] Preferably, the method for methyl esterification of fatty acids is as follows: the fermentation broth obtained above is separated into solid and liquid phases, the solid is collected to obtain oil cells, 0.5% (v / v) of lysozyme is added, and after enzymatic hydrolysis at 55°C for 1 h, 30 mL of n-hexane is added for extraction, and the mixture is allowed to stand for layering. The upper organic phase liquid is taken, and after repeated extraction, the solvent is evaporated, and the total oil content is determined.

[0092] Preferably, the gas chromatography detection method involves taking 30 μL of the oil obtained in the previous step and methylating the fatty acids: adding 500 μL of a 1 mol / L NaOH methanol solution and centrifuging at 1200 rpm for 30 min at room temperature; then adding 40 μL of sulfuric acid and 500 μL of chromatographic grade n-hexane to extract the fatty acid methyl esters; finally, centrifuging at 8000 rpm for 2 min, taking the upper layer, filtering it through a microporous membrane to remove impurities, injecting it into a gas chromatograph, and analyzing and calculating the total oil content.

[0093] Preferably, the specific detection method of the gas chromatography is as follows:

[0094] Detection conditions: Injector temperature 100℃, injection volume 1.0 μL, split ratio 69.8:1; Column:

[0095] DB-23 (60.0m × 0.25mm × 0.25μm); Chromatographic conditions: initial temperature 100℃, increased to 196℃ at 25℃ / min, then increased to 220℃ at 2℃ / min and held for 6 min; detector temperature: 280℃. Qualitative and quantitative analysis were performed using heptadecanoglycerate triglyceride (C17:0) methyl ester standard.

[0096] The fifth aspect of the present invention provides a method for cell disruption of the fermentation broth of the above three recombinant strains.

[0097] Preferably, the cell wall disruption method of the fermentation broth is a biological enzymatic method. The fermentation broth is centrifuged at 10,000 rpm to precipitate the cells, and then the following processing steps are performed: 2g of cells are added to water to adjust the concentration to 10-14% by volume → autolysis at room temperature and natural pH for 1.5-2 hours → pH is adjusted to 5.5-6.5 → temperature is adjusted to 54-56℃ → 0.5% (v / v) of cell wall lysis enzyme is added → hydrolysis is carried out by stirring for 18-24 hours → enzyme is inactivated by heating to 85-95℃ for 10-15 minutes → the precipitate is collected by centrifugation at 10,000 rpm.

[0098] The fifth aspect of this invention provides a method for calculating the cell wall breakage rate of the fermentation broth of the above three recombinant strains.

[0099] Preferably, the cell wall disruption rate is calculated as follows: The precipitates from centrifugation at 10,000 rpm before and after treatment are resuspended in water until OD600 = 1. 1 mL of the suspension is taken and observed under a microscope. Cells with intact morphology are counted using a hemocytometer. Each sample is counted 5 times, and the average value is taken. The cell wall disruption rate is the percentage difference between the number of cells with intact morphology after treatment and the number of cells before treatment.

[0100] The present invention will be described in detail below through embodiments. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. In the following embodiments, unless otherwise stated, the experimental methods used are conventional methods well known to those skilled in the art.

[0101] In the following examples, *Yersinia lipolyticis* po1f is a derivative strain of *Yersinia lipolyticis* ATCC20460, directly provided by Professor Peng Xu of the University of Maryland, and purchased from Yeastern Biotech (Taiwan); it was used as the CRISPR-Cas9 recombinant vector pCRISPRyl-ura1-BEM1. — pCRISPRyl-ura1-MHY1 — pCRISPRyl-ura1-CLA4— The series of plasmids were constructed in-house; unless otherwise specified, all reagents and culture media used were commercially available, and all methods used were conventional.

[0102] Example 1

[0103] The recombinant engineered strain is the *Yarrowia lipophila* strain po1f-BEM1, which has the BEM1 gene knocked out. — Construction:

[0104] (1) The plasmid pCRISPRyl-ura1 (nucleotide sequence as shown in SEQ ID NO. 5) was linearized by PacI digestion. Then, using primers BEM1_Up_F / BEM1_Up_R (nucleotide sequences as shown in SEQ ID NO. 6 and SEQ ID NO. 7) and BEM1_Dn_F / BEM1_Dn_R (nucleotide sequences as shown in SEQ ID NO. 8 and SEQ ID NO. 9) as templates, the upstream arm BEM1_Up and the downstream arm BEM1_Dn (nucleotide sequences as shown in SEQ ID NO. 10 and SEQ ID NO. 11, 1500bp and 1508bp, respectively) of BEM1 were amplified. Subsequently, the linearized pCRISPRyl-ura1, BEM1_Up, and BEM1_Dn were assembled using Gibson assembly to obtain the recombinant plasmid pCRISPRyl-ura1-BEM1. — The recombinant plasmid pCRISPRyl-ura1-BEM1 — After being verified by Shanghai Sangon Biotech sequencing, it was used for gene knockout.

