Yarrowia lipolytica yl590 and use thereof

Abstract

Yarrowia lipolytica Yl590 and the use thereof. The Yarrowia lipolytica Yl590, taxonomically classified as Yarrowia lipolytica, was deposited with the China General Microbiological Culture Collection Center on 4 June 2024, with a deposit address of Beijing, China and a deposit number of CGMCC No. 30855. The present invention can solve the problem of the slow growth rate of Yarrowia lipolytica in the prior art, and is suitable for the technical field of microorganisms.

Description

Yarrowia lipolytica Y1590 and application thereof

[0001] This application is based on and claims priority to Chinese application No. 202411372939.0, filed on September 29, 2024, the disclosure of which is incorporated herein in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of microbial technology, in particular, to a Yarrowia lipolytica Y1590 and application thereof. BACKGROUND

[0003] Yarrowia lipolytica is a microorganism with important industrial application potential, which has been certified by the US Food and Drug Administration (FDA) as "Generally Recognized as Safe" (GRAS). This yeast is known for its ability to synthesize a variety of valuable metabolites, but its growth rate and tolerance in actual fermentation processes limit the improvement of yield.

[0004] Current technology has proven that geraniol can be synthesized from scratch by Saccharomyces cerevisiae and Yarrowia lipolytica, and some engineered strains have shown high production efficiency. For example, the engineered strain in patent application CN114989997A can achieve a yield of 919 mg / L in 5 days; while the strain in patent application CN117264792A can achieve a yield of up to 8510 mg / L in 8 days in a 5L fermenter. Increasing the tolerance of the strain to enable it to proliferate continuously under harsh conditions such as high concentration of glucose will further increase the yield. At the same time, accelerating the growth rate of the strain can shorten the fermentation period, thereby improving the fermentation yield.

[0005] Therefore, in terms of industrial application and strain modification, it is crucial to screen a chassis strain with fast growth, strong tolerance and heritable modification ability. This not only helps to shorten the fermentation period and reduce production costs, but also has important significance for realizing the efficient synthesis of microbial cell factories. Currently, there is no report showing that any Yarrowia lipolytica has a significant improvement in growth rate, which indicates that this field still needs further research and development.

[0006] SUMMARY

[0007] The main purpose of the present application is to provide a Yarrowia lipolytica Y1590 and application thereof, to solve the problem of slow growth of Yarrowia lipolytica in the prior art.

[0008] In order to achieve the above-mentioned purpose, according to a first aspect of the present application, there is provided a Yarrowia lipolytica Yl590, classified as Yarrowia lipolytica, which was preserved in the China General Microbiological Culture Collection Center on June 4, 2024, located in Beijing, China, and has a preservation number of CGMCC No. 30855.

[0009] Further, the Yarrowia lipolytica Yl590 contains the nucleic acid sequence shown in SEQ ID NO: 1.

[0010] Further, the Yarrowia lipolytica Yl590 has the abilities of fast growth, multi-carbon source utilization, and fast carbon source consumption; preferably, the fast growth includes that, under the same conditions, the Yarrowia lipolytica Yl590 and the Yarrowia lipolytica Po1f with the same initial cell concentration are proliferated to a specific cell concentration, and the required time of the Yarrowia lipolytica Yl590 is 30%-90% of that of the Yarrowia lipolytica Po1f; preferably, the carbon source is independently selected from any one or more of the following: ethanol, methanol, xylose, glycerol, sodium acetate, malonic acid, galactose, sucrose, or glucose; more preferably, the carbon source is glucose.

[0011] Further, the Yarrowia lipolytica Yl590 has the ability to resist any one or more of the following conditions: high-concentration glucose, low pH, and high-concentration sodium chloride; the concentration of the high-concentration glucose is 100-600 g / L; preferably, the concentration of the high-concentration glucose is 100 g / L or 200 g / L; the concentration of the high-concentration sodium chloride is 50-100 g / L; preferably, the concentration of the high-concentration sodium chloride is 50 g / L, 60 g / L, or 70 g / L; the pH value of the low pH is 2-6; preferably, the pH value is 3, 3.5, 4, 5, or 6.

