Yarrowia lipolytica Y1590 and its application

Abstract

The present invention provides a Yarrowia lipolytica Y1590 and its application. The Yarrowia lipolytica Y1590 is classified and named as Yarrowia lipolytica, and was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on June 4, 2024, with a deposit address in Beijing, China, and a deposit number of CGMCC No. 30855. It can solve the problem of slow growth of Yarrowia lipolytica in the prior art and is applicable to the field of microbial technology.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to Yarrowia lipolytica Y1590 and applications thereof. Background Art

[0002] Yarrowia lipolytica is a microorganism with important industrial application potential and has been certified as "Generally Recognized as Safe" (GRAS) by the U.S. Food and Drug Administration (FDA). This yeast is well known for its ability to synthesize a variety of valuable metabolites, but its growth rate and tolerance in actual fermentation processes limit the increase in production.

[0003] Current technology has proven that sclareol 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 reach a yield of 919 mg / L within 5 days; and the strain in patent application CN117264792A can produce up to 8510 mg / L in a 5 L fermenter within 8 days. Improving the tolerance of the strain so that it can continue to proliferate under harsh conditions such as high concentrations of glucose will further increase the yield. At the same time, accelerating the growth rate of the strain can shorten the fermentation cycle, thereby increasing the fermentation yield.

[0004] Therefore, in terms of industrial applications and strain modification, it is crucial to screen out chassis strains with fast growth, strong tolerance and genetic modification capabilities. This not only helps to shorten the fermentation cycle and reduce production costs, but is also of great significance for achieving efficient synthesis of microbial cell factories. At present, there are no reports showing that any Yarrowia lipolytica has significantly improved growth rate and other aspects, which indicates that this field still needs further research and development. Summary of the invention

[0005] The main purpose of the present invention is to provide a Yarrowia lipolytica Y1590 and its application to solve the problem of slow growth rate of Yarrowia lipolytica in the prior art.

[0006] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a Yarrowia lipolytica Yl590 is provided, which is classified and named as Yarrowia lipolytica Yarrowia lipolytica, and is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 4, 2024, with a deposit address in Beijing, China, and a deposit number of CGMCC No.30855.

[0007] Furthermore, Yarrowia lipolytica Y1590 contains the nucleic acid sequence shown in SEQ ID NO:1.

[0008] Furthermore, Yarrowia lipolytica Yl590 has the ability to grow rapidly, utilize multiple carbon sources and consume carbon sources rapidly; preferably, rapid growth includes: Yarrowia lipolytica Yl590 and Yarrowia lipolytica Po1f with the same initial bacterial concentration proliferate to a specific bacterial concentration under the same conditions, and the time required for Yarrowia lipolytica Yl590 is 30%-90% of that for Yarrowia lipolytica Po1f; preferably, the carbon sources are each 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.

[0009] Furthermore, Yarrowia lipolytica Yl590 has the ability to tolerate any one or more of the following conditions: high glucose concentration, low pH and high sodium chloride concentration; the concentration of high glucose concentration is 100-600 g / L; preferably, the concentration of high glucose concentration is 100 g / L or 200 g / L; the concentration of high sodium chloride concentration is 50-100 g / L; preferably, the concentration of high sodium chloride concentration is 50 g / L, 60 g / L or 70 g / L; the pH value of low pH is 2-6; preferably, the pH value is 3, 3.5, 4, 5 or 6.

[0010] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, an engineered strain for producing sclareol is provided, wherein the starting strain of the engineered strain is the above-mentioned Yarrowia lipolytica Yl590, and the genome of the engineered strain is inserted with the lysostigmine diol pyrophosphate synthase gene CLS, the sclareol synthase gene TP1132 and the geranylgeranyl pyrophosphate synthase gene CrtE by a gene editing method.

[0011] Furthermore, the protein expressed by the lysine diol pyrophosphate 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.