[0105] (2) Using Yersinia lipolyticis po1f as the original strain, po1f strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h). 1 mL of po1f cells were collected from the fermentation broth, centrifuged, and the supernatant was discarded. Then, 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (2 M), 5 μL of single-stranded DNA (salmon sperm), and 5 μL of the recombinant plasmid pCRISPRyl-ura1-BEM1 obtained in step (1) were added. — After mixing, the mixture was incubated at 39°C for 1 hour, then spread on uracil-deficient plates to screen for single colonies of recombinant strains in which the uracil selection marker URA (nucleotide sequence as shown in SEQ ID NO.4) replaced the BEM1 gene (amino acid sequence as shown in SEQ ID NO.1).

[0106] (3) Select single colonies from the uracil-deficient plates in step (2) for colony PCR verification. Use primer pairs BEM1_testUp_F / BEM1_testUp_R (nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.13) and BEM1_testDn_F / BEM1_testDn_R (nucleotide sequences as shown in SEQ ID NO.14 and SEQ ID NO.15) to verify the recombinant plasmid pCRISPRyl-ura1-BEM1. — If both the upstream and downstream sites of the integration are correct, then the recombinant plasmid pCRISPRyl-ura1-BEM1 is confirmed. — Knockout of the upstream and downstream sites of the box integration correctly validated the recombinant plasmid pCRISPRyl-ura1-BEM1. — The recombinant engineered strain obtained by integrating the knockout site into the po1f cells was the BEM1 gene knockout Yersinia lipophila strain po1f-BEM1. — .

[0107] Example 2

[0108] The recombinant engineered strain is the *Yarrowia lipolyticis* strain po1f-MHY1 with the MHY1 gene knocked out. — Construction:

[0109] (1) The plasmid pCRISPRyl-ura1 (nucleotide sequence as shown in SEQ ID NO. 8) was linearized by PacI digestion. Then, using primers MHY1_Up_F / MHY1_Up_R (nucleotide sequences as shown in SEQ ID NO. 16 and SEQ ID NO. 17) and MHY1_Dn_F / MHY1_Dn_R (nucleotide sequences as shown in SEQ ID NO. 18 and SEQ ID NO. 19) as templates, the upstream arm MHY1_Up and the downstream arm MHY1_Dn (nucleotide sequences as shown in SEQ ID NO. 20 and SEQ ID NO. 21, each 1000 bp) of MHY1 were amplified. Subsequently, the linearized pCRISPRyl-ura1, BEM1_Up, and BEM1_Dn were assembled using Gibson assembly to obtain the recombinant plasmid pCRISPRyl-ura1-MHY1. — The recombinant pCRISPRyl-ura1-BEM1 — After being verified by Shanghai Sangon Biotech sequencing, it was used for gene knockout.

[0110] (2) Using Yersinia lipolyticis po1f as the original strain, po1f strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h). 1 mL of po1f cells were collected from the fermentation broth, centrifuged, and the supernatant was discarded. Then, 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (2 M), 5 μL of single-stranded DNA (salmon sperm), and 5 μL of the recombinant plasmid pCRISPRyl-ura1-BEM1 obtained in step (1) were added. — After mixing, the mixture was incubated at 39°C for 1 hour, then spread on uracil-deficient plates to screen for single colonies of recombinant strains in which the uracil selection marker URA (nucleotide sequence as shown in SEQ ID NO.4) replaced the MHY1 gene (amino acid sequence as shown in SEQ ID NO.2).