[0012] In order to achieve the above-mentioned purpose, according to a second aspect of the present application, there is provided an engineered strain for producing sclareol, wherein the starting strain of the engineered strain is the Yarrowia lipolytica Yl590 described above, and the pyrophosphate lycophellandrenediol ester synthase gene CLS, the sclareol synthase gene TP1132, and the geranylgeranyl pyrophosphate synthase gene CrtE are inserted into the genome of the engineered strain by a gene editing method.

[0013] Further, the protein expressed by the pyrophosphate lycophellandrenediol ester synthase gene CLS has the amino acid sequence shown in SEQ ID NO: 4; the protein expressed by the sclareol synthase gene TP1132 has the amino acid sequence shown in SEQ ID NO: 5; and the protein expressed by the geranylgeranyl pyrophosphate synthase gene CrtE has the amino acid sequence shown in SEQ ID NO: 6.

[0014] Further, the Ku70, URA3 and LEU2 genes in the genome of the engineered strain are knocked out by a gene editing method; preferably, the gene editing method comprises inserting, deleting or replacing sequences of the chromosome of Yarrowia lipolytica Y1590 by using CRISPR, homologous recombination, non-homologous end joining or Cre / lox system.

[0015] To achieve the above-mentioned object, according to a third aspect of the present application, a method for producing sclareol is provided, which comprises: preparing seed liquid and fermentation culture by using the above-mentioned engineered strain producing sclareol as the fermentation strain, and obtaining sclareol from the fermentation broth and / or the bacterial cells.

[0016] Further, the method for preparing seed liquid comprises: inoculating the engineered strain producing sclareol into seed culture medium, and culturing at 28-32℃ and 150-250rpm for 16-36h; preferably, the seed culture medium is YPD liquid medium.

[0017] Further, the method for fermentation culture comprises: inoculating the seed liquid containing the engineered strain producing sclareol into fermentation culture medium, obtaining initial OD 600 of 0.01-0.04; and fermenting at 28-32℃ and 150-280rpm for 36-72h to obtain fermentation broth and / or bacterial cells; preferably, the fermentation culture medium is YPD liquid medium.

[0018] Further, the method for obtaining sclareol from fermentation broth and / or bacterial cells comprises: mixing the fermentation broth and / or bacterial cells with an organic reagent selected from any one or more of the following: n-dodecane, anhydrous ethanol, methanol, isopropanol, acetone, n-heptane, n-hexane or acetonitrile; preferably, the organic reagent is n-dodecane.

[0019] By applying the technical solution of the present application, a Yarrowia lipolytica Y1590 strain is screened and obtained, which has the following advantages: fast growth, efficient utilization of substrate, strong tolerance, and can be genetically manipulated. This not only can save the time required for culture and fermentation, but also can be used as a chassis strain for further application in synthesis of different products. BRIEF DESCRIPTION OF DRAWINGS

[0020] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description of the exemplary embodiments of the present application and the explanation thereof serve to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0021] FIG. 1 shows the phylogenetic tree of 16S rDNA gene of the strain Y1590 according to the embodiment 1 of the present application and the standard strain of Yarrowia lipolytica.

[0022] Figure 2 shows the Yl590 and Po1f strain plate culture result graph according to embodiment 1 of the present application, wherein Figure 2A shows the results of Yl590 and Po1f strain plate culture for 24h, and Figure 2B shows the results of Yl590 and Po1f strain plate culture for 48h.

[0023] Figure 3 shows the microscope morphology graph of Yl590 strain according to embodiment 1 of the present application.

[0024] Figure 4 shows the carbon source utilization result graph of Yl590 and Po1f strains according to embodiment 2 of the present application.

[0025] Figure 5 shows the tolerance result graph of Yl590 and Po1f strains according to embodiment 3 of the present application.

[0026] Figure 6 shows the growth curve result graph of Yl590 and Po1f strains according to embodiment 3 of the present application.

[0027] Figure 7 shows the fermentation result graph of Yl590 strain producing sclareol according to embodiment 5 of the present application.