[0012] Furthermore, the Ku70, URA3 and LEU2 genes are knocked out of the genome of the engineered strain by a gene editing method; preferably, the gene editing method includes: using CRISPR, homologous recombination, non-homologous end joining or Cre / lox system to insert, delete or replace sequences in the chromosome of Yarrowia lipolytica Yl590.

[0013] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, a method for producing sclareol is provided, which comprises: preparing seed liquid and fermenting the above-mentioned engineered strain producing sclareol as a fermentation strain, and obtaining sclareol from the fermentation liquid and / or bacterial cells.

[0014] Furthermore, the method for preparing the seed solution comprises: inoculating the engineered strain producing sclareol into a seed culture medium, and culturing at 28-32° C. and 150-250 rpm for 16-36 h; preferably, the seed culture medium is a YPD liquid culture medium.

[0015] Furthermore, the fermentation culture method comprises: inoculating the seed liquid containing the engineered strain producing sclareol into the fermentation medium to obtain an initial OD 600 The starting fermentation culture medium is 0.01-0.04; the fermentation is carried out at 28-32°C and 150-280 rpm for 36-72 h to obtain fermentation culture medium and / or bacterial cells; preferably, the fermentation medium is YPD liquid culture medium.

[0016] Furthermore, 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, wherein the organic reagent is 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.

[0017] By applying the technical solution of the present invention, a strain of Yarrowia lipolytica Y1590 was screened and obtained, which has the following advantages: rapid growth, efficient use of substrates, strong tolerance, and gene manipulation. This can not only save the time required for cultivation and fermentation, but also can be used as a chassis strain for further application in the synthesis of different products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0019] Figure 1 The 16SrDNA gene phylogenetic tree of strain Y1590 and standard strain of Yarrowia lipolytica according to Example 1 of the present invention is shown.

[0020] Figure 2 The results of plate culture of Y1590 and Po1f strains according to Example 1 of the present invention are shown, wherein Figure 2 A shows the results of Y1590 and Po1f strains cultured on plates for 24 hours. Figure 2 Middle B shows the results of Yl590 and Po1f strains cultured on plates for 48 hours.

[0021] Figure 3 The microscopic morphology of the Y1590 strain according to Example 1 of the present invention is shown.

[0022] Figure 4 The graph shows the carbon source utilization results of the Yl590 and Po1f strains according to Example 2 of the present invention.

[0023] Figure 5 The figure shows the tolerance results of Y1590 and Po1f strains according to Example 3 of the present invention.

[0024] Figure 6 The growth curve results of the Y1590 and Po1f strains according to Example 3 of the present invention are shown.

[0025] Figure 7 The figure shows the fermentation result of the Y1590 strain producing sclareol according to Example 5 of the present invention.

[0026] Biological material information: A Yarrowia lipolytica Yl590, classified and named Yarrowia lipolytica, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 4, 2024, with the deposit address in Beijing, China, and the deposit number is CGMCC No. 30855. DETAILED DESCRIPTION

[0027] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0028] As mentioned in the background technology, Yarrowia lipolytica is a commonly used fermentation tray strain in the prior art, but the growth rate of Yarrowia lipolytica in the prior art limits its industrial fermentation capacity. In the present application, the inventors have found a faster growing Yarrowia lipolytica Y1590, and based on this, a series of protection schemes of the present application are proposed.

[0029] In the first typical embodiment of the present application, a Yarrowia lipolytica Yl590 is provided, which is classified and named as Yarrowia lipolytica Yarrowia lipolytica, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on June 4, 2024, with a storage address in Beijing, China, and a storage number of CGMCC No. 30855.

[0030] The growth rate of the above-mentioned Yarrowia lipolytica Y1590 is faster than that of other Yarrowia lipolytica in the prior art, has the ability to grow rapidly, can quickly consume carbon sources, and can shorten the growth cycle, thereby shortening the time required for cultivation and fermentation.