[0111] (3) Select single colonies from the uracil-deficient plate in step (2) for colony PCR verification. Use primer pairs MHY1_testUp_F / MHY1_testUp_R (nucleotide sequences as shown in SEQ ID NO.22 and SEQ ID NO.23) and MHY1_testDn_F / MHY1_testDn_R (nucleotide sequences as shown in SEQ ID NO.24 and SEQ ID NO.25) to verify the recombinant plasmid pCRISPRyl-ura1-MHY1. — If both the upstream and downstream sites of the integration are correct, then the recombinant plasmid pCRISPRyl-ura1-MHY1 is confirmed to be correct. — Knockout of both upstream and downstream sites of the box integration correctly validated the recombinant plasmid pCRISPRyl-ura1-MHY1. — The recombinant engineered strain obtained by integrating into the knockout site of po1f cells is the Yersinia lipophila strain po1f-MHY1 with the MHY1 gene knocked out. — .

[0112] Example 3

[0113] The recombinant engineered strain is the CLA4 gene knocked-out Yersinia lipophila strain po1f-CLA4. — Construction:

[0114] (1) The plasmid pCRISPRyl-ura1 (nucleotide sequence as shown in SEQ ID NO. 8) was linearized by PacI digestion. Then, using primers CLA4_Up_F / CLA4_Up_R (nucleotide sequences as shown in SEQ ID NO. 26 and SEQ ID NO. 27) and CLA4_Dn_F / CLA4_Dn_R (nucleotide sequences as shown in SEQ ID NO. 28 and SEQ ID NO. 29), the upstream arm CLA4_Up and the downstream arm CLA4_Dn (nucleotide sequences as shown in SEQ ID NO. 30 and SEQ ID NO. 31, 1502bp and 1503bp, respectively) of CLA4 were amplified using the Yersinia lipolyticis genome as a template. Subsequently, the linearized pCRISPRyl-ura1, CLA4_Up, and CLA4_Dn were assembled using Gibson assembly to obtain the recombinant plasmid pCRISPRyl-ura1-CLA4. — Recombinant pCRISPRyl-ura1-CLA4 — After being verified by Shanghai Sangon Biotech sequencing, it was used for gene knockout.

[0115] (2) Using Yersinia lipolyticis po1f as the original strain, po1f strain was cultured in 2 mL of YPD medium to the exponential growth phase (16-24 h). 1 mL of po1f cells were collected from the fermentation broth, centrifuged, and the supernatant was discarded. Then, 90 μL of 50% PEG4000 solution, 5 μL of lithium acetate (2 M), 5 μL of single-stranded DNA (salmon sperm), and 5 μL of the recombinant plasmid pCRISPRyl-ura1-CLA4 obtained in step (1) were added. — After mixing, the mixture was incubated at 39°C for 1 hour, then spread on uracil-deficient plates to screen for single colonies of recombinant strains in which the uracil selection marker URA (nucleotide sequence as shown in SEQ ID NO.4) replaced the CLA4 gene (amino acid sequence as shown in SEQ ID NO.3).

[0116] (3) Select single colonies from the uracil-deficient plate in step (2) for colony PCR verification. Use primer pairs CLA4_testUp_F / CLA4_testUp_R (nucleotide sequences as shown in SEQ ID NO. 32 and SEQ ID NO. 33) and CLA4_testDn_F / CLA4_testDn_R (nucleotide sequences as shown in SEQ ID NO. 34 and SEQ ID NO. 35) to verify the recombinant plasmid pCRISPRyl-ura1-CLA4. — If both the upstream and downstream sites are correctly integrated, it proves that the recombinant plasmid pCRISPRyl-ura1-CLA4 is correct.— Knockout of both upstream and downstream sites of the box integration correctly validated the recombinant plasmid pCRISPRyl-ura1-CLA4. — The recombinant engineered strain obtained by integrating into the knockout site of po1f cells was the CLA4 gene knockout Yersinia lipophila strain po1f-CLA4. — .

[0117] Example 4: Bio-oil fermentation and analysis of recombinant strains

[0118] The recombinant Yersinia lipophila strain po1f-BEM1 obtained in Examples 1, 2, and 3 was selected. — po1f-MHY1 — po1f-CLA4 — The cells were inoculated into 100 mL of seed culture medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured at 28 °C and 220 rpm for 48 h to obtain recombinant Yersinia lipolytica seed culture. Another 2 mL of the seed culture was centrifuged at 4500 rpm to obtain bacterial cells, which were then prepared for microscopic examination and photographed using a TEM (transmission electron microscope) to observe morphological changes (see details). Figure 2 As can be seen, the cell walls (outermost layer) of the recombinant Yersinia lipophila strains obtained in Examples 1, 2, and 3 have a lighter, thinner, and more irregular cell wall color in the imaging.