[0028] Biological material information: a Yarrowia lipolytica Yl590, classified as Yarrowia lipolytica, was preserved in the China General Microbiological Culture Collection Center on June 4, 2024, and the address of the preservation unit is No. 3, Institute of Microbiology, Chinese Academy of Sciences, 1 North Chengxian Road, Chaoyang District, Beijing, China, 100101, and the preservation number is CGMCC No. 30855. DETAILED DESCRIPTION

[0029] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0030] As mentioned in the background, Yarrowia lipolytica is a commonly used fermentation chassis strain in the prior art, but the growth speed of Yarrowia lipolytica in the prior art limits its fermentation capacity in industry. In the present application, the inventors found a Yarrowia lipolytica Yl590 with faster growth speed, and on this basis, a series of protection schemes of the present application are proposed.

[0031] In a first typical embodiment of the present application, a Yarrowia lipolytica Yl590 is provided, which is classified as Yarrowia lipolytica and was preserved in the China General Microbiological Culture Collection Center on June 4, 2024, and the address of the preservation unit is Beijing, China, and the preservation number is CGMCC No. 30855.

[0032] The growth speed of the Yarrowia lipolytica Y1590 is faster than other Yarrowia lipolytica in the prior art, and the Yarrowia lipolytica Y1590 has a fast growth ability, can quickly consume carbon sources, and can shorten the growth cycle, thereby shortening the time required for culture and fermentation.

[0033] The Yarrowia lipolytica Y1590 has strong tolerance, and can tolerate lower pH, higher salt (such as NaCl) concentration, and higher substrate (such as glucose) concentration compared to other Yarrowia lipolytica in the prior art. Such tolerance facilitates increasing the concentration of the Yarrowia lipolytica Y1590 in fermentation, and can reduce the risk of contamination by other bacteria. The molecular biology methods (including but not limited to genetic modification methods) of Yarrowia lipolytica in the prior art are also applicable to the Yarrowia lipolytica Y1590 in the present application, that is, the Yarrowia lipolytica Y1590 can be conveniently genetically modified and the like, and has a high application prospect.

[0034] In a preferred embodiment, the Yarrowia lipolytica Y1590 contains the nucleic acid sequence shown in SEQ ID NO: 1.

[0035] SEQ ID NO: 1:

[0036] In a preferred embodiment, the Yarrowia lipolytica Y1590 has the ability of fast growth, multi-carbon source utilization, and fast consumption of the carbon source.

[0037] In a preferred embodiment, the fast growth includes that the Yarrowia lipolytica Y1590 and Yarrowia lipolytica Po1f with the same initial cell concentration are proliferated to a specific cell concentration under the same conditions, and the required time of the Yarrowia lipolytica Y1590 is 30%-90% of that of the Yarrowia lipolytica Po1f, including but not limited to 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0038] In a preferred embodiment, the carbon source is independently selected from any one or more of the following: ethanol, methanol, xylose, glycerol, sodium acetate, malonic acid, galactose, sucrose, or glucose; more preferably, the carbon source is glucose.

[0039] In a preferred embodiment, the Yarrowia lipolytica Y1590 has the ability to tolerate any one or more of the following conditions: high glucose concentration, low pH, and high sodium chloride concentration; the high glucose concentration is 100-600 g / L (including but not limited to 100, 125, 150, 175, 200, 250, 300, 400, 500, or 600 g / L); preferably, the high glucose concentration is 100 g / L or 200 g / L; the high sodium chloride concentration is 50-100 g / L (including but not limited to 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / L); preferably, the high sodium chloride concentration is 50 g / L, 60 g / L, or 70 g / L; the low pH has a pH value of 2-6 (including but not limited to 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6); preferably, the pH value is 3, 3.5, 4, 5, or 6.

[0040] The Yarrowia lipolytica Y1590 described above has the characteristics of fast growth and fast substrate utilization, and in different stages of culture and proliferation, the time required by this culture method is less than that of other Yarrowia lipolytica in the prior art, which facilitates the saving of the time required in the entire culture period, such as faster reaching of the target OD 600 in the fermentation stage, and reduces the time required for fermentation.

[0041] The Yarrowia lipolytica Y1590 described above can utilize various carbon sources, and those skilled in the art can select carbon sources according to actual conditions and needs, including but not limited to using carbon sources as energy sources and / or using carbon sources as precursors of target metabolites in engineering bacteria. In different carbon sources, the growth rate of the Yarrowia lipolytica Y1590 is faster than that of the Yarrowia lipolytica in the prior art.