[0031] And the tolerance of Yarrowia lipolytica Y1590 is strong. Compared with other Yarrowia lipolytica in the prior art, it can tolerate lower pH, higher salt (such as NaCl) concentration, and higher substrate (such as glucose) concentration. This tolerance is convenient for increasing the concentration of Yarrowia lipolytica Y1590 in fermentation, and can also reduce the risk of contamination by foreign bacteria. And the molecular biological methods (including but not limited to genetic modification methods) for Yarrowia lipolytica in the prior art are also applicable to Yarrowia lipolytica Y1590 in the present application, that is, Yarrowia lipolytica Y1590 can be conveniently subjected to genetic modification and other operations, and has a high application prospect.

[0032] In a preferred embodiment, the Yarrowia lipolytica Y1590 contains the nucleic acid sequence shown in SEQ ID NO:1.

[0033] SEQ ID NO: 1:

[0034] .

[0035] In a preferred embodiment, the Yarrowia lipolytica Y1590 has the ability to grow rapidly, utilize multiple carbon sources and quickly consume the carbon sources.

[0036] In a preferred embodiment, the rapid growth includes: the Yarrowia lipolytica Yl590 and the Yarrowia lipolytica Po1f with the same initial bacterial concentration proliferate to a specific bacterial concentration under the same conditions, and the time required for the Yarrowia lipolytica Yl590 is 30%-90% of that of the Yarrowia lipolytica Po1f, including but not limited to 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0037] In a preferred embodiment, the carbon sources are each independently selected from any one or more of the following: ethanol, methanol, xylose, glycerol, sodium acetate, malonate, galactose, sucrose or glucose; more preferably, the carbon source is glucose.

[0038] In a preferred embodiment, 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 concentration of 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 concentration of high glucose concentration is 100 g / L or 200 g / L; the concentration of high sodium chloride 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 concentration of high sodium chloride is 50 g / L, 60 g / L or 70 g / L; the pH value of low pH is 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.

[0039] The above-mentioned Yarrowia lipolytica Y1590 has the characteristics of fast growth rate and fast substrate utilization. In the culture and proliferation at different stages, the time required for this culture method is less than that of other Yarrowia lipolytica in the prior art, which is convenient for saving the time required for the entire culture cycle. For example, the target OD can be reached faster in the fermentation stage. 600 , reducing the time required for fermentation.

[0040] Moreover, the above-mentioned Yarrowia lipolytica Y1590 can realize the utilization of multiple 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 engineered bacteria. Moreover, among different carbon sources, the growth rate of Yarrowia lipolytica Y1590 is faster than that of Yarrowia lipolytica in the prior art.

[0041] The above-mentioned Yarrowia lipolytica Y1590 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. This ability enables Yarrowia lipolytica Y1590 to amplify and ferment faster and at higher densities in high-concentration substrates, and other bacteria are difficult to survive or compete with Yarrowia lipolytica Y1590 in such a culture environment, reducing the risk of bacterial contamination and reducing the need for aseptic conditions for fermentation.

[0042] In a second typical embodiment of the present application, an engineered strain for producing sclareol is provided, wherein the starting strain of the engineered strain is the above-mentioned Yarrowia lipolytica Y1590, and the genome of the engineered strain is inserted with a gene editing method into which a lysyl pyrophosphate synthase gene CLS, a sclareol synthase gene TP1132, and a geranylgeranyl pyrophosphate synthase gene CrtE;

[0043] In a preferred embodiment, the Ku70, URA3 and LEU2 genes are knocked out in the genome of the engineered strain by a gene editing method; preferably, the gene editing method includes: using CRISPR, homologous recombination, non-homologous end joining or Cre / lox system to insert, delete or replace sequences in the chromosome of Yarrowia lipolytica Yl590.

[0044] Preferably, the engineered strain is Y1590ΔKu70 (URA3 - ), siteC6::CrtE, siteE16::CLS, siteB4::TP1132, △LEU2.

[0045] In the above-mentioned engineered strain, the amino acid sequence expressed by the pyrophosphate lysine diol synthase gene CLS is SEQ ID NO: 4, the amino acid sequence expressed by the sclareol 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.