[0119] The three recombinant Yeast lipolyticis seed cultures were inoculated into a 5L fermenter at a volume percentage of 10%. The fermenter contained 3L of fermentation medium (0.1g / L peptone, 3-1.3g / L ammonium sulfate, 3-3.4g / L YNB, and 5g / L yeast extract). Under conditions of 28℃ and a rotation speed of 300-1000rpm, 52.6g / L ammonium sulfate feed, 200g / L sodium hydroxide, 20g / L glucose solution, and a high-efficiency fermentation-specific defoamer were added to the fermentation broth in real time to maintain the pH in the range of 5.4-5.6, the glucose content at 20ml / L, and the bubble level below 4L. Fermentation was carried out for 168 hours to obtain the fermentation broth.

[0120] The fermentation broth was subjected to fatty acid methyl esterification treatment, and the relative content of each fatty acid was detected by gas chromatography. The total oil content was then calculated. The fermentation broth obtained above was separated into solid and liquid phases. The solid was collected to obtain oil cells. 0.5% (v / v) of lysozyme was added, and the cells were enzymatically hydrolyzed at 55℃ for 1 h. Then, 30 mL of n-hexane was added for extraction. The cells were allowed to stand for separation. The upper organic phase liquid was taken, and the extraction was repeated. The solvent was evaporated, and the total oil content was determined.

[0121] Take another 30 μL of the oil obtained in the previous step and methyl esterify the fatty acids: add 500 μL of 1 mol / L NaOH in methanol solution and centrifuge at 1200 rpm for 30 min at room temperature; then add 40 μL of sulfuric acid and 500 μL of chromatographic grade n-hexane to extract fatty acid methyl esters; finally, centrifuge at 8000 rpm for 2 min, take the upper layer, filter it through a microporous membrane to remove impurities, inject it into a gas chromatograph, and analyze and calculate the total oil content.

[0122] The specific detection method using gas chromatography is as follows:

[0123] Detection conditions: Inlet temperature 100℃, injection volume 1.0μL, split ratio 69.8:1;

[0124] Column: DB-23 (60.0m × 0.25mm × 0.25μm);

[0125] Chromatographic conditions: Initial temperature 100℃, temperature increased to 196℃ at 25℃ / min. Subsequently, temperature increased to 220℃ at 2℃ / min and held for 6 min;

[0126] Detector temperature: 280℃.

[0127] Qualitative and quantitative analysis was performed using heptadecanoglycerate triglyceride (C17:0) methyl ester standard;

[0128] Starting strain po1f and recombinant Yersinia lipophila strain po1f-BEM1 — po1f-MHY1 — po1f-CLA4 — The graph shows the oil content in the fermentation broth after fermentation. Figure 3 As shown, considering the total lipid content of the fermentation broth, the three recombinant Yersinia lipophila strains are superior to the original strain po1f. Calculations show that in 1L of fermentation broth, po1f-BEM1... — The total fat content is 952 mg, po1f-MHY1 — The total fat content is 1180mg, po1f-CLA4 — The total lipid content of the three recombinant Yersinia lipolytica strains was 1120 mg. The average total lipid content was 1050.67 mg, all higher than the 900 mg of the original strain. Therefore, the modification of these three genes did not have a negative impact on the bio-lipid fermentation of the original strain; in fact, there may even be a beneficial effect to be explored. Specifically, the po1f-MHY1 gene modification of the MHY1 gene... — The total lipid content of the strain was significantly increased. Further analysis of fermentation data revealed that po1f-MHY1 at the same time point... —The biomass of the fermentation broth of this strain was significantly higher than that of the other three strains, suggesting that the deletion of the MHY1 gene leads to a lack of C2H2-type zinc finger protease, which is related to cell morphology transformation. This, in turn, shortens the cell cycle, alters cell morphology, reduces nutrient consumption during cell growth, and increases lipid synthesis. Example 5: Cell wall disruption of the fermentation broth of the recombinant strain.