[0042] The Yarrowia lipolytica Y1590 described above can also tolerate higher osmotic pressure conditions and lower pH conditions, including but not limited to proliferation in higher concentrations of glucose and / or sodium chloride and lower pH. Such ability can enable the Yarrowia lipolytica Y1590 to be faster and higher-density expanded and fermented in high-concentration substrates, and in such culture environment, other miscellaneous bacteria are difficult to survive or compete with the Yarrowia lipolytica Y1590, reducing the risk of contamination by miscellaneous bacteria and reducing the need for sterile conditions for fermentation.

[0043] In a second typical embodiment of the present application, an engineered strain producing sclareol is provided, and the starting strain of the engineered strain is the Yarrowia lipolytica Y1590 described above, and the pyrophosphate lysadecadienol synthase gene CLS, the sclareol synthase gene TP1132, and the geranylgeranyl pyrophosphate synthase gene CrtE are inserted into the genome of the engineered strain by gene editing methods;

[0044] In a preferred embodiment, the Ku70, URA3 and LEU2 genes in the genome of the engineered strain are knocked out by gene editing methods; preferably, the gene editing methods comprise insertion, deletion or substitution of sequences in the chromosome of Y. lipolytica Y1590 using CRISPR, homologous recombination, non-homologous end joining or Cre / lox system.

[0045] Preferably, the engineered strain is Y. lipolytica Y1590AKu70(URA3 - ), siteC6::CrtE, siteE16::CLS, siteB4::TP1132, ALLEU2.

[0046] In the engineered strain, the amino acid sequence expressed by the pyrophosphorly-lycoballene diol synthase gene CLS is SEQ ID NO: 4, the amino acid sequence expressed by the trichocarol synthase gene TP1132 is SEQ ID NO: 5, and the amino acid sequence expressed by the geranylgeranyl pyrophosphate synthase gene CrtE is SEQ ID NO: 6. Those skilled in the art can also flexibly modify or replace the genes and the expressed proteins according to the prior art.

[0047] Trichocarol is a diterpene compound with a labdane skeleton, mainly used as a raw material in the perfume industry. Trichocarol can be used as a raw material for synthesizing ambergris chemical agents such as ambrox, norambrox, methylambrox, etc. In addition, trichocarol is also a potential anti-tumor drug, which has cytotoxicity to various human cancer cells such as leukemia cells, tumor cells and colon cancer cells.

[0048] The methods for producing trichocarol include extraction from plants, biofermentation, etc., among which constructing microbial cell factories is a promising way to efficiently synthesize trichocarol and other high-value terpenoids. Traditionally, trichocarol is obtained by extraction from plant extracts of trichocarpus flower spikes. However, due to the influence of environmental and climatic factors in the planting area, the plant extraction method is limited. In contrast, the use of microbial fermentation has the advantages of short growth cycle, all-weather production and stable yield, which can meet the growing market demand. It is crucial to select a suitable microorganism for the successful implementation of the biological process, which requires consideration of its production capacity, substrate utilization rate and tolerance, and mastery of relevant technologies to achieve strain modification. Using the above Y. lipolytica Y1590 as a starting strain, by introducing exogenous genes, an engineered strain producing trichocarol can be obtained.

[0049] In a third typical embodiment of the present application, a method for producing sclareol is provided, which comprises: preparing seed liquid and fermentation culture by using the above-mentioned engineered strain producing sclareol as the fermentation strain, and obtaining sclareol from the fermentation broth and / or the bacterial cells.

[0050] In a preferred embodiment, the method for preparing seed liquid comprises: inoculating the engineered strain producing sclareol into seed culture medium, culturing at 28-32°C (including but not limited to 28, 29, 30, 31 or 32°C), 150-250rpm (including but not limited to 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250rpm) for 16-36h (including but not limited to 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 22, 25, 28, 30, 32, 34 or 36h); preferably, the seed culture medium is YPD liquid medium.

[0051] In the prior art, the conventional Yarrowia lipolytica needs to be cultured for at least 20h in the process of inoculation and proliferation to obtain seed culture medium, while the Yarrowia lipolytica Y1590 of the present application has a fast proliferation speed and can obtain seed culture medium with suitable concentration and activity after 16h of culture.

