Construction method and application of yeast engineering strain for producing oil and single-cell protein by using ethanol

Abstract

The invention discloses a construction method and application of a yeast engineering strain for producing oil and single-cell protein by using ethanol, and belongs to the technical field of biology. According to the invention, an ethanol-tolerant mutant strain is obtained through adaptive domestication, then adh2 and ada genes are integrated into the mutant strain, and DGA1 and DGA2 genes are overexpressed at the same time, so that the yeast engineering strain capable of producing oil and producing single-cell protein is obtained, and the yeast engineering strain capable of producing oil and producing single-cell protein can effectively improve the ethanol consumption and reduce the production cost. After fermentation for 72 hours, the consumption of ethanol is increased by 5 times, overexpression of DGA1 and DGA2 genes significantly increases the yield of grease, the ethanol utilization rate and grease synthesis ability are significantly improved, and the accumulation amount of single-cell protein reaches 73.8% of the dry cell weight. The constructed yeast engineering strain for producing oil and single-cell protein provides a new technical strategy for the production of ethanol-based oil and single-cell protein, and assists green manufacturing and carbon neutralization targets.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for constructing an oil-producing and single-cell protein-producing yeast engineering strain utilizing ethanol and application thereof. BACKGROUND

[0002] Under the background of increasing global attention to sustainable development, the development and utilization of third-generation biorefining technology has become a key strategy to alleviate food security pressure and reduce greenhouse gas emissions. Currently, global oil production mainly relies on agricultural planting, which has limitations such as low land utilization and long production cycle. In contrast, microbial oil, as a resource to replace traditional vegetable oil, has great development potential due to its short production cycle, diverse raw material sources, and ease of industrialization. At the same time, single-cell protein synthesized by microorganisms is becoming an important solution for the global food and feed industry as a high-efficiency and sustainable protein source. By utilizing carbon dioxide-derived carbon sources, cells can extend carbon chains to synthesize oil and protein, effectively reducing competition for food resources and reducing resource waste, providing new protection for global food security.

[0003] Yarrowia lipolytica, as a microorganism certified by the US FDA as a safe level, is recognized as an ideal oil synthesis cell factory due to its natural strong oil-producing capacity, wide substrate spectrum, and excellent stress resistance. In addition to oil synthesis, Yarrowia lipolytica also has great potential in single-cell protein synthesis. However, the synthesis of oil and single-cell protein using traditional sugar carbon sources competes with food resources and faces many challenges. At the same time, although renewable carbon sources such as lignocellulose are abundant, their pretreatment costs are high and their conversion efficiency is low, limiting their large-scale application.

[0004] Ethanol, as a product of one-carbon fixation technology, has significant advantages such as simple metabolic pathway, high energy density, and pH stability, making it a very potential alternative carbon source. The biological conversion process driven by ethanol as the core substrate is promoting the development of third-generation biorefining technology based on non-food single-carbon / waste carbon sources. Therefore, the development of non-food carbon sources such as ethanol for microbial oil synthesis and single-cell protein accumulation can not only realize the high-value utilization of waste resources, but also promote green biological manufacturing and help achieve the carbon neutralization goal. SUMMARY

[0005] The object of the present application is to provide a construction method of an oil-producing and single-cell protein-producing yeast engineering strain utilizing ethanol and its application, so as to solve the problems existing in the prior art. The present application obtains an ethanol-tolerant mutant strain through adaptive domestication, and then integrates adh2 and ada genes into the mutant strain, and overexpresses DGA gene, so as to obtain a yeast engineering strain, which can effectively increase ethanol consumption, increase oil production, and increase intracellular protein accumulation.

[0006] In order to achieve the above object, the present application provides the following solutions.

[0007] In a first aspect, the present application provides an oil-producing and single-cell protein-producing yeast engineering strain utilizing ethanol, wherein the oil-producing yeast engineering strain is obtained by cloning DGA1 and DGA2 genes into a plasmid to obtain a recombinant overexpression plasmid, and transforming the recombinant overexpression plasmid into Yarrowia lopolytica ETHO1 to obtain a Yarrowia lopolytica engineering strain; cloning adh2 and ada genes into a plasmid to obtain a recombinant plasmid, and transforming the recombinant plasmid into the Yarrowia lopolytica engineering strain to obtain the oil-producing and single-cell protein-producing yeast engineering strain.

[0008] The Yarrowia lopolytica ETHO1 is preserved in the China Center for Type Culture Collection on September 8, 2025, and the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M20251984.