[0129] The three recombinant *Yersinia lipophila* fermentation broths from Example 4 were centrifuged at 10,000 rpm to precipitate the cells, which were then subjected to the following treatment steps: 2g of cells were added to water to adjust the concentration to 10-14% (v / v) → autolysis was allowed at room temperature and natural pH for 1.5-2 hours → pH was adjusted to 5.5-6.5 → temperature was adjusted to 54-56℃ → 0.5% (v / v) of cell-lysing enzyme was added → hydrolysis was carried out with stirring for 18-24 hours → enzyme inactivation was performed at 85-95℃ for 10-15 minutes → the precipitate was collected by centrifugation at 10,000 rpm. The precipitates before and after treatment were resuspended in water to OD600 = 1. 1mL of the suspension was taken and observed under a microscope. Cells with intact morphology were counted using a hemocytometer. Each sample was counted 5 times, and the average value was taken. The cell disruption rate was the percentage of the difference in the number of cells with intact morphology after treatment relative to the number of cells before treatment (see details). Figure 4 After enzymatic cell disruption treatment commonly used in bioproduction, the average cell disruption efficiency of the starting strain *Yarrowia lipophila* po1f was 25%, the average cell disruption efficiency of po1f-BEM1 was 55%, and that of po1f-MHY1 was... — The average cell wall disruption efficiency of strain po1f-CLA4 was 72%. — The average cell wall disruption efficiency of the strain was 68%, indicating that the cell walls of recombinant strains lacking the three cell wall formation-related genes BEM1, MHY1, or CLA4 are more easily destroyed by cell wall lysing enzymes, which can significantly improve the cell wall disruption effect.

[0130] Comparative Example 1

[0131] Biological oil fermentation was carried out according to the method of Example 3, except that the recombinant *Yersinia lipolyticis* was replaced with the starting strain *Yersinia lipolyticis* po1f obtained in Example 1. The total oil content of the fermentation broth obtained by the detection method of Example 3 and the treatment method of Example 4 was 900 mg / L, and the cell wall disruption rate was 25%.

[0132] Furthermore, due to the thick cell walls and difficulty in disrupting wild-type *Yarrowia lipolytica* cells, numerous studies have developed various cell disruption methods to effectively obtain intracellular lipids. These methods can be broadly categorized into mechanical and non-mechanical methods based on whether an external force is applied. Mechanical disruption is energy-intensive and prone to product contamination; non-mechanical disruption methods include physical treatments such as pyrolysis and freeze-thaw cycles, acid / alkali or chemical solvent treatments, and enzymatic treatments, which are time-consuming but simple and easy to implement. While enzymatic disruption offers advantages such as mild conditions and minimal product loss, it is relatively expensive. This invention focuses on the morphological engineering of *Yarrowia lipolytica*, exploring the technical effects and production applications achievable by knocking out three genes related to cell wall formation. The genetic engineering method provided by this invention can serve as an upstream technology for bioproduction. *Yarrowia lipolytica* strains with the three cell wall formation-related genes BEM1, MHY1, or CLA4 knocked out can be further modified and used in chassis strains for production, fundamentally solving the technical challenge of thick cell walls and difficulty in disruption. Only one modification is needed to achieve sustained technical effects, resulting in low cost and high returns. Meanwhile, the production strain based on this technology, combined with various existing methods such as mechanical and non-mechanical cell disruption used in downstream production, can provide new ideas for solving technical problems.

[0133] SEQ ID NO.1

[0134] GSNIDPARKCQSMRGV*TQPRWRSSSMFASAGRK*LR*MES*LCMTRKQPPADSRDSTPHLPLPRDISGESQIRAWHG*VVVCMHRSTLI*THRYLCSTTA*QIMTPRT*K*GKLQLPVGVVKIK*GQHVAHGAVWLYKGGIRRMRERGKRGSENIEAKFVFKNK*TRQAPFTHPPSLLGFPEIHTSYTHYLPPTCTPLIIPVVLRFQRQSANTVSVSVVPTV*EMIKVSINGCLEAVCGLLRVCDGSTKLAGHPACLQMSLHP*EGAVA*VETPCCHRGNMNGNGERAMVDMGTVRTGDYVGIKCSASLFSSFLCGFGVALGLSQCDCHVHDKQLNR ALVRGMDRVFSGPIFSVDYAIIDQIEAPSSFCFCFQAWGIRAETPFQPELPHRYTHHFTNPS*RLFYDSLH PCHHRLGVVMGLFFTS*HKKPAAVSS*RTAPRFRILTQGIRRSLKGEKTTSSGSANSLVSDKNPLHTNPP*EWPLGVLESNFAFLAVQSGLQTVCLVLSV*TAVRTIIDRSLCIERRASCTQPV*KLEPPPGGASEDDVGIVYFRCS IYF*GCVMI*GIGGPYG*LYVLHWLAYRVVSLGVIHVYHLIGIQSMLLQYVQLVDAHFRRAD*CNEFLLVPSMGASSII*SGSVCPTTVSGLVKSLVP*KVYPGRSSSISSISSISPLMASEWCLVPVFDHVSNPNWF GDC*LWNRPAC*FWNWFENWSED*FAN*FGN*FGN*FGNCWP*NCCCWLMVVSSLLG*SPSHHRQNL SHTIV*GPPW*GTSASCV*PSNTF*KCL*EMRARISGVGGSVGSI*NCPFC*LGIISTGSGLSSKYRSMLGMKIVSLLEVSM*SLSSI*WGFSSNRSSSKES*AVAGTPPLLCRDSEPSSIRSPRLRRPKASIRRARLTFDCSSRDRVRLSRVPSIRIGFLSIFRAATEICLGL*LYWSKLLQADSA*LMHEPERLDVFTRWFEGISGVFLRL*

