Leucosporidium giganthum MYL-12 and method and application of transforming exogenous DNA thereof
By using isolation, identification, and electroporation techniques, a method for transforming exogenous DNA into *Saccharomyces cerevisiae* MYL-12 was established, solving the transformation problem of unconventional oil-producing yeasts, achieving efficient oil production and gene editing, and promoting the development of the microbial oil industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MAIYUAN LABORATORY
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-29
AI Technical Summary
The lack of efficient conversion methods in current technologies hinders the development of lipid production in unconventional oil-producing yeasts such as marine red yeast, limits genome editing and metabolic engineering, and restricts the improvement of microbial lipid synthesis technology.
A strain of *Saccharomyces cerevisiae* MYL-12 was isolated and identified, and a simple and efficient method for exogenous DNA transformation was established. The hygromycin B phosphotransferase gene (Hygr) was introduced into the strain via electroporation. A genetic manipulation platform was constructed, and voltage and conditions were optimized to improve transformation efficiency.
It has achieved efficient exogenous gene conversion, improved oil production capacity, enriched the types of chassis cells for oil synthesis, and promoted the large-scale and green development of the microbial oil industry.
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Figure CN122104450A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a yeast strain, and more particularly to a method for transforming a yeast strain with exogenous genes. Background Technology
[0002] Oil-producing yeasts are yeasts with an oil content exceeding 20% of their cell dry weight. They possess advantages such as rapid growth, strong resistance to adverse conditions, a broad substrate utilization spectrum, and the ability to utilize waste lignocellulose, making them ideal hosts for microbial oil production. The oils synthesized by these yeasts are rich in long-chain fatty acids such as palmitic acid, oleic acid, stearic acid, and linoleic acid, with a fatty acid composition very similar to vegetable oils, showing potential applications in the food and other fields. Currently, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) produced by oil-producing yeasts are already being used as new food resources in infant formula and health products. Discovering new oil-producing yeast germplasm resources from samples of different environmental sources, and based on this, developing genetic manipulation platform technologies for oil-producing yeast strains with industrial production potential, and further utilizing synthetic biology techniques and metabolic engineering methods to increase oil yield or directionally synthesize high-value-added single-cell oils with different fatty acid compositions and functional uses, is of great significance for achieving green, low-cost, and efficient synthesis of animal and plant alternative oils.
[0003] Genetic transformation of exogenous genes is one of the most common experimental techniques in molecular biology and a fundamental method in synthetic biology, enabling the stable expression of exogenous proteins in the target host. Common transformation methods for yeast include lithium acetate transformation, electroporation transformation, and Agrobacterium-mediated transformation. However, the applicable transformation methods, operating parameters, and transformation efficiencies vary significantly among different yeast species. Transformation methods for traditional model yeasts (such as Saccharomyces cerevisiae) cannot be directly applied to unconventional wild-type oil-producing yeasts with unique physiological characteristics. For example, the unconventional oil-producing yeast *Rhodotorula rubra* is rich in lipids and carotenoids, but the lack of efficient transformation methods hinders the development of lipid production. Sun Wenyi et al. successfully inserted exogenous genes into its genome by establishing a new transformation method, providing a basis for the modification of metabolic processes (Sun Wenyi, Zhang Sufang, Lin Xinping, et al. A genetic transformation method in *Rhodotorula rubra*). Rhodotorula mucilaginosa Applications of [J]. China Biotechnology Journal, 2016, 36(06):81-86.). Currently, the scarcity of genetic tools severely restricts the rational metabolic engineering modification and basic biological research of strains with industrial production potential. Therefore, developing efficient and stable genetic transformation methods for unconventional oil-producing yeasts and establishing targeted transformation systems are prerequisites for successfully introducing exogenous genes, realizing genome editing, and carrying out metabolic engineering. This has significant scientific and industrial implications for breaking through the current bottlenecks in microbial lipid synthesis technology, significantly improving lipid production capacity, and achieving chassis cell diversification. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention proposes a strain of *Saccharomyces cerevisiae* MYL-12 and its method and application for transforming exogenous DNA.