[0009] Preferably, the nucleotide sequence of the DGA1 gene is shown as SEQ ID NO. 25; and the nucleotide sequence of the DGA2 gene is shown as SEQ ID NO. 27.

[0010] Preferably, the nucleotide sequence of the adh2 gene is shown as SEQ ID NO. 7; and the nucleotide sequence of the ada gene is shown as SEQ ID NO. 8.

[0011] In a second aspect, the present application further provides a construction method of the yeast engineering strain, which comprises the steps of cloning DGA1 and DGA2 genes into a plasmid to obtain a recombinant overexpression plasmid, and transforming the recombinant overexpression plasmid into Yarrowia lopolytica ETHO1 to obtain a Yarrowia lopolytica engineering strain; cloning adh2 and ada genes into a plasmid to obtain a recombinant plasmid, and transforming the recombinant plasmid into the Yarrowia lopolytica engineering strain to obtain the oil-producing and single-cell protein-producing yeast engineering strain.

[0012] Preferably, the method for constructing the engineered *Yersinia lipophila* strain is as follows: constructing a recombinant overexpression plasmid overexpressing the DGA1 gene and a recombinant overexpression plasmid overexpressing the DGA2 gene; transforming the recombinant overexpression plasmid overexpressing the DGA1 gene into the strain to obtain recombinant bacteria; and then transforming the recombinant overexpression plasmid overexpressing the DGA2 gene into the recombinant bacteria to obtain the engineered *Yersinia lipophila* strain.

[0013] Preferably, the recombinant overexpression plasmid for overexpressing the DGA1 gene uses the Cas9-A08 plasmid as its base plasmid; and the recombinant overexpression plasmid for overexpressing the DGA2 gene uses the Cas9-AXP plasmid as its base plasmid.

[0014] Thirdly, the present invention also provides a method for producing oil and single-cell protein using ethanol, comprising the steps of fermenting the oil-producing yeast engineered strain in a culture medium with ethanol as the carbon source to obtain cells containing oil and single-cell protein, and separating and purifying the oil and single-cell protein.

[0015] Preferably, the fermentation culture method includes the following steps: taking the yeast engineered strain and culturing it in a culture medium with ethanol as the sole carbon source for 120 h.

[0016] Fourthly, the present invention also provides the application of the described yeast engineered strain in the production of oils and single-cell proteins using ethanol as a carbon source.

[0017] The present invention discloses the following technical effects:

[0018] This invention obtains an ethanol-tolerant mutant strain through adaptive domestication. Based on this, the DGA1 and DGA2 genes are overexpressed, and then the adh2 and ada genes are integrated into the mutant strain, thereby obtaining an oleogenous yeast engineered strain. This oleogenous yeast engineered strain can effectively increase ethanol consumption, with a 5-fold increase in ethanol consumption within 72 hours after fermentation. Overexpression of the DGA1 and DGA2 genes significantly increases oil yield, and its ethanol utilization rate, single-cell protein accumulation, and oil synthesis capacity are significantly improved. The oleogenous yeast engineered strain constructed in this invention provides a new technical strategy for the production of ethanol-based oils and single-cell proteins, contributing to green manufacturing and carbon neutrality goals.

[0019] Preservation Information: Yarrowia lopolytica ETHO1 was deposited at the China Center for Type Culture Collection (CCTCC) on September 8, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20251984. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0021] Figure 1 The growth of the strains under different ethanol concentrations;

[0022] Figure 2 The growth of the strain under different concentrations of glucose and ethanol;

[0023] Figure 3 The results of the ethanol tolerance evolution experiment of Yersinia lipophila; where (a) is the OD before the second generation after generations 1-29 of tolerance evolution. 600 (b) shows the growth of a single colony on an ethanol plate, and (c) shows the OD value of the bacterial culture after 60 h of growth at a concentration of 25 g / L ethanol. 600 value;

[0024] Figure 4 The results of the experiment evaluating the ethanol utilization and lipid synthesis performance of evolved and wild-type strains at a ethanol concentration of 20 g / L are shown. Among them, (a) shows the changes in ethanol concentration of the two strains at each time point during the culture process, (b) shows the ethanol consumption of the two strains at each time point during the culture process, and (c) shows the OD of the strains at each node during the culture process. 600 The changes are shown in (d), which shows the lipid synthesis of the two strains at different time points.

[0025] Figure 5 The images show the plasmid map of pINA1269-adh2-ada and the colony PCR electrophoresis results; where (a) is the plasmid map and (b) is the electrophoresis result (M is the marker, and lanes 1-8 are eight single colonies randomly selected after being transformed into the pINA1269-adh2-ada plasmid).