[0052] In a preferred embodiment, the method for fermentation culture comprises: inoculating seed liquid containing the engineered strain producing sclareol into fermentation culture medium to obtain initial OD 600 The initial fermentation bacterial liquid is 0.01-0.04 (including but not limited to 0.01, 0.015, 0.02, 0.025, 0.03, 0.035 or 0.04); the fermentation is carried out at 28-32°C (including but not limited to 28, 29, 30, 31 or 32°C), 150-280rpm (including but not limited to 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 280rpm) for 36-72h (including but not limited to 36, 42, 48, 54, 60, 66 or 72h) to obtain fermentation broth and / or bacterial cells; preferably, the fermentation culture medium is YPD liquid medium.

[0053] In a preferred embodiment, the method for obtaining sclareol from fermentation broth and / or bacterial cells comprises: mixing the fermentation broth and / or bacterial cells with organic reagent, which includes but is not limited to any one or more of the following: n-dodecane, anhydrous ethanol, methanol, isopropanol, acetone, n-heptane, n-hexane or acetonitrile; preferably, the organic reagent is n-dodecane.

[0054] The beneficial effects of the present application will be further explained in detail below with specific embodiments.

[0055] Example 1 Screening and identification of Yarrowia lipolytica Y1590

[0056] 1. Sampling and separation:

[0057] Dilution method was used to isolate Yarrowia lipolytica. 9 g of dairy products purchased from different regions of Inner Mongolia Autonomous Region were weighed and placed in a 250 mL conical flask containing 90 mL of sterile water. The mixture was mixed in a shaker at 28°C and 200 rpm for 30 min, then diluted 10 and 100 times with sterile water, respectively, and spread on LB solid medium containing tributyrin. The plates were incubated in a 30°C incubator until colonies grew.

[0058] The composition of LB solid medium containing tributyrin is as follows: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, tributyrin 2 mL, and agar powder 20 g / L.

[0059] 2. Strain purification:

[0060] Among the final colonies, the sample with round, smooth, flat and light yellow colony morphology was selected for three-zone streak separation on YPD solid medium, and incubated at 30°C for 24 h to obtain purified monoclonal. The strain was activated in YPD liquid medium and incubated at 30°C for 20 h, then preserved and stored in a -80°C refrigerator.

[0061] The composition of YPD solid medium is as follows: yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L, and agar powder 20 g / L. The glucose needs to be sterilized separately before adding.

[0062] The composition of YPD liquid medium is as follows: yeast powder 10 g / L, peptone 20 g / L, and glucose 20 g / L. The glucose needs to be sterilized separately before adding.

[0063] 3. Identification of Yarrowia lipolytica:

[0064] The 26S ribosomal RNA gene of Yarrowia lipolytica was amplified by PCR. The primer sequences are shown in Table 1.

[0065] Table 1

[0066] The 26S ribosomal RNA gene sequence of the strain was obtained by gene amplification, and the nucleotide sequence was sequenced by Anshengda Company. The strain information was obtained by NCBI website (https: / / www.ncbi.nlm.nih.gov / ) BLAST, and the results showed that the strain Yl590 had the highest homology with Yarrowia lipolytica WSYC 781 (FR856618.1) with 99% consistency. The 26S ribosomal RNA sequence with high similarity to the sequence was selected to construct a phylogenetic tree, as shown in Figure 1. According to the above results, Yl590 belongs to Yarrowia lipolytica.

[0067] The 26S ribosomal RNA gene sequence of the strain Yl590 is the sequence shown in SEQ ID NO: 1.

[0068] SEQ ID NO: 1:

[0069] The comparison of plate culture of strain Yl590 and Yarrowia lipolytica standard strain Po1f for 24h and 48h is shown in Figure 2A and Figure 2B. The left side of the figure is Po1f plate, and the right side is Yl590 plate.

[0070] The microscope morphology of strain Yl590 (×100) is shown in Figure 3.

[0071] Example 2 Growth and substrate utilization of Yarrowia lipolytica Yl590

[0072] Yarrowia lipolytica Yl590 and control Po1f strain were activated into test tubes containing 5mL YPD liquid, and cultured at 30℃, 220rpm for 16h.