[0005] The technical solution of this invention is implemented as follows:
[0006] This invention isolates and identifies a cocoa butter-producing yeast strain, *Saccharomyces cerevisiae* MYL-12, and determines resistance screening markers that can be used to construct genetic manipulation platform technologies. In particular, it establishes a simple and efficient method for exogenous gene transformation, which lays an important foundation for further development of gene editing technology, metabolic engineering, and synthetic biology research of this strain.
[0007] On the one hand, the present invention provides a strain of *Saccharomyces cerevisiae* MYL-12, which is classified and named as follows: Naganishia albima It was deposited on January 14, 2026 at the China Center for Type Culture Collection, with accession number CCTCC M 2026106, and the deposit address is Wuhan University, Wuhan, China.
[0008] Secondly, the present invention provides the application of the above-mentioned *Saccharomyces cerevisiae* MYL-12 in the fermentation production of microbial oils.
[0009] Preferably, the above application steps are as follows: inoculate the seed culture of *Saccharomyces cerevisiae* MYL-12 into a nitrogen-limiting medium, and ferment it at 28-32℃ and 200-220 rpm for 72-120 h to obtain microbial oil.
[0010] Preferably, the microbial oil is a cocoa butter-like substance; the OD of the seed liquid 600 The nitrogen content was 0.8-1.0; the nitrogen sources in the nitrogen-limiting medium were peptone, yeast extract, ammonium sulfate and ammonium chloride, and the carbon source was glucose.
[0011] After freeze-drying, the fermented cells were methylated and analyzed by gas chromatography. The content of the main C16-C18 fatty acids in the cells accounted for 21.56% of the cell dry weight. The fatty acid composition was very similar to that of cocoa butter (23% C16:0, 35% C18:0, 35% C18:1), indicating that it has cocoa butter production potential. It was identified as an oil-producing yeast with cocoa butter production potential.
[0012] Thirdly, the present invention also provides a method for transforming exogenous DNA using the above-mentioned *Saccharomyces cerevisiae* MYL-12, the steps of which are as follows:
[0013] (1) Take the bacterial culture of Yeast MYL-12, wash and centrifuge, and resuspend it in EBbuffer solution containing dithiothreitol. The precipitate obtained by centrifugation is the competent cells.
[0014] (2) Competent cells are mixed with exogenous DNA containing resistance genes, and the resulting mixture is added to a pre-cooled electroporation vessel. After standing on ice, electroporation is performed.
[0015] (3) After electroporation, the cells were resuspended in YPD medium containing sorbitol and incubated. The cells were collected, spread on YPD medium containing selection markers, and verified by colony PCR to obtain positive transformants that were transformed with exogenous DNA.
[0016] Preferably, the OD of the bacterial solution in step (1) above is... 600 The concentration of dithiothreitol is 0.8-1.0 mM; the EB buffer solution contains 1-50 mM Tris-HCl buffer, 0.1-10 mM MgCl2 and 50-500 mM sucrose, pH 7.5.
[0017] Preferably, in step (2) above, the volume ratio of competent cells to exogenous DNA is 10:0.5-1, and the mass of exogenous DNA is 500 ng-1 μg; the resistance gene is the hygromycin B phosphotransferase gene (…). Hygr The codon-optimized sequence is shown in SEQ ID NO.1; the ice resting time was 5 min, and the electroconversion parameters were voltage 0.8-2.6 kV, capacitance 25 μF, and resistance 200 Ω. When the voltage was 1.8-2.2 kV, the number of convertants was relatively large. Preferably, when the voltage was 2 kV, the conversion efficiency was the highest at 40 cfu / µg.
[0018] Preferably, the concentration of sorbitol in step (3) above is 0.5 M; the incubation is carried out at 28-32℃ and 150-200 rpm for 2-4 h, and the screening marker is hygromycin B with a minimum resistance concentration of 50 mg / L; the primers used for colony PCR verification are those with sequences as shown in SEQ ID NO.2. Hyg -F and the sequence as shown in SEQ ID NO.3 Hyg -R.