[0046] Sclareol is a diterpene compound with a hemi-florin skeleton, mainly used as a raw material in the perfume industry. Sclareol can be used as a raw material for the synthesis of ambergris chemicals, such as ambroxol, ambroxol, methyl ambroxol, etc. In addition, sclareol is also a potential anti-tumor drug, which is cytotoxic to various human cancer cells such as leukemia cells, tumor cells and colon cancer cells.

[0047] The methods for producing sclareol include plant extraction, biological fermentation, etc., among which the construction of a microbial cell factory is a very promising way to efficiently synthesize high-value terpenoid compounds such as sclareol. Traditionally, sclareol is obtained by extracting from the plant extract of sclareol inflorescence. However, due to the influence of environmental and climatic factors in the planting area, the plant extraction method is limited. In contrast, the method of using microbial fermentation has the advantages of short growth cycle, all-weather production and stable yield, which can meet the growth of market demand. Selecting a suitable microorganism is crucial to the successful implementation of a biological process. It is necessary to consider its production capacity, substrate utilization and tolerance, and master relevant technologies to achieve strain transformation. Using the above-mentioned Yarrowia lipolytica Yl590 as the starting strain, an engineered strain producing sclareol can be obtained by introducing exogenous genes.

[0048] In a third typical embodiment of the present application, a method for producing sclareol is provided, which comprises: preparing seed liquid and fermenting the above-mentioned engineered strain producing sclareol as a fermentation strain, and obtaining sclareol from the fermentation liquid and / or bacterial cells.

[0049] In a preferred embodiment, the method for preparing the seed solution comprises: inoculating the engineered strain producing clarysol into a seed culture medium, and culturing at 28-32° C. (including but not limited to 28, 29, 30, 31 or 32° C.) and 150-250 rpm (including but not limited to 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 rpm) for 16-36 h (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 36 h); preferably, the seed culture medium is YPD liquid culture medium.

[0050] In the prior art, conventional Yarrowia lipolytica needs to be cultured for at least 20 hours during the process of inoculation and proliferation to obtain seed culture medium, while the Yarrowia lipolytica Y1590 of the present application has a fast proliferation rate and a seed culture medium with suitable concentration and activity can be obtained after 16 hours of cultivation.

[0051] In a preferred embodiment, the fermentation culture method comprises: inoculating the seed liquid containing the engineered strain producing sclareol into the fermentation medium to obtain an initial OD 600The starting fermentation bacteria 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.) and 150-280 rpm (including but not limited to 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 280 rpm) for 36-72 h (including but not limited to 36, 42, 48, 54, 60, 66 or 72 h) to obtain fermentation liquid and / or bacteria; preferably, the fermentation medium is YPD liquid medium.

[0052] In a preferred embodiment, the method for obtaining sclareol from a fermentation broth and / or a bacterial cell comprises: mixing the fermentation broth and / or the bacterial cell with an organic reagent, wherein the organic reagent 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.

[0053] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0054] Example 1 Screening and identification of Yarrowia lipolytica Y1590

[0055] 1. Sampling and separation:

[0056] The 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, and then diluted 10 and 100 times with sterile water, respectively, and spread on LB solid medium plates containing tributyrin, and cultured in a 30°C incubator until colonies grew.

[0057] The components of LB solid medium containing tributyrin are as follows: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 2 mL tributyrin and 20 g / L agar powder.

[0058] 2. Bacteria purification:

[0059] Among the colonies that eventually grew, samples with round, smooth, flat and light yellow morphology were selected for three-zone separation in YPD solid culture medium, cultured at 30°C for 24 h to obtain purified monoclonal clones, activated in YPD liquid culture medium, cultured at 30°C for 20 h, and preserved in a -80°C refrigerator.

[0060] The components of YPD solid culture medium are as follows: yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L, agar powder 20 g / L, of which glucose needs to be sterilized separately before addition.