[0073] The cultured bacterial liquid was inoculated into 50mL YPD liquid flask at initial OD 600 =0.2, and cultured at 30℃, 220rpm. Samples at different times 0h, 5h, 23h, 29h, 47h, 53h, 71h were taken to detect OD 600 value and substrate consumption to compare the growth state of the strains.

[0074] The growth and carbon source consumption are shown in Figure 4. During the whole growth cycle, the OD 600The growth rate of Y1590 strain was higher than that of the control PoIf strain, with the highest being 3 times, indicating that the growth rate of Y1590 strain was faster. In addition, the glucose consumption of Y1590 strain was close to complete at 47 h, while the glucose of PoIf strain was still remaining at 72 h, indicating that the substrate utilization rate of Y1590 strain was faster.

[0075] Example 3 Performance analysis of Yarrowia lipolytica Y1590

[0076] 1. Substrate carbon source utilization analysis

[0077] In each well of a 96-well plate, different substrate carbon sources were added, 5 g / L ethanol, 5 g / L methanol, 5 g / L xylose, 5 g / L glycerol, 5 g / L sodium acetate, 5 g / L malonic acid, 5 g / L galactose, 5 g / L sucrose, 5 g / L glucose, and the strain PoIf (TasteBiotech, TS153400) was used as a control, with a total volume of 500 μL, 30°C, 700 rpm overnight culture.

[0078] The formula of SD medium: yeast basic nitrogen source (YNB) 1.7 g / L, ammonium sulfate 5 g / L, L-arginine 0.1 g / L, L-cysteine 0.1 g / L, L-lysine 0.1 g / L, L-threonine 0.1 g / L, L-asparagine 0.05 g / L, L-isoleucine 0.05 g / L, L-phenylalanine 0.05 g / L, L-proline 0.05 g / L, L-serine 0.05 g / L, L-tyrosine 0.05 g / L, L-valine 0.05 g / L, L-methionine 0.05 g / L, L-tryptophan 0.1 g / L, L-histidine 0.1 g / L, L-leucine 0.1 g / L, uracil 0.1 g / L, adenine 0.1 g / L, pH 7.0 ± 0.2.

[0079] Yeast basic nitrogen source (YNB) components: inositol 2.0 mg / L, nicotinic acid (vitamin B3) 0.4 mg / L, thiamine hydrochloride (vitamin B1) 0.4 mg / L, copper sulfate 0.04 mg / L, potassium dihydrogen phosphate 1000 mg / L, boric acid 0.5 mg / L, pyridoxal hydrochloride (vitamin B6) 0.4 mg / L, calcium pantothenate (vitamin B5) 0.4 mg / L, p-aminobenzoic acid 0.2 mg / L, magnesium sulfate 500 mg / L, manganese sulfate 0.4 mg / L, zinc sulfate 0.4 mg / L, ferric chloride 0.2 mg / L, riboflavin (vitamin B2) 0.2 mg / L, calcium chloride 100 mg / L, potassium iodide 0.1 mg / L, sodium molybdate 0.2 mg / L, biotin (vitamin B7, H) 0.002 mg / L, sodium chloride 100 mg / L.

[0080] The results are shown in Figure 5. Y1590 strain can utilize multiple carbon sources, and the obvious ability to utilize ethanol, glycerol, sodium acetate, malonic acid, galactose or glucose is superior to Polf, and the growth of Y1590 strain is best when glycerol or glucose is used as carbon source.

[0081] 2. Tolerance analysis

[0082] In each well of a 96-well plate, different glucose concentrations (100, 200, 300, 400, 500, 600 g / L), pH values (2, 3, 3.5, 4, 5, 6), and sodium chloride concentrations (50, 60, 70, 80, 100 g / L) were added to the SD medium, and strain Polf (TasteBiotech, TS153400) was used as a control, with a total volume of 500 μL, 30°C, 700 rpm overnight culture. The results are shown in Figure 6. Under the conditions of glucose concentration 100, 200 g / L or pH 3, 4, 5, 6 or sodium chloride concentration 50, 60, 70 g / L, the growth of Y1590 strain is significantly better than that of Polf.