[0019] Hygromycin B resistance gene with optimized codons Hygr The promoter pTEF1 and terminator T were constructed into the lipophilic yeast expression vector pYLXP'. XPR2 An expression cassette for the resistance gene was constructed. The pTEF1- expression cassette was then loaded with the expression cassette. Hygr -T XPR2The plasmid and linearized fragment were electrotransformed into *Saccharomyces cerevisiae* MYL-12, and positive transformants were obtained after incubation for 2-4 h. This established an efficient method for transforming exogenous genes into wild-type *Saccharomyces cerevisiae* MYL-12.
[0020] Fourthly, the above methods are applied to the modification of chassis strains for microbial oil production.
[0021] Preferably, the strain in the chassis is *Saccharomyces cerevisiae* MYL-12, with the preservation number CCTCC M 2026106.
[0022] The present invention has the following beneficial effects:
[0023] This invention provides a strain of *Saccharomyces cerevisiae* MYL-12, which is classified and named as follows: Naganishia albida The strain MYL-12 was deposited on January 14, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026106, located at Wuhan University, Wuhan, China. The ratios of the main fatty acids synthesized by strain MYL-12 in nitrogen-limited medium to cell dry weight were: C16:0 = 45.5 mg / g, C18:0 = 32.3 mg / g, C18:1 = 64.8 mg / g, C18:2 = 67.2 mg / g, and C18:3 = 5.8 mg / g. The total intracellular C16-C18 fatty acid content was 215.6 mg / g, accounting for 21.56% of the cell dry weight, and the C16-C18 fatty acid yield was 2.5 g / L. Based on these findings, it was identified as an oil-producing yeast and can serve as a chassis strain for the synthesis of functional oils such as cocoa butter.
[0024] This invention also established a method for effectively transforming exogenous genes into strain MYL-12, screened for antibiotics with hygromycin B as the selection marker, and a minimum resistance concentration of 50 mg / L. A method for constructing a vector containing... Hygr Resistance gene expression cassette pTEF1- Hygr -T XPR2 The plasmid was amplified by PCR to obtain the linearized fragment pTEF1- to be transformed. Hygr -T XPR2 Electroporation into strain MYL-12 achieved a maximum conversion efficiency of 40 cfu / μg at an optimal voltage of 2 kV. This invention lays a technological foundation for the efficient expression of exogenous genes in *Saccharomyces cerevisiae*, the construction of gene editing technology systems, precise metabolic engineering, and synthetic biology research. It plays a crucial role in overcoming current bottlenecks in microbial lipid synthesis technology, significantly improving lipid production capacity, enriching the types of lipid synthesis chassis cells, and promoting the large-scale and green development of the microbial lipid industry. Attached Figure Description
[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0026] Picture 1 Morphological characteristics of *Saccharomyces cerevisiae* MYL-12; where A represents colony morphology on YPD agar plates and B represents cell morphology under a 40× microscope.
[0027] Picture 2 It is the fatty acid composition of the fat synthesized from cocoa butter and MYL-12 yeast fermentation.
[0028] Picture 3 The growth inhibition of *Cyclomycin B* at different concentrations on *Saccharomyces cerevisiae* MYL-12 is shown in the figure.
[0029] Picture 4 pTEF1-, a gene expression cassette carrying the hygromycin B resistance marker gene. Hygr -T XPR2 A schematic diagram of the plasmid and linearized fragment.
[0030] Picture 5 Colony PCR was used to verify the successful transformation of the exogenous gene into yeast cells.
[0031] Picture 6 The effect of different voltages on the electroconversion efficiency of Yeast MYL-12. Detailed Implementation
[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.
[0034] The culture medium formula used in this invention is as follows:
[0035] (1) YPD medium: glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L.
[0036] (2) LB medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.
[0037] (3) Nitrogen-limiting medium (pH 6.0): glucose 40 g / L, yeast extract 1 g / L, ammonium sulfate 0.3 g / L, magnesium sulfate heptahydrate 1.5 g / L, potassium dihydrogen phosphate 2.4 g / L, disodium hydrogen phosphate 0.91 g / L, calcium chloride monohydrate 0.22 g / L, trace elements 10 mL / L (v / v).