[0135] SEQ ID NO.2

[0136] MDLELEIPVLHSMDSHHQVVDSHRLAQQQFQYQQIHMLQQTLSQQYPHTPSTTPPIYMLSPADYEKDAVSISPVMLWPPSAHSQASYHYEMPSVISPSPSPTRSFCNPRELEVQDELEQLEQQPAALSVEHLFDIENSSIEYAHDELHDTSSCSDSQSSFSPQQSPASPASTYSPLEDEFLNLAGSELKSEPSADDEKDDVDTELPQQPEIIIPVSCRGRKPSIDDSKKTFVCTHCQRRFRRQEHLKRHFRSLHTREKPFNCDTCGKKFSRSDNLAQHMRTHPRD*

[0137] SEQ ID NO.3

[0138] *

[0139] SEQ ID NO.4

[0140] atgccctcctacgaagctcgagctaacgtccacaagtccgcctttgccgctcgagtgctcaagctcgtggcagccaagaaaaccaacctgtgtgcttctctggatgttaccaccaccaaggagctcattgagcttgccgataaggtcggaccttatgtgtgcatgatcaagacccatatcgacatcattgacgacttcacctacgccggcactgtgctccccctcaaggaacttgctcttaagcacggtttcttcctgttcgaggacagaaagttcgcagatattggcaacactgtcaagcaccagtacaagaacggtgtctaccgaatcgccgagtggtccgatatcaccaacgcccacggtgtacccggaaccggaatcattgctggcctgcgagctggtgccgaggaaactgtctctgaacagaagaaggaggacgtctctgactacgagaactcccagtacaaggagttcctggtcccctctcccaacgagaagctggccagaggtctgctcatgctggccgagctgtcttgcaagggctctctggccactggcgagtactccaagcagaccattgagcttgcccgatccgaccccgagtttgtggttggcttcattgcccagaaccgacctaagggcgactctgaggactggcttattctgacccccggggtgggtcttgacgacaagggagacgctctcggacagcagtaccgaactgttgaggatgtcatgtctaccggaacggatatcataattgtcggccgaggtctgtacggccagaaccgagatcctattgaggaggccaagcgataccagaaggctggctgggaggcttaccagaagattaactgttag

[0141] SEQ ID NO.5

[0142]

[0143] SEQ ID NO.6 Artificially synthesized BEM1_Up_F CCACCTGACGTCTTAATTAATCCATATACTCACGCAACTA SEQ ID NO.7 Artificially synthesized BEM1_Up_R

[0144] CCACAGAACACACCCCGGTACGACTGGCACTTTCGGGCTG

[0145] SEQ ID NO.8 Artificially synthesized BEM1_Dn_F

[0146] CACTGCACTACCACTACACCGGTAGATCTGGGTTATCTGG

[0147] SEQ ID NO.9 Artificially synthesized BEM1_Dn_R

[0148] TTTTGCAACTGGGGCATATGCCTTCAAGCTTGGGTCATCT

[0149] SEQ ID NO.10BEM1_Up

[0150]

[0151]

[0152] SEQ ID NO.12BEM1_testUp_F is artificially synthesized.

[0153] GGTGGGTTAAATTGCAGCCG

[0154] SEQ ID NO.13BEM1_testUp_R is artificially synthesized.

[0155] GAAGGGGTAAATGAGGAGTG

[0156] SEQ ID NO.14BEM1_testDn_F artificially synthesized

[0157] GCAACAGAAATCTGTCTCGg

[0158] SEQ ID NO.15BEM1_testDn_R is artificially synthesized.

[0159] GTGCGGTTAGAAGACGCAGA

[0160] SEQ ID NO.16MHY1_Up_F is artificially synthesized.

