A transformation method of Rhodosporidium toruloides

Abstract

The present invention relates to the field of biotechnology and specifically discloses a method for transforming Rhodosporidium toruloides. The method comprises the following steps: 1) obtaining a single clone of Rhodosporidium toruloides, inoculating it into a culture medium for cultivation, and collecting the cells; 2) adding a transformation solution to the cells in step 1), resuspending the cells, and allowing them to stand; the transformation solution comprises PEG, a mediator, and salmon sperm DNA; 3) adding the DNA to be transformed to the cells in step 2), and incubating the cells; 4) adding a dithiothreitol solution or a dimethyl sulfoxide solution to the cell solution in step 3), followed by heat shock, resuspending the collected cells, and incubating them. The present invention constructs an efficient and reliable method for transforming Rhodosporidium toruloides with exogenous DNA. The transformation method of the present invention for Rhodosporidium toruloides has a simple process and high transformation efficiency, with a maximum transformation efficiency of 1938 transformants / μg DNA.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a transformation method of Rhodosporidium toruloides. Background Art

[0002] Transformation is a common experimental technique in molecular biology and a fundamental tool in synthetic biology. Transformation allows exogenous DNA to be stably expressed in a host cell. Common transformation methods for yeast include lithium acetate, electroporation, and Agrobacterium infection. The efficiency and specific operating parameters of these transformation methods vary significantly depending on the yeast species.

[0003] Rhodosporidium toruloides belongs to the Fungi, subkingdom Dikaryomycetes, phylum Basidiomycetes, orders Desmocerales, genus Rhodosporidium. Compared to Escherichia coli, Rhodosporidium toruloides possesses a more comprehensive set of gene expression regulatory mechanisms, the ability to process, modify, and secrete expressed products, and exhibits strong stress resistance and robustness. As a typical model strain for oil production, its oil accumulation can reach over 70% of its dry cell weight. Furthermore, Rhodosporidium toruloides has significant industrial potential for the production of carotenoids, amino acid derivatives, and enzymes. However, due to its thick cell wall, traditional transformation methods are not ideal. Currently, the mainstream transformation method for Rhodosporidium toruloides is Agrobacterium-mediated transformation, developed by Professor Zhao Zongbao's laboratory and published in FEMS yeast research (Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides). However, this method is time-consuming and requires specific steps for the cell connection between Agrobacterium and yeast. Tully and Gilbert developed protoplast transformation of Rhodosporidium toruloides in 1985 ("Transformation of Rhodosporidium toruloides"). This method is relatively cumbersome and requires specific steps for β-1,3-glucan and cell wall regeneration. In 2017, Professor Zhao Zongbao's team published "Fast and efficient genetic transformation of oleaginous yeast Rhodosporidium toruloides by using electroporation," proposing to optimize electroporation efficiency through pretreatment. However, this method achieved high transformation efficiency for haploid Rhodosporidium toruloides NP11 and, in our laboratory, was not suitable for engineered Rhodosporidium toruloides strains. Tsai et al. developed a lithium acetate / polyethylene glycol-mediated chemical transformation ("Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16"), but its extremely low transformation efficiency (20 transformants / μg DNA) limits its application in genetic manipulation. Low transformation efficiency and cumbersome procedures remain important issues restricting the development of genetic modification of Rhodosporidium toruloides. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a method for transforming Rhodosporidium toruloides. The method provides a simple process and high transformation efficiency, significantly improving the transformation efficiency from 20 transformants / μg DNA to ~2000 transformants / μg DNA compared to traditional lithium acetate transformation.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a method for transforming Rhodosporidium toruloides, comprising the following steps:

[0007] 1) A single colony of Rhodosporidium toruloides was inoculated into a culture medium and the cells were collected;

[0008] 2) adding a transformation solution to the cells in step 1), resuspending the cells, and allowing to stand; the transformation solution comprises PEG, mediator, and salmon sperm DNA;

[0009] 3) adding the DNA to be transformed to the bacterial cells in step 2) and incubating;

[0010] 4) adding dithiothreitol solution or dimethyl sulfoxide solution to the bacterial solution in step 3), then heat shocking the solution, collecting the bacterial cells, resuspending the cells, and incubating the cells;

[0011] The PEG is PEG1000 and / or PEG8000; the mediator is lithium chloride or lithium acetate.