[0061] The components of YPD liquid culture medium are as follows: 10 g / L yeast powder, 20 g / L peptone, and 20 g / L glucose, of which glucose needs to be sterilized separately before addition.

[0062] 3. Identification of Yarrowia lipolytica:

[0063] The 26S ribosomal RNA gene of Yarrowia lipolytica was amplified by PCR. The primer sequences are shown in Table 1 below.

[0064] Table 1

[0065] .

[0066] After obtaining the 26S ribosomal RNA gene sequence of the strain through gene amplification, the nucleotide sequence was obtained by sequencing by Anshengda Company, and then the strain information was obtained through BLAST on the NCBI official website (https: / / www.ncbi.nlm.nih.gov / ). The results showed that strain Yl590 had the highest homology with Yarrowia lipolytica WSYC 781 (FR856618.1), with a consistency of 99%. The 26S ribosomal RNA sequence with high similarity to the sequence was selected to construct a phylogenetic tree, such as Figure 1 According to the above results, Y1590 belongs to Yarrowia lipolytica.

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

[0068] SEQ ID NO: 1:

[0069] .

[0070] The comparison of strain Yl590 and the standard strain Po1f of Yarrowia lipolytica cultured on plates for 24 h and 48 h is shown in the figure below. Figure 2 Middle A and Figure 2 As shown in B, the left side of the figure are all Po1f plates, and the right side are all Yl590 plates.

[0071] Microscopic morphology of strain Yl590 (×100 times) Figure 3 shown.

[0072] Example 2 Growth and substrate utilization of Yarrowia lipolytica Y1590

[0073] Yarrowia lipolytica Yl590 and the control Po1f strain were activated in test tubes containing 5 mL of YPD liquid and cultured at 30°C and 220 rpm for 16 h.

[0074] The culture solution was adjusted according to the initial OD 600=0.2 was inoculated into a 50 mL YPD liquid shake flask and cultured at 30°C and 220 rpm. Samples were taken at different times of 0 h, 5 h, 23 h, 29 h, 47 h, 53 h, and 71 h to test OD 600 Values ​​and substrate consumption were used to compare the growth status of the strains.

[0075] Growth and carbon source consumption Figure 4 As shown, during the entire growth period, the OD of the Yl590 strain 600 The results showed that the Yl590 strain had a higher growth rate than the control Po1f strain, with the highest being 3 times higher. In addition, the Yl590 strain consumed almost all glucose at 47 h, while the Po1f strain still had glucose remaining at 72 h, indicating that the Yl590 strain utilized the substrate faster.

[0076] Example 3 Performance Analysis of Yarrowia lipolytica Y1590

[0077] 1. Analysis of substrate carbon source utilization

[0078] Different substrate carbon sources, 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, and 5 g / L glucose SD medium were added to each well of a 96-well plate, and the strain Po1f (Testo Biotechnology, TS153400) was used as a control. The total volume was 500 μL and the culture was carried out overnight at 30°C and 700 rpm.

[0079] The formula of SD medium was as follows: yeast basal nitrogen base (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.

[0080] Yeast basal nitrogen source (YNB) ingredients: inositol 2.0 mg / L, niacin (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, pyridoxine 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.

[0081] The results are as follows Figure 5 As shown, the Yl590 strain can utilize a variety of carbon sources, and its ability to utilize ethanol, glycerol, sodium acetate, malonate, galactose or glucose is significantly better than that of Po1f. Among them, the Yl590 strain grows best when glycerol or glucose is used as the carbon source.

[0082] 2. Tolerance Analysis

[0083] SD medium with 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) was added to each well of a 96-well plate, and strain Po1f (Testo Bio, TS153400) was used as a control. The total volume was 500 μL, and the culture was carried out overnight at 30°C and 700 rpm. The results are shown in Figure 2. Figure 6 As shown, under the conditions of glucose concentration of 100, 200 g / L or pH of 3, 4, 5, 6 or sodium chloride concentration of 50, 60, 70 g / L, the growth of Yl590 strain was significantly better than that of Po1f.