[0038] The trace element composition is as follows: zinc sulfate 100 mg / L, ferrous sulfate 550 mg / L, copper sulfate 76 mg / L, and manganese sulfate 76 mg / L.
[0039] Hygromycin B is added to YPD medium as needed, and ampicillin is added to LB medium as needed. Solid culture media require the addition of 1.5 g / L agar powder during preparation.
[0040] The sterilization conditions for the culture media used in this invention are all high-pressure steam sterilization at 115°C for 20 min.
[0041] Unless otherwise specified, the percentage concentrations mentioned in the embodiments of the present invention are mass / mass percentage concentrations (w / w, unit g / 100 g), mass / volume percentage concentrations (w / v, unit g / 100 mL), or volume / volume percentage concentrations (v / v, unit mL / 100 mL).
[0042] The primers used in this invention were synthesized by Beijing Qingke Biotechnology Co., Ltd.
[0043] The genes used in this invention were synthesized by Genscript Biotech Inc. after codon optimization.
[0044] The yeast *Leptochloa chinensis* belongs to the Basidiomycota phylum of the Kingdom Fungi. Its cells are spherical to oval and reproduce by budding. It usually does not produce hyphae or pseudohyphae. Currently, there are no reports on the construction of the genetic operating system and genetic engineering modification of *Leptochloa chinensis*.
[0045] Example 1: Isolation, purification and identification of *Saccharomyces cerevisiae* MYL-12
[0046] (1) Sample collection and processing
[0047] Take 2-4 g of soil sample from Ulanhot City, Inner Mongolia, place it in a centrifuge tube, add 10 mL of sterile water, and vortex for 30 s to mix the sample. In a sterile laminar flow hood, serially dilute the sample 10-fold to 1×10⁻⁶. -3 Use a pipette to aspirate 100 μL of 1×10⁻⁶ solution.-1 1×10 -2 1×10 -3 Three dilutions of the sample were spread on plates containing Bengal red medium, with three plates for each dilution. The plates were then inverted and incubated at 30°C for three days.
[0048] Preparation of Bengal Red medium: 5.0 g / L peptone, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 10.0 g / L glucose, 0.1 g / L chloramphenicol, 0.033 g / L Bengal Red, and 20.0 g / L agar powder.
[0049] (2) Isolation and purification of strains
[0050] Select several typical single colonies grown on the above-mentioned Bengal Red medium and inoculate them into test tubes containing 5 mL of YPD liquid medium. Incubate them in a shaker at 30℃ and 220 rpm for 1-2 days. Repeatedly streak the culture in new solid and liquid YPD medium to purify it until a pure culture of single colonies is obtained. Preserve the purified strain by adding sterile glycerol to a final concentration of 15% (v / v) and storing it at -80℃ for subsequent experiments.
[0051] (3) Microscopic observation of colony morphology and strains
[0052] The yeast was streaked onto YPD solid medium plates and incubated at 30°C for 3 days. Colony morphology was then observed. The yeast colonies were milky white, round, raised, opaque, smooth, glossy, thick, and had neat edges. Picture 1 A). Under a 40× microscope, yeast cells appear round or oval, uniform in size, dispersed, do not form pseudohyphae, have clear outlines, and intact structures, and reproduce asexually through budding. Picture 1 B).
[0053] (4) Identification of strains
[0054] The ITS gene of this yeast was amplified using colony PCR. Template preparation: 30 μL of cell lysis buffer was added to a PCR tube. A suitable amount of single colony was picked and added to the lysis buffer, and the mixture was incubated at 85℃ for 15 min to lyse the yeast cell wall. After brief centrifugation of the lysis buffer, 1 μL of the supernatant was used as a template for PCR amplification. The primers used for PCR amplification were universal primers for fungal ITS sequence amplification: the forward primer was ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and the reverse primer was ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 56℃ annealing for 20 s, 72℃ extension for 5 s, for 30 cycles; 72℃ extension for 5 min.