[0012] In the technical solution of the present invention, the addition of PEG1000 and / or PEG8000 can protect Rhodosporidium toruloides from high concentrations of lithium chloride or lithium acetate, and can also neutralize the surface electrons of Rhodosporidium toruloides, promoting closer contact between the DNA to be transformed and the cell membrane of the neutralized Rhodosporidium toruloides. Experiments have shown that when PEG1000 and PEG8000 are used, more transformants can be obtained, with the highest transformation efficiency reaching 1938 transformants / μg DNA. This invention solves the problems of complex procedures and low transformation efficiency of traditional Rhodosporidium toruloides transformation methods. Compared with the mainstream Agrobacterium-mediated transformation, the technical solution of the present invention has a simpler process, shorter experimental time, and significantly improved transformation efficiency.

[0013] Compared with adding a dimethyl sulfoxide solution, adding a dithiothreitol solution in the transformation method of the present invention can increase the number of transformants obtained, thereby effectively improving the transformation efficiency.

[0014] As a preferred embodiment of the transformation method of Rhodosporidium toruloides of the present invention, in step 1), a single clone of Rhodosporidium toruloides was taken and inoculated into YPD medium for culture. At this time, the OD 600 <1 transferred to YPD medium and cultured to OD 600 The concentration of nitric oxide was 0.2, and the culture was continued for 4-6 hours.

[0015] In the technical solution of the present invention, the culture time after transfer was adjusted, and the OD 600 When the concentration is less than 1, the Rhodosporidium toruloides yeast is more suitable for receiving the DNA to be transformed.

[0016] As a preferred embodiment of the Rhodosporidium toruloides transformation method of the present invention, when the PEG is PEG1000 and PEG8000, the mass concentration of PEG1000 and PEG8000 is 50%, and the volume ratio of PEG1000 to PEG8000 is 1:1.

[0017] When PEG is a combination of PEG1000 and PEG8000, the transformation efficiency of Rhodosporidium toruloides can be improved, and a larger number of transformants are obtained.

[0018] As a preferred embodiment of the transformation method of Rhodosporidium toruloides of the present invention, the concentration of the lithium chloride or lithium acetate is 100 mM-1M.

[0019] As a preferred embodiment of the Rhodosporidium toruloides transformation method of the present invention, the mass of the DNA to be transformed is 500 ng-1 μg.

[0020] The present invention uses a smaller amount of DNA to be transformed, and only 500 ng of DNA to be transformed is needed in a 500 μL mixed system, so the cost is low.

[0021] As a preferred embodiment of the transformation method of Rhodosporidium toruloides of the present invention, the concentration of the dithiothreitol solution is 1 M, and the concentration of the DMSO solution is 10%.

[0022] As a preferred embodiment of the transformation method of Rhodosporidium toruloides of the present invention, in step 4), the heat shock temperature is 40-42° C., and the heat shock time is 30-60 min; preferably, the heat shock temperature is 40° C., and the heat shock time is 30 min.

[0023] The 37°C heat shock conditions used by Tsai and Peter Otoupal had limited transformation efficacy for engineered Rhodosporidium toruloides yeast. No transformants emerged when the heat shock temperature was 37°C and the heat shock duration was 60 minutes. The present invention significantly improves the transformation efficacy of Rhodosporidium toruloides yeast by varying the heat shock temperature and duration.

[0024] As a preferred embodiment of the transformation method of Rhodosporidium toruloides of the present invention, in step 4), the incubation time is 4 hours.

[0025] The post-culture time selected by Tsai or Peter Otoupal is too long, which causes the transformants to lose their reproductive advantage in the culture medium. The present invention controls the post-culture time to 4 hours, thereby preventing the transformants from losing their reproductive advantage in the culture medium and improving the transformation effect of Rhodosporidium toruloides.