[0083] Example 4 Gene editing ability of Yarrowia lipolytica Y1590

[0084] Yarrowia lipolytica Y1590 was subjected to gene editing. According to the prior art, URA3 and LEU2 genes of Y1590 strain were knocked out by CRISPR technology to facilitate gene editing, and Ku70 gene was knocked out to enhance its homologous recombination ability. Three heterologous genes that can synthesize sclerol, pyrophosphate lybadiene diol synthase gene CLS (amino acid sequence SEQ ID NO: 4), sclerol synthase gene TP1132 (amino acid sequence SEQ ID NO: 5) and geranylgeranyl pyrophosphate synthase gene CrtE (amino acid sequence SEQ ID NO: 6) were integrated into the genome of Yarrowia lipolytica by homologous recombination technology. As a result, Yarrowia lipolytica Y1590 correctly knocked out genes and overexpressed genes, indicating that the strain can be subjected to gene editing operation and can be used as a chassis strain for modification.

[0085] SEQ ID NO: 4

[0086] SEQ ID NO: 5

[0087] SEQ ID NO: 6

[0088] The specific steps are as follows:

[0089] Preparation of competent cells: pick activated single colony from YPD solid medium plate into test tube, incubate at 30°C for 24h; centrifuge at 5000rpm to collect bacterial cells, resuspend in 1 mL 1x TE Buffer, centrifuge again to collect bacterial cells, resuspend in 1 mL 0.1 mol / L LiAC (OD 600 ~4), incubate at 30°C for 1h; centrifuge at 5000rpm for 1 min to collect bacterial cells, resuspend in appropriate amount of 0.1 mol / L LiAC, which is the competent cells.

[0090] Construction of engineering bacteria: add 15 μL salmon sperm DNA (which needs to be heated and then cooled), 1-5 μg DNA to be transformed or 2 μg plasmid into 1.5 mL centrifuge tube, mix well, then add 40 μL prepared competent cells (OD 600 ~4), incubate at 30°C for 15 min; add 280 μL 50% PEG4000, 70 μL 0.5 mol / L LiAC and 16 μL 1 mol / L DTT into the mixture of previous step, mix well, incubate at 30°C for 1h; add 40 μL DMSO into the mixture of previous step, incubate at 39°C for 10 min, add 600 μL 0.1 mol / L LiAC, mix well, incubate at 30°C for 1h; centrifuge at 5000rpm for 2 min to collect bacterial cells, resuspend in water, then spread on medium containing corresponding antibiotic or auxotrophic medium, incubate at 30°C for 2-3 days until single colony grows, the Yl590 engineering bacteria is named Yl590△Ku70 (URA3 - ), siteC6::CrtE, siteE16::CLS, siteB4::TP1132, △LEU2. For comparison at the same level, the Po1f engineering bacteria is named Po1f△Ku70, siteB1::CLS, siteC6::CrtE, siteB4::TP1132.

[0091] Example 5 Yarrowia lipolytica Yl590 shake flask level of squalene production

[0092] Seed medium: scrape one ring of Yl590△Ku70 (URA3 - ), siteC6::CrtE, siteE16::CLS, siteB4::TP1132, △LEU2 and Po1f△Ku70, siteB1::CLS, siteC6::CrtE, siteB4::TP1132 plate bacteria into test tube containing 5 mL YPD liquid medium, incubate at 30°C, 200 rpm shaker for 16h.

[0093] Fermentation medium: 50 mL of YPD medium (pH 6) was inoculated with 0.02 OD of seed liquid and incubated at 30℃, 220 rpm for 72-96 h. 600 Fermentation medium: 50 mL of YPD medium (pH 6) was inoculated with 0.02 OD of seed liquid and incubated at 30℃, 220 rpm for 72-96 h.

[0094] Sclareol detection: 1 mL of fermentation liquid was taken at 48 h of fermentation, added with 1 mL of n-dodecane and shaken for 1 h, and the supernatant was taken for gas chromatography detection. The result showed that the Yl590 produced 1.37 mg / L of sclareol, while the Po1f produced only 0.96 mg / L of sclareol. In addition, the OD of Yl590 was 1.7 times of that of Po1f, indicating that the growth and sugar (glucose) consumption speed were faster. The sclareol production, growth and sugar consumption of Yl590 strain in a shake flask are shown in Figure 7. 600 Sclareol detection: 1 mL of fermentation liquid was taken at 48 h of fermentation, added with 1 mL of n-dodecane and shaken for 1 h, and the supernatant was taken for gas chromatography detection. The result showed that the Yl590 produced 1.37 mg / L of sclareol, while the Po1f produced only 0.96 mg / L of sclareol. In addition, the OD of Yl590 was 1.7 times of that of Po1f, indicating that the growth and sugar (glucose) consumption speed were faster. The sclareol production, growth and sugar consumption of Yl590 strain in a shake flask are shown in Figure 7.