[0055] The PCR amplification product was identified as approximately 500 bp in length by agarose gel electrophoresis. Sequencing analysis by Beijing Qingke Biotechnology Co., Ltd. yielded the ITS gene sequence of strain MYL-12, as shown in SEQ ID NO.4. This sequence was then compared with the NCBI database using BLAST analysis. Based on the analysis results, the strain was identified as *Saccharomyces cerevisiae*. Naganishia albida The strain was named *Saccharomyces cerevisiae* MYL-12 and was identified on November 28, 2024.
[0056] Example 2: Fermentation of Microbial Oils by *Saccharomyces cerevisiae* MYL-12
[0057] Yeast rice (MYL-12) stored at -80℃ was streaked onto YPD solid medium for strain activation. Single colonies were picked and inoculated into test tubes containing 5 mL of YPD liquid medium, and cultured at 30℃ and 220 rpm for 15 h as the seed culture. 1 mL of the seed culture was inoculated into a 500 mL shake flask containing 50 mL of nitrogen-limiting medium and fermented at 30℃ and 220 rpm for 72 h. After fermentation, the bacterial culture was centrifuged (12000 rpm for 2 min), and the collected cells were washed twice with sterile water and then freeze-dried. The freeze-dried cells were then subjected to fatty acid methyl esterification treatment, and the intracellular C16-C18 major fatty acid content and fermentation titer were analyzed by gas chromatography to evaluate its lipid synthesis capacity.
[0058] The fatty acid methyl esterification method is as follows: Weigh 1-2 mg of the freeze-dried bacterial cells, add methyl heptadecanate as an internal standard, add 500 μL of methyl esterification reagent (0.5 N sodium hydroxide-methanol solution), and methyl esterify for 2 h at room temperature and in a shaker at 1200 rpm; then add 40 μL of 98% concentrated sulfuric acid to each reaction solution to neutralize the sample; then add 400 μL of n-hexane to each sample, and react for 10 min at room temperature and in a shaker at 1200 rpm to extract fatty acid methyl esters; finally, centrifuge the sample at 12000 rpm for 2 min, and collect the upper n-hexane phase for gas chromatography analysis.
[0059] The test results showed that after 72 h of fermentation in nitrogen-limited medium, the proportions of the main fatty acids synthesized by *Saccharomyces cerevisiae* MYL-12 to the cell dry weight were as follows: C16:0 = 45.5 mg / g, C18:0 = 32.3 mg / g, C18:1 = 64.8 mg / g, C18:2 = 67.2 mg / g, and C18:3 = 5.8 mg / g. The total intracellular C16-C18 fatty acid content was 215.6 mg / g, accounting for 21.56% of the cell dry weight. Furthermore, the fatty acid composition was very similar to that of cocoa butter (23% C16:0, 35% C18:0, 35% C18:1) (e.g., ...). Picture 2 As shown in the figure, it has cocoa butter-like production potential, with a C16-C18 fatty acid yield of 2.5 g / L, thus identifying it as an oil-producing yeast with cocoa butter-like production potential.
[0060] Example 3: Antibiotic screening markers for *Saccharomyces cerevisiae* MYL-12
[0061] The growth inhibition of wild-type *Chrysomyces cerevisiae* MYL-12 by three commonly used antibiotics (hygromycin B, neomycin sulfate, and G418) was tested. Specifically, single colonies from YPD solid medium plates were inoculated into test tubes containing 5 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 15 h. 100 μL of the bacterial solution was then spread onto YPD solid medium plates containing different concentrations (0, 25, 50, 75, 100, 125, 150 mg / L) and different types of antibiotics (hygromycin B, neomycin sulfate, and G418). Three parallel experiments were performed for each antibiotic concentration. The plates were then inverted and incubated at 30℃ for 3 days to observe for yeast colony growth.
[0062] The lowest concentration of an antibiotic that completely inhibits yeast growth is taken as the minimum effective resistance concentration of that antibiotic. This is then used in antibiotic concentration gradient screening experiments. Picture 3The study determined that 50 mg / L hygromycin B can serve as an effective antibiotic selection marker. Therefore, the hygromycin B resistance gene can be used as an resistance selection marker gene for constructing the Yeast Extract MYL-12 genetic operating system.