[0026] The present invention also provides the application of the above-mentioned transformation method of Rhodosporidium toruloides in the transformation of chassis cells of ergothioneine-producing strains. The transformation method of Rhodosporidium toruloides of the present invention can greatly improve the transformation efficiency.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides an efficient and reliable method for transforming Rhodosporidium toruloides with exogenous DNA. This method features a simple process and high transformation efficiency, reaching a maximum of 1938 transformants per μg of DNA. The addition of PEG1000 and / or PEG8000 protects Rhodosporidium toruloides from damage by high concentrations of lithium chloride or lithium acetate, neutralizes surface electrons in the yeast, and promotes closer contact between the DNA to be transformed and the neutralized Rhodosporidium toruloides cell membrane. Experiments have demonstrated that using PEG1000 and PEG8000 yields a higher number of transformants. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram of the number of transformants under different experimental conditions;

[0030] Figure 2 is the number of transformants under different experimental conditions. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0033] Example 1: A method for transforming Rhodosporidium toruloides

[0034] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0035] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0036] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0037] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0038] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0039] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0040] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0041] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0042] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0043] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0044] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0045] In this embodiment, the mediator used in steps 4-6 is lithium acetate, the mixed solution used in step 6 is 50% PEG1000, and the additive used in step 9 is 1 M DTT.

[0046] Example 2: A method for transforming Rhodosporidium toruloides

[0047] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0048] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0049] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0050] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0051] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0052] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0053] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0054] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0055] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0056] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0057] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0058] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium acetate, the mixed solution used in step 6 is 50% PEG1000, and the additive used in step 9 is 10% DMSO. The other parameters are the same as in Example 1.

[0059] Example 3: A method for transforming Rhodosporidium toruloides

[0060] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0061] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0062] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0063] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0064] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0065] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0066] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0067] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0068] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0069] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0070] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0071] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium chloride, the mixed solution used in step 6 is 50% PEG1000, and the additive used in step 9 is 1M DTT. The other parameters are the same as in Example 1.

[0072] Example 4: A method for transforming Rhodosporidium toruloides

[0073] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0074] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0075] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0076] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0077] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0078] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0079] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0080] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0081] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0082] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0083] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0084] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium acetate, the mixed solution used in step 6 is 50% PEG8000, and the additive used in step 9 is 1M DTT. The other parameters are the same as in Example 1.

[0085] Example 5: A method for transforming Rhodosporidium toruloides

[0086] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0087] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0088] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0089] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0090] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0091] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0092] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0093] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0094] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0095] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0096] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0097] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium acetate, the mixed solution used in step 6 is 50% PEG8000, and the additive used in step 9 is 10% DMSO. The other parameters are the same as in Example 1.

[0098] Example 6: A method for transforming Rhodosporidium toruloides

[0099] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0100] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0101] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0102] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0103] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0104] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0105] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0106] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0107] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0108] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0109] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0110] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium chloride, the mixed solution used in step 6 is 50% PEG8000, and the additive used in step 9 is 1M DTT. The other parameters are the same as in Example 1.

[0111] Example 7: A method for transforming Rhodosporidium toruloides

[0112] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0113] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0114] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0115] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0116] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0117] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0118] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0119] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0120] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0121] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0122] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0123] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium acetate, the mixed solution used in step 6 is 50% PEG1000 and 50% PEG8000, and the additive used in step 9 is 1M DTT. The other parameters are the same as in Example 1.

[0124] Example 8: A method for transforming Rhodosporidium toruloides

[0125] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0126] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0127] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0128] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0129] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0130] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0131] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0132] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0133] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0134] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0135] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0136] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium acetate, the mixed solution used in step 6 is 50% PEG1000 and 50% PEG8000, and the additive used in step 9 is 10% DMSO. The other parameters are the same as in Example 1.

[0137] Example 9: A method for transforming Rhodosporidium toruloides

[0138] A method for transforming Rhodosporidium toruloides comprises the following steps:

[0139] 1. Pick a single colony of Rhodosporidium toruloides from a fresh YPD plate, inoculate it into 5 mL YPD, and culture it overnight at 30°C and 200 rpm (OD 600 <1);

[0140] 2. Transfer to 50 mL of fresh YPD medium until OD 600 =0.2, culture for 4-6h until OD 600 Approximately equal to 0.6;

[0141] 3. Centrifuge the bacterial solution obtained in step 2 at 4000g for 5 min, remove the supernatant, and wash twice with sterile water;

[0142] 4. Wash once with 10 mL of 100 mM mediator (pH 7.6) (centrifuge at 5000 g for 5 min).