[0084] Example 4 Gene Editing Capability of Yarrowia lipolytica Y1590

[0085] Gene editing was performed on Yarrowia lipolytica Yl590. According to the prior art, the URA3 and LEU2 genes of the Yl590 strain were knocked out by CRISPR technology to facilitate gene editing, and the knockout of the Ku70 gene enhanced its homologous recombination ability. The three heterologous genes that can synthesize sclareol, the pyrophosphate lysine diol synthase gene CLS (amino acid sequence SEQ ID NO: 4), the sclareol synthase gene TP1132 (amino acid sequence SEQ ID NO: 5) and the geranylgeranyl pyrophosphate synthase gene CrtE (amino acid sequence SEQ ID NO: 6) were integrated into the Yarrowia lipolytica genome by homologous recombination technology. As a result, the knockout and overexpression genes of Yarrowia lipolytica Yl590 were correct, indicating that the strain can be subjected to gene editing operations and can be transformed as a chassis strain.

[0086] SEQ ID NO: 4

[0087] MAFTFTSAHLFLPVTENHSVHVNYSIPPGNWRLWSTAKGGSNKLDIRRLRCSARRTPEPLAQGSNGGRDGVEAIQRLQTIADDKIDGGANELGIVVWDLIRDGVDAVKSMFDSMGDGDISISAYDTAWVALVKDVNGSGGPQFPSSLQWIVDNQLPDGSWGDSEVFSAYDRLLKTLACVVALKSWNIRPDKCQKGLKFFRDNISKLEKENVEASAQMLSGFEVVFLSLIEVARRLDIQIPLHSPVFEDLIARRNLKFAKIPLDLMHNVPTSLLNSLEGMTGVELDWEKLLKLQSQDGSFITSPSSTAFALMQTNDTKCLGYLKFVVQKFNGGAPGQYPVEIFERIWVVDRLQRLGISRYFQLEIKECCLDYAFKHWTQYGSSWARNTPVYDLDDTCMAFRILRLHGYDVSAEAFRHFEKNGVFFCFGWETTQSVTVNFNLYRATQVAFPGENILKEAKQFSFNFLMKKQAAREFQDKWVILKDFPGELKYALEFPWYASLPRVETRFYVEQYGGDNDVWIGKTLYRMPYINNNVYLELAKLDFNNCQALHRKEWETMQKWFMESKLDEFGVSSKTLLESYFLAAASIFEPERSTERLAWAKTAFLMETIGSYFDDEMNSKDLRKAFVQEFKNIYERRMEAKGTKWNLIIILLTTLNHLTEVCGRDINSYLCHSWEKWMMMWEPEGDRYKGAAELLSNSINLSSGRLFSNDTLSHPNYEKLVTLSNKLCHQLGNSRRGNHNEDSDIKDTKIEIAMQELVQLVHQNSSDDISMDLKQTFFAVVRSFYYAAHCDRGTINSHIVKVLFESVV。

[0088] SEQ ID NO:5

[0089] MSLAFNVGVTPFSGQRVGSRKEKFPVQGFPVTTPNRSRLIVNCSLTTIDFMAKMKENFKREDDKFPTTTTLRSEDIPSNLCIIDTLQRLGVDQFFQYEINTILDNTFRLWQEKHKVIYGNVTTHAMAFRLLRVKGYEVSSEELAPYGNQEAVSQQTNDLPMIIELYRAANERIYEEERSLEKILAWTTIFLNKQVQDNSIPDKKLHKLVEFYLRNYKGITIRLGARRNLELYDMTYYQALKSTNRFSNLCNEDFLVFAKQDFDIHEAQNQKGLQQLQRWYADCRLDTLNFGRDVVIIANYLASLIIGDHAFDYVRLAFAKTSVLVTIMDDFFDCHGSSQECDKIIELVKEWKENPDAEYGSEELEILFMALYNTVNELAERARVEQGRSVKEFLVKLWVEILSAFKIELDTWSNGTQQSFDEYISSSWLSNGSRLTGLLTMQFVGVKLSDEMLMSEECTDLARHVCMVGRLLNDVCSSEREREENIAGKSYSILLATEKDGRKVSEDEAIAEINEMVEYHWRKVLQIVYKKESILPRRCKDVFLEMAKGTFYAYGINDELTSPQQSKEDMKSFVF。