[0161] AGTGCCACCTGACGTCTTAATATTATGTCGTCGAAAAAGG

[0162] SEQ ID NO.17MHY1_Up_R is artificially synthesized.

[0163] CCACAGAACACACCCCGGTATTTGGCGATAATGTACTAT

[0164] SEQ ID NO.18MHY1_Dn_F artificially synthesized

[0165] CACTGCACTACCACTACACCAGGTAGAGCGCCTTCTAGTC

[0166] SEQ ID NO.19MHY1_Dn_R artificially synthesized

[0167] CAACTGGGGCATATGTTAAGCGACGGGAGGGGTCTACAAG

[0168] SEQ ID NO.20MHY1_Up

[0169] Tattatgtcgtcgaaaaaggaagtcggttttgagtcttgggagtggcagccgaattagattggtacagtagagccgagtccggggagtggcaggtgtcagcgaaagctcaaaggacgagaggggacccctgagcagacgggcggctaggtctcgacagatgaggtgcgcttctctcgtctcggtaaccctgtcaggacgtccagacactcttgccagcttgcaccacctcccttcgtggtttatgacgaaacacgcttagctaaagttctaaaggctaatattacgaactctgtgaaaaggcgaaaaaccacaaaagggacgtgtggtgttgaaaaggtgaaaaagagaaaacgaaaagtctggccgaagtcaatggtgccaaacccagagaaaactctatccaaactctgtccaaactctgttgctcatgtctgtctcactatcagtcgtaaacgtaagcgggtgttacggttagcacagcttcatgggacaagtacaaaagcaccacatatccctctctccagcttttttttctggtaggcaaaaactatcttggggtccgaccagcttcctcaccctcaccctcacactcaccagcactccccccttcaaccgctccaccaactaccctttctaaccacgctcttttactaaacacacacatctctggctgcgccaaaaagtgtccactcttccctccactcttattcctttccagtcactcctttgcctgttatttttgttgataccccttcgagtcattcaacatctttcccaaccacttctttcaaccgtcccacacccctaccacgctttccccacgaccaccacaacttgtcaccactaactttggcccattcattaaactcttgtcggccgttataatacttctttgtgtgtcgcatcgcatctaatcaggaaaacccaaacccggattagccaatttcgccaaaaccatcccaatcgaaaaacaaaaagtcatagtacattatcgccaaa。

[0170] SEQ ID NO.21MHY1_Dn

[0171] Aggtagagcgccttctagtctccgctccatttttttattgtaaccagttaatagagagtttttgaatgaatgttttaatttaatgtctgctttgagagtgtctattccttgtattccgtcctaagtatgtcgccttgttctcagtaaaccaaatctacgttagccatacctgacatcggcacgatcccttcagatcgacatgccaaaattccggataaggactttctgattgcgtgacagagtttcgaatctggctgtggatcggcgcagctggggaaacgacttttagttatacgcaacacacggcactgttggtgacgagtggttaacctgttctaggatgagtactttccggtacaagtagtggaagagaaattgagggtaaaaaaagagacgtagaagccaatgatgcagcatcttcatacacactaaatctcctgcagtcgcttgtccactttgtaagctagtcgcttgtacctacctgggacaggtgtactgtacaccgtgctattactcagctccaattggataccgaatgtttcccgcgcccagatacggcagctaaatcttacgtctcttattgtgactgtctggagaaatacccccggcagtgtgtgtgtatgcggtctccaagacctaaaagttaaagtgggtatgccaaccttagagcagccccgaaatacatacatgaggtttcgtactcgcctagtgaaacacaaaaacgacgcttgaaaccgatggctgcctatgggagagagacagagctaagcccggaccccggactttctgcctggtttggcagtgtagattcacgtatgtccagtctagatggtcgcctgtcgagcccgtcacgccatgccgcctattttctttttcccttccagaatttgactaaggattaacacgtcgatgtctcacaatcgagaaaagtgaagagcccaggacgagacgtcataagagcagctgtggcccgtacaagcttgtagacccctcccgtcgc。

[0172] SEQ ID NO.22MHY1_testUp_F artificially synthesized

[0173] GGCTCTTCAGTCTCATCTGG

[0174] SEQ ID NO.23MHY1_testUp_R is artificially synthesized.

[0175] CCACAGCATTACCGGTGAGA

[0176] SEQ ID NO.24MHY1_testDn_F artificially synthesized

[0177] CGAGATCATCATCCCTGTGT

[0178] SEQ ID NO.25MHY1_testDn_R is artificially synthesized.