[0026] Figure 6 The image shows the validation results of yeast after transforming pINA1269-adh2-ada plasmid into po1f-Δku70; where M is the marker, C is the control strain (starting strain Po1f-Δku70), and lanes 1-8 are eight single colonies randomly selected after transforming pINA1269-adh2-ada plasmid.

[0027] Figure 7The growth of single colonies of *Yarrowia lipophila* under different concentrations of ethanol was shown. Among them, (a) shows the growth of single colonies at a concentration of 10 g / L ethanol, (b) shows the growth of single colonies at a concentration of 15 g / L ethanol, (c) shows the growth of single colonies at a concentration of 20 g / L ethanol, (d) shows the growth of single colonies at a concentration of 25 g / L ethanol, (e) shows the growth of single colonies at a concentration of 30 g / L ethanol, and (f) shows the growth of single colonies at a concentration of 35 g / L ethanol.

[0028] Figure 8 The images show the plasmid spectra of recombinant plasmids Cas9-A08-DGA1 and Cas9-AXP-DGA2; where (a) is the plasmid spectra of Cas9-A08-DGA1 and (b) is the plasmid spectra of Cas9-AXP-DGA2.

[0029] Figure 9 The images show the PCR electrophoresis results; (a) is the yeast verification PCR electrophoresis image after successful DGA1 knock-in, (b) is the yeast verification PCR electrophoresis image after successful DGA2 knock-in, and (c) is the yeast verification electrophoresis image after DGA2 knock-in in the DGA1 knock-in bacteria; 1-2 in (a), 1-10 in (b), and 1-8 in (c) are all knock-in bacteria, and C is the control bacteria (starting strain Po1f-Δku70).

[0030] Figure 10 The results of the performance evaluation experiment of the engineered strains integrating ethanol utilization and lipid synthesis are shown. Among them, A is the growth status of each strain at each node during the culture process, B is the change of ethanol concentration of each strain at each time point during the culture process, C is the lipid synthesis status of each strain at different time points, and D is the single-cell protein accumulation of the engineered bacteria at different time points. Detailed Implementation

[0031] The experimental materials used in the embodiments of this invention are as follows:

[0032] (1) The Yarrowia lipolytica Po1f-Δku70 strain was constructed by knocking out the ku70 gene in the Yarrowia lipolytica Po1f strain using CRISPR-Cas9 technology. The construction method was based on the literature "Metabolically engineering of Yarrowia lipolytica for the biosynthesis of naringenin from a mixture of glucose and xylose".

[0033] (2) The names, characteristics and sources of the plasmids used in this experiment are shown in Table 1.

[0034] Table 1 Gene plasmids and their sources

[0035]

[0036] (3) MM-Leu-Ura medium:

[0037] ① The formula for MM-Leu-Ura is as follows: (NH4)2SO4 5 g / L, KH2PO4 3 g / L, MgSO4·7H2O 0.5 g / L, Leu 0.5 g / L, Ura 0.5 g / L. Dissolve in ultrapure water and autoclave at 121℃ for 20 min. The above concentrations are for use. In actual preparation, to avoid the impact of subsequent addition of ethanol on the total volume of the system, the concentration should be twice that used. In addition, vitamin solution, trace metal element solution, and FeSO4·7H2O solution need to be prepared separately, added immediately upon use, at concentrations of 1 mL / L, 2 mL / L, and 2 mL / L respectively. The formulas for the vitamin solution and trace metal element solution are as follows.

[0038] ② Vitamin Solution (200 mL): Biotin 0.01 g, para-aminobenzoic acid 0.01 g, niacin 0.2 g, calcium pantothenate (vitamin B5) (4℃) 0.2 g, zinc pyridoxine hydrochloride (vitamin B1) (4℃) 0.2 g, inositol 5 g. First, dissolve 0.04 g of NaOH in 50 mL of water to adjust the pH to 6.5, then add the remaining vitamins. Readjust the pH to 6.5 before and after adding the inositol. Finally, add the vitamins and bring the volume to 200 mL. Filter the solution using a 0.22 μm sterile aqueous filter membrane for sterilization and store at 4℃ for later use.

[0039] ③ Trace metal element solution (250 mL): CuSO4·5H2O 1.5 g, KI 0.022 g, MnSO4·H2O 0.75 g, Na2MoO4·2H2O 0.05 g, H3BO3 0.005 g, CoCl2·6H2O 0.125 g, ZnCl2 5 g, EDTA-Na 14.89 g, H2SO4 (≥98%) 1.25 mL. Dissolve in 250 mL of ultrapure water, filter sterilize using a 0.22 μm sterile aqueous filter membrane, and store at 4℃ for later use.