[0090] SEQ ID NO: 6

[0091] MVSGSKAGVSPHREIEVMRQSIDDHLAGLLPETDSQDIVSLAMREGVMAPGKRIRPLLMLLAARDLRYQGSMPTLLDLACAVELTHTASLMLDDMPCMDNAELRRGQPTTHKKFGESVAILASVGLLSKAFGLIAATGDLPGERRAQAVNELSTAVGVQGLVLGQFRDLNDAALDRTPDAILSTNHLKTGILFSAMLQIVAIASASSPSTRETLHAFALDFGQAFQLLDDLRDDHPETGKDRNKDAGKSTLVNRLGADAARQKLREHIDSADKHLTFACPQGGAIRQFMHLWFGHHLADWSPVMKIA。

[0092] The specific steps are as follows:

[0093] Preparation of competent cells: Pick activated monoclones from the YPD solid medium plate into a test tube and incubate at 30 °C for 24 h; collect the cells by centrifugation at 5000 rpm, resuspend in 1 mL 1xTE Buffer, collect the cells by centrifugation again, and resuspend in 1 mL 0.1 mol / L LiAC (OD 600 3-6), incubate at 30 °C for 1 h; collect the cells by centrifugation at 5000 rpm for 1 min, and resuspend in an appropriate amount of 0.1 mol / L LiAC to obtain competent cells.

[0094] Construct engineered bacteria: Add 15 µL salmon sperm DNA (need to be heated in advance and then cooled), 1-5 µg DNA to be transformed or 2 µg plasmid to a 1.5 mL centrifuge tube, mix well, and then add 40 µL of the prepared competent cells (OD 600 ~4), incubate at 30 ℃ for 15 min; add 280 μL 50% PEG4000, 70 μL 0.5 mol / L LiAC and 16 μL 1 mol / L DTT to the mixture in the previous step, mix well, and incubate at 30 ℃ for 1 h; add 40 μL DMSO to the mixture in the previous step, incubate at 39 ℃ for 10 min, add 600 μL 0.1 mol / L LiAC, mix well, and resuscitate at 30 ℃ for 1 h; collect the bacteria by centrifugation at 5000 rpm for 2 min, resuspend in water, and spread on a culture medium containing the corresponding antibiotics or a nutrient-deficient culture medium, and culture at 30 ℃ for 2-3 days until a single colony grows. The Yl590 engineered bacteria was named Yl590△Ku70 (URA3 - ), siteC6::CrtE, siteE16::CLS, siteB4::TP1132, △LEU2. For comparison at the same level, Po1f was genetically manipulated using the same method as above, and the engineered bacteria were named Po1f△Ku70, siteB1::CLS, siteC6::CrtE, siteB4::TP1132.

[0095] Example 5 Sclareol production by Yarrowia lipolytica Y1590 at shake flask level

[0096] Seed culture medium: scrape Yl590△Ku70(URA3 -), one loop each of siteC6::CrtE, siteE16::CLS, siteB4::TP1132, △LEU2 and Po1f△Ku70, siteB1::CLS, siteC6::CrtE, siteB4::TP1132 plate bacteria were transferred to test tubes containing 5 mL YPD liquid culture medium and cultured at 30°C, 200 rpm shaking for 16 h.

[0097] Fermentation medium: according to the initial OD 600 The seed solution was transferred to a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium (YPD medium, pH 6) with an inoculum size of 0.02, and fermented at 30°C and 220 rpm for 72-96 h.