[0179] TGGACCCTGCTTGGCAGATC

[0180] SEQ ID NO.26CLA4_Up_F is artificially synthesized.

[0181] CCACCTGACGTCTTAATTAAGAAAACTTGGTTAGGGAGAG

[0182] SEQ ID NO.27CLA4_Up_R is artificially synthesized.

[0183] CCACAGAACACACCCCGGTAAGTCGGGATGTGCAATTAAC

[0184] SEQ ID NO.28CLA4_Dn_F artificially synthesized

[0185] CACTGCACTACCACTACACCGACATGGGTACGGTATAGGT

[0186] SEQ ID NO.29CLA4_Dn_R is artificially synthesized.

[0187] TTTTGCAACTGGGGCATATGCAAGATCCCCGGCTTTGTGC

[0188] SEQ ID NO.30CLA4_Up

[0189]

[0190] SEQ ID NO.31CLA4_Dn

[0191]

[0192] SEQ ID NO.32CLA4_testUp_F artificially synthesized

[0193] AGCCCTTGATACACAGCCCA

[0194] SEQ ID NO.33CLA4_testUp_R is artificially synthesized.

[0195] AGAGCAGCAGTTGTagcagc

[0196] SEQ ID NO.34CLA4_testDn_F artificially synthesized

[0197] GATAACAACAGTCTGAGTGA

[0198] SEQ ID NO.35CLA4_testDn_R is artificially synthesized.

[0199] ATCTCCACTGCTCCCTGGTC

[0200] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for improving the cell wall disruption rate of *Yersinia lipolytica* using recombinant strains, characterized in that: Includes the following steps: The method is a biological enzymatic method, in which the fermentation broth of *Yarrowia lipolyticis* is centrifuged at 10,000 rpm to precipitate the bacterial cells, and then subjected to the following processing steps: Adjust the bacterial cell concentration to 10-14% by volume with water, and then allow it to autolyze at room temperature and natural pH for 1.5-2 hours. Adjust the pH to 5.5-6.5, adjust the temperature to 54-56℃, add 0.5% lysozyme, stir and hydrolyze for 18-24 hours, then heat to 85-95℃ to inactivate the enzyme for 10-15 minutes, and then centrifuge at 10000rpm to collect the precipitate. The recombinant strain of *Yersinia lipolytica* was constructed by genetically modifying the *Yersinia lipolytica* strain by knocking out the cell wall formation-related gene. The cell wall formation-related gene is BEM1, which encodes actin cytoskeleton-related protein, and its amino acid sequence is shown in SEQ ID NO.1; Alternatively, the cell wall formation-related gene is a C2H2 type zinc finger protease MHY1 that is related to cell morphology transformation, and its amino acid sequence is shown in SEQ ID NO.2; Alternatively, the cell wall formation-related gene is a positive gene effector CLA4 that encodes hyphae formation and influences cell size, and its amino acid sequence is shown in SEQ ID NO.3; The genetic modification method is homologous recombination or CRISPR-Cas9 gene editing; The starting strain of the *Yarrowia lipolytica* strain is *Yarrowia lipolytica* po1f; The method for constructing the recombinant *Yarrowia lipophila* strain as described above includes the following steps: Gene knockout was performed using Yersinia lipophila po1f as the starting strain. The gene knockout process includes: first, constructing a CRISPR-Cas9 gene knockout recombinant vector pCRISPRyl-ura1-BEM1, pCRISPRyl-ura1-MHY1, or pCRISPRyl-ura1-CLA4 containing the BEM1, MHY1, or CLA4 gene, and then transforming it into competent cells of Yersinia lipolytica po1f. The uracil selection marker URA in the recombinant vector pCRISPRyl-ura1 replaces the BEM1, MHY1, or CLA4 gene in Yersinia lipolytica po1f. Subsequently, positive recombinant strains are screened using YNB solid medium. The nucleotide sequence of the uracil selection marker URA is shown in SEQ ID NO.

4.

2. The method according to claim 1, characterized in that: The method for preparing the fermentation broth of the lipophilic yeast includes the following steps: A recombinant strain was constructed by knocking out the cell wall formation-related gene of the *Yersinia lipolytica* strain through genetic modification. The recombinant strain was then inoculated into a fermentation medium for fermentation to obtain the fermentation broth of *Yersinia lipolytica*.

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