[0063] Example 4: Electroconversion method for *Saccharomyces cerevisiae* MYL-12
[0064] Constructing a pTEF1- expression cassette carrying resistance marker genes Hygr -T XPR2 The plasmid was obtained and the linearized fragment pTEF1- to be transformed was obtained by PCR amplification. Hygr -T XPR2 ( Picture 4 Based on the codon usage preference of *Saccharomyces cerevisiae* MYL-12, the hygromycin B resistance gene was identified. Hygr Codon optimization and synthesis were performed, and the optimized nucleic acid sequence is shown in SEQ ID NO.1. This sequence was then further constructed into the Yersinia lipophila expression vector pYLXP', using the promoter pTEF1 and terminator T. XPR2 In between, pTEF1-, carrying the resistance gene expression cassette, was obtained. Hygr -T XPR2 The plasmid was used to amplify the linearized fragment pTEF1- to be transformed by PCR. Hygr -T XPR2 .
[0065] The pTEF1- expression cassette containing the resistance marker gene was used. Hygr -T XPR2 The plasmid and linearized fragment were electrotransformed into *Saccharomyces cerevisiae* MYL-12, and the specific steps are as follows:
[0066] 1) Activate the yeast by streaking YPD solid medium plates with MYL-12 stored at -80℃, pick a single colony and inoculate it into a test tube containing 5 mL of YPD liquid medium, and incubate overnight at 30℃ and 220 rpm in a shaker.
[0067] 2) Transfer the overnight culture to a 250 mL shake flask containing 50 mL of fresh YPD liquid medium, and initially inoculate with OD. 600 The concentration was 0.15-0.2, and the cells were cultured for another 4-5 hours at 30℃ and 220 rpm in a shaker until the bacterial cells reached the OD value. 600 When the value reaches 0.8-1.0, stop the culture.
[0068] 3) After placing the bacterial cells in ice water for 10 min, centrifuge at 4℃ and 5000×g for 5 min to collect the bacterial cells. Discard the supernatant. Wash the collected bacterial cells twice with 50 mL of pre-cooled sterile ddH2O, and then wash once with 25 mL of pre-cooled EB buffer (10 mM Tris-HCl buffer, 1 mM MgCl2, 270 mM sucrose, pH 7.5). Then resuspend in 25 mL of pre-cooled EB buffer and add DTT to a final concentration of 1 mM.
[0069] 4) Incubate the above cell mixture on ice for 60 min, centrifuge at 4℃ and 5000×g for 5 min to collect the cells, discard the supernatant, wash once with 25 mL of pre-cooled EB buffer, and finally resuspend the cells in 250 µL of EB buffer, aliquot and prepare competent cells.
[0070] 5) Take 50-100 µL of competent cells, add 5-10 µL of plasmid or linearized DNA fragment, mix in a pre-cooled 2 mm gap electroporation cuvette, and incubate on ice for 5 min before electroporation.
[0071] 6) Set the electric shock device to V=0.8-2.6 kV, 200 Ω, 25 μF and perform the electric shock.
[0072] 7) Immediately after electroporation, the cells were resuspended in 1 mL of YPD medium containing 0.5 M sorbitol and incubated at 30°C and 200 rpm for 2-3 h. The cells were then collected, spread onto YPD resistance selection plates containing 50 mg / L hygromycin B, and inverted in a 30°C incubator for 2-3 days. The number of transformants was counted, and the hygromycin B resistance gene was evaluated. Hygr Colony PCR amplification and sequencing analysis were performed to verify whether it was a positive transformant.
[0073] Transformation efficiency = total number of transformants / amount of DNA added (µg).
[0074] The electroporation method established above can successfully transform pTEF1-, which carries the resistance marker gene expression cassette. Hygr -T XPR2 Plasmids and linearized fragments were introduced into *Saccharomyces cerevisiae* MYL-12 competent cells to transform the transformants. Hygr Gene amplification by colony PCR ( Picture 5 The forward primer used is Hyg -F (5'-ATGAAGAAGCCCGAGTTGACAGC-3') and the reverse primer is HygThe PCR amplification products were sequenced and analyzed using the formula -R(5'-GACGACGGTTGCCGCTATCTGC-3'), verifying that all transformants successfully transformed the exogenous gene. The transformation efficiency was 40 cfu / µg at an electroporation voltage of 2 kV. Therefore, this invention establishes a simple and effective method for transforming wild-type *Saccharomyces cerevisiae* MYL-12 with exogenous genes.