[0098] Detection of Scl: After 48 h of fermentation, 1 mL of fermentation broth was added with 1 mL of n-dodecane and shaken for 1 h. The supernatant was centrifuged and sent to gas chromatography for detection. Results The scl yield of Yl590 was 1.37 mg / L, while the scl yield of the control Po1f was only 0.96 mg / L. In addition, the OD of Yl590 was 600 The sclareol production, growth and sugar consumption of strain Y1590 in shake flasks are shown in Figure 2. Figure 7 shown.

[0099] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the growth rate of the above-mentioned Yarrowia lipolytica Y1590 is faster than that of other Yarrowia lipolytica in the prior art, and has the ability to grow rapidly and consume substrates, and can shorten the growth cycle, thereby shortening the time required for cultivation and fermentation. And the tolerance of Yarrowia lipolytica Y1590 is strong, and compared with other Yarrowia lipolytica in the prior art, it can tolerate lower pH, higher salt (such as sodium chloride) concentration, and higher substrate (such as glucose) concentration. This tolerance is convenient for increasing the concentration of Yarrowia lipolytica Y1590 in fermentation, and can also reduce the risk of contamination by miscellaneous bacteria. And the molecular biological methods (including but not limited to genetic modification methods) for Yarrowia lipolytica in the prior art are also applicable to Yarrowia lipolytica Y1590 in the present application, that is, Yarrowia lipolytica Y1590 can be conveniently subjected to genetic modification and other operations, and has a high application prospect.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Yarrowia lipolytica Y1590, classified and named as Yarrowia lipolytica Yarrowia lipolytica , deposited on June 4, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address in Beijing, China, and the deposit number is CGMCC No. 30855.

2. An engineered strain producing sclareol, characterized in that: The starting strain of the engineering strain is the Yarrowia lipolytica Y1590 described in claim 1, and the genome of the engineering strain is inserted with the lysyl pyrophosphate synthase gene by a gene editing method. CLS , sclareol synthase gene TP1132 Geranylgeranyl pyrophosphate synthase gene CrtE ; The lysyl pyrophosphate synthase gene CLS The expressed protein is the polypeptide shown in SEQ ID NO: 4; The sclareol synthase gene TP1132 The expressed protein is the polypeptide shown in SEQ ID NO: 5; The geranylgeranyl pyrophosphate synthase gene CrtE The expressed protein is the polypeptide shown in SEQ ID NO:

6.

3. The engineered strain according to claim 2, characterized in that The genome of the engineered strain is knocked out by the gene editing method Ku70 , URA3 and LEU2 Gene.

4. The engineered strain according to any one of claims 2-3, characterized in that The gene editing method comprises: inserting, deleting or replacing a sequence on the chromosome of the Yarrowia lipolytica Y1590 using CRISPR, homologous recombination, non-homologous end joining or Cre / lox system.

5. A method for producing sclareol, characterized in that: The production method comprises: The engineered strain producing sclareol according to any one of claims 2 to 4 is used as a fermentation strain to prepare a seed solution and perform fermentation culture, and obtain the sclareol from the fermentation solution and / or bacterial cells.

6. The production method according to claim 5, characterized in that The preparation method of the seed solution comprises: The sclareol-producing engineered strain is inoculated into a seed culture medium and cultured at 28-32° C. and 150-250 rpm for 16-36 h.

7. The production method according to claim 5, characterized in that: The fermentation culture method comprises: The seed liquid containing the engineered strain producing sclareol was inoculated into the fermentation medium to obtain an initial OD 600 The starting fermentation liquid is 0.01-0.04; The fermentation is carried out at 28-32° C. and 150-280 rpm for 36-72 h to obtain the fermentation liquid and / or the bacterial cell.

8. The production method according to claim 5, characterized in that: The method for obtaining the sclareol from the fermentation broth and / or the bacterial cell comprises: The fermentation liquid and / or the bacterial cells are mixed with an organic reagent, wherein the organic reagent is selected from any one or more of the following: n-dodecane, anhydrous ethanol, methanol, isopropanol, acetone, n-heptane, n-hexane or acetonitrile.

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