[0040] Example 1: Ethanol Adaptation Evolution of Yersinia lipophila

[0041] 1. Ethanol-adaptive evolution of Yersinia lipophila

[0042] Wild-type strain Po1f-Δku70 was used as the starting strain (hereinafter referred to as strain YL). The culture was performed using twice-concentrated MM-Leu-Ura medium. The volume of ethanol required to achieve the target ethanol concentration was calculated and added, with the remainder supplemented with ultrapure water. The initial pH was adjusted to 7.0 ± 0.2 using 6 M NaOH solution.

[0043] (1) Determination of the initial concentration of evolution

[0044] Additional glucose and ethanol were added, with the glucose concentration controlled at 10 g / L. Yeast strain YL was cultured at ethanol concentration gradients of 2 g / L, 5 g / L, 10 g / L, and 15 g / L, and then inoculated with yeast to induce OD. 600 The value was 0.4. The OD of the bacterial culture was measured at 15 h, 24 h, and 35 h post-inoculation. 600 Value and record.

[0045] The results are as follows Figure 1 As shown, in the early stage of cultivation (0-30 h), the growth of the yeast strain exhibited an ethanol concentration-dependent inhibitory effect, manifested as an decrease in OD with increasing ethanol concentration. 600 The value decreased significantly. However, at 35 h of culture, the OD values ​​among different ethanol concentration treatment groups showed no significant difference. 600 The values ​​tended to be consistent, indicating that although the YL strain was significantly inhibited by ethanol stress in the initial stage, it was able to establish a new metabolic homeostasis after a certain period of adaptive regulation, and eventually achieved tolerance to higher concentrations of ethanol. This phenomenon suggests that the YL strain may have an ethanol stress adaptation period of about 5 hours.

[0046] Based on the above experimental results, five experimental groups were set up. Three groups increased the ethanol concentration in the culture medium containing 10 g / L glucose, while the other two groups used only ethanol as the sole carbon source for the strain. Specific conditions are shown in Table 2. Samples were taken at 24, 48, 60, 72, and 96 h after culture to measure the OD of the bacterial culture medium. 600 Value and record.

[0047] Table 2. Design of bacterial growth groups at different glucose and ethanol concentrations.

[0048]

[0049] Figure 1 , 2 The results showed that when ethanol and glucose were added to the culture medium simultaneously as carbon sources, the growth rate of strain YL decreased significantly compared to other groups with increasing ethanol concentration. Figure 1Under conditions where ethanol is the sole carbon source, yeast growth was inhibited in the early stages at a concentration of 20 g / L ethanol, but gradually began to grow in the later stages (72 h). Figure 2 This demonstrates that yeast growth is significantly stressed at this ethanol concentration, but it can still maintain basic growth. Based on this, 20 g / L was determined as the initial screening concentration for ethanol tolerance evolution. In subsequent subcultures, a stepwise concentration escalation strategy was adopted, dynamically adjusting the ethanol concentration (increment by 5-10 g / L per generation) according to changes in the strain's growth kinetic parameters and metabolic characteristics, in order to obtain evolved strains with significantly improved ethanol tolerance.

[0050] (2) Evolutionary adaptation to ethanol tolerance

[0051] Take a sterile 25 mL Erlenmeyer flask, add 2.5 mL of twice-concentrated sterile MM-Leu-Ura medium, and add a certain amount of ampicillin and chloramphenicol (to a final concentration of 50 μg / mL). Calculate the amount of anhydrous ethanol to be added and add it to the medium (to a final ethanol concentration of 20 g / L). Set up three replicates. For each flask, use sterile 6 M NaOH solution to correct the initial pH to 7.0 ± 0.2, and then add sterile water to bring the medium volume to 5 mL. Measure the OD before subculturing. 600 Value makes the initial OD 600 The concentration was fixed at 0.30. The evolved strain was cultured at 30℃ with constant temperature shaking at 220 rpm. At the 10th generation, the ethanol concentration was increased to 25 g / L for further subculturing. At the 25th generation, the ethanol concentration was increased to 30 g / L. Subculturing was performed every 60 h (when the biomass reached the late logarithmic phase). OD was measured before each subculture. 600 Value, and preserve the seed every 5-10 generations.

[0052] Results of the evolutionary experiment on ethanol tolerance in Yersinia lipophila: Figure 3 As shown in (a), during subculturing, unstable growth or significant intragroup variability occurred. Tolerance evolution was observed when the ethanol concentration was increased to 30 