[0075] The effect of different voltages on transformation efficiency was investigated using the aforementioned electroconversion method. Under the conditions of constant competent cell preparation and exogenous DNA concentration, the number of transformants was counted when the voltage during electroporation was controlled at 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, and 2.6 kV. Among these, *Saccharomyces cerevisiae* MYL-12 showed a higher number of transformants within the voltage range of 1.5–2.2 kV. Picture 6 Preferably, at the optimal voltage of 2 kV, the conversion efficiency is up to 40 cfu / μg.
[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A strain of *Saccharomyces cerevisiae* MYL-12, its classification name is... Naganishia albida It was deposited on January 14, 2026 at the China Center for Type Culture Collection, with accession number CCTCC M 2026106, and the deposit address is Wuhan University, Wuhan, China.
2. The application of the light-colored yeast MYL-12 as described in claim 1 in the fermentation production of microbial oils.
3. The application according to claim 2, characterized in that, The steps are as follows: Inoculate the seed culture of *Saccharomyces cerevisiae* MYL-12 into a nitrogen-limiting medium and ferment it at 28-32℃ and 200-220 rpm for 72-120 h to obtain microbial oil.
4. The application according to claim 3, characterized in that: The microbial oil is a cocoa butter-like substance; the OD of the seed liquid... 600 The nitrogen content was 0.8-1.0; the nitrogen sources in the nitrogen-limiting medium were peptone, yeast extract, ammonium sulfate and ammonium chloride, and the carbon source was glucose.
5. The method for transforming exogenous DNA into *Saccharomyces cerevisiae* MYL-12 according to claim 1, characterized in that, The steps are as follows: (1) Take the bacterial culture of Yeast MYL-12, wash and centrifuge, resuspend in EB buffer solution containing dithiothreitol, and the precipitate obtained by centrifugation is the competent cells; (2) Competent cells are mixed with exogenous DNA containing resistance genes, and the resulting mixture is added to a pre-cooled electroporation vessel. After standing on ice, electroporation is performed. (3) After electroporation, the cells were resuspended in YPD medium containing sorbitol and incubated. The cells were collected, spread on YPD medium containing selection markers, and verified by colony PCR to obtain positive transformants that were transformed with exogenous DNA.
6. The method according to claim 5, characterized in that: The OD of the bacterial solution in step (1) 600 The concentration of dithiothreitol is 0.8-1.0 mM; the EB buffer solution contains 1-50 mM Tris-HCl buffer, 0.1-10 mM MgCl2 and 50-500 mM sucrose, pH 7.
5.
7. The method according to claim 6, characterized in that: In step (2), the volume ratio of competent cells to exogenous DNA is 10:0.5-1, and the mass of exogenous DNA is 500 ng-1 μg; the resistance gene is the hygromycin B phosphotransferase gene, and its optimized codon sequence is shown in SEQ ID NO.1; the electroporation parameters are voltage 0.8-2.6 kV, capacitance 25 μF, and resistance 200 Ω.
8. The method according to claim 7, characterized in that: In step (3), the concentration of sorbitol was 0.5 M; the incubation conditions were 28-32℃ and 150-200 rpm for 2-4 h; the screening marker was hygromycin B with a minimum resistance concentration of 50 mg / L; the primers used for colony PCR verification were those with sequences as shown in SEQ ID NO.
2. Hyg -F and the sequence as shown in SEQ ID NO.3 Hyg -R.
9. The application of the method according to any one of claims 5-8 in the chassis strain modification of microbial oil production strains.
10. The application according to claim 9, characterized in that: The strain in the chassis was *Saccharomyces cerevisiae* MYL-12, with the preservation number CCTCC M 2026106.