[0143] 5. Resuspend in 1 mL of 100 mM mediator (pH 7.6);

[0144] 6. Centrifuge the resuspended bacteria from step 5 at 8000g for 1 min, remove the supernatant, and resuspend the bacteria in 250 μl of the pre-prepared mixture, 80 μl of 1M mediator, and 20 μl of 2 mg / ml salmon sperm DNA.

[0145] 7. Let the resuspended cells obtained in step 6 stand at room temperature for 5 minutes;

[0146] 8. Then, add 500 ng of the DNA to be transformed to the system in step 7, dilute the volume to 400 μL with sterile water, and incubate at 30°C and 200 rpm for 30 min;

[0147] 9. Add 100 μL of the supplement to the bacterial solution obtained in step 8, heat shock at 40°C for 30 min, collect the cells, wash once with YPD medium, resuspend in 5 mL of YPD medium, and incubate at 30°C, 200 rpm for 4 h;

[0148] 10. Collect the bacteria obtained by incubation in step 9, resuspend them in 200 μL YPD medium, spread them on YPD plates with corresponding antibiotics, and culture them at 30°C for 2-3 days.

[0149] Compared with Example 1, the difference is that the mediator used in steps 4-6 is lithium chloride, the mixed solution used in step 6 is 50% PEG1000 and 50% PEG8000, and the additive used in step 9 is 1M DTT. The other parameters are the same as in Example 1.

[0150] The number of transformants obtained by the transformation method of Rhodosporidium toruloides in Example 1-9 is as follows: Figure 1-2 shown.

[0151] The number of transformants = colony number × dilution factor × total volume of transformation reaction solution / volume of plated bacterial solution;

[0152] Frequency of transformation (number of transformants / μg plasmid DNA) = total number of transformants / amount of plasmid DNA added (μg)

[0153] Results: Comparing Examples 1-9, while Examples 4 and 7-9 yielded a greater number of transformants, Example 7 was the optimal example, achieving a maximum transformation efficiency of 1938 transformants / μg DNA. The PEG in Examples 7-9, using 50% PEG1000 and 50% PEG8000, protected Rhodosporidium toruloides from high concentrations of lithium chloride or lithium acetate. It also neutralized surface electrons in Rhodosporidium toruloides, promoting closer contact between the DNA to be transformed and the neutralized Rhodosporidium toruloides cell membrane, resulting in a greater number of transformants. This demonstrates the high transformation efficiency of this method.

[0154] By comparing Example 7 and Example 9, compared with adding dimethyl sulfoxide solution, adding dithiothreitol solution in the transformation method of the present invention can significantly increase the number of obtained transformants, thereby effectively improving the transformation efficiency.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for transforming Rhodosporidium toruloides, characterized in that: The following steps are involved: 1) Take a single colony of Rhodosporidium toruloides, inoculate it into culture medium, and collect the cells; 2) adding a transformation solution to the cells in step 1), resuspending the cells, and allowing to stand; the transformation solution comprises PEG, mediator, and salmon sperm DNA; 3) adding the DNA to be transformed to the bacterial cells after standing in step 2) and incubating to obtain a bacterial solution; 4) Adding dithiothreitol solution to the bacterial solution in step 3), followed by heat shock, collecting the bacterial cells, resuspending them, and incubating them; The PEG is PEG1000 and PEG8000; the mediator is lithium acetate; The mass concentrations of PEG1000 and PEG8000 were both 50%.

2. The transformation method according to claim 1, wherein In step 1), a single colony of Rhodosporidium toruloides was taken and inoculated into YPD medium for culture. 600 <1, transfer to YPD medium and culture until the OD of the bacterial solution 600 The concentration was set to 0.2 and culture was continued for 4-6 h.

3. The transformation method according to claim 1, wherein The concentration of the lithium acetate is 100 mM-1 M.

4. The transformation method according to claim 1, wherein The mass of the DNA to be transformed is 500 ng-1 µg.

5. The transformation method according to claim 1, wherein The concentration of the dithiothreitol solution is 1 M.

6. The transformation method according to claim 1, wherein In the step 4), the heat shock temperature is 37-42° C., and the heat shock time is 30-60 min.

7. The transformation method according to claim 1, wherein In step 4), the incubation time is 4 hours.

8. the application of the transformation method as described in any one of claim 1-7 in producing bacterial strain chassis cells transformation for thioneine.

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