[0095] As can be seen from the above description, the above-mentioned embodiments of the present application achieve the following technical effects: the growth speed of the above-mentioned Yarrowia lipolytica Yl590 is faster than that of other Yarrowia lipolytica in the prior art, has fast growth ability and substrate consumption ability, can shorten the growth cycle, and thus shorten the time required for culture and fermentation. In addition, the Yarrowia lipolytica Yl590 has strong tolerance, can tolerate lower pH, higher salt (such as sodium chloride) concentration and higher substrate (such as glucose) concentration compared with other Yarrowia lipolytica in the prior art, which facilitates to increase the concentration of Yarrowia lipolytica Yl590 in fermentation and reduce the risk of contamination by other bacteria. In addition, the molecular biology methods (including but not limited to genetic modification methods) of Yarrowia lipolytica in the prior art are also applicable to the Yarrowia lipolytica Yl590 in the present application, that is, the Yarrowia lipolytica Yl590 can be conveniently subjected to genetic modification and other operations, and has high application prospect.

[0096] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Yarrowia lipolytica Y1590, classified as Yarrowia lipolytica, deposited with the China General Microbiological Culture Collection Center on June 4, 2024, located in Beijing, China, and assigned accession number CGMCC No. 30855.

2. The Yarrowia lipolytica Yl 590 of claim 1, wherein, The Yarrowia lipolytica Y1590 contains a nucleic acid sequence as shown in SEQ ID NO:

1.

3. An engineered strain producing sclareol, characterized in that, The starting strain of the engineered strain is the Yarrowia lipolytica Y1590 of claim 1 or 2, and the pyrophosphate lysadalenediol synthase gene CLS, the sclareol synthase gene TP1132, and the geranylgeranyl pyrophosphate synthase gene CrtE are inserted into the genome of the engineered strain by a gene editing method.

4. The engineered bacterial strain of claim 3, characterized in that, The Ku70, URA3, and LEU2 genes are knocked out from the genome of the engineered strain by the gene editing method.

5. The engineered bacterial strain of claim 3, characterized in that, The protein expressed by the pyrophosphate lysadalenediol synthase gene CLS has an amino acid sequence as shown in SEQ ID NO: 4; The protein expressed by the sclareol synthase gene TP1132 has an amino acid sequence as shown in SEQ ID NO: 5; The protein expressed by the geranylgeranyl pyrophosphate synthase gene CrtE has an amino acid sequence as shown in SEQ ID NO:

6.

6. The engineered strain of any one of claims 3-5, characterized in that, The gene editing method comprises inserting, deleting, or replacing sequences of the chromosome of the Yarrowia lipolytica Y1590 using CRISPR, homologous recombination, non-homologous end joining, or Cre / lox system.

7. A method for producing sclareol, characterized by, The production method comprises: The engineered strain producing sclareol of any one of claims 3-6 is used as a fermentation strain to prepare a seed solution and perform fermentation culture, and the sclareol is obtained from a fermentation broth and / or a bacterial body.

8. The production method according to claim 7, characterized by, The preparation method of the seed solution comprises: The engineered strain producing sclareol is inoculated into a seed culture medium, and cultured at 28-32°C and 150-250 rpm for 16-36 h.

9. The production method according to claim 7, characterized by, The fermentation culture method comprises: The seed liquid containing the engineered strain producing the fragrant perillyl alcohol is inoculated into a fermentation medium to obtain an initial OD 600 The initial fermentation bacterial liquid is 0.01-0.

04. The fermentation broth and / or the bacterial body is mixed with an organic reagent selected from any one or more of the following: n-dodecane, anhydrous ethanol, methanol, isopropanol, acetone, n-heptane, n-hexane, or acetonitrile.

10. The production method according to claim 7, characterized by, ​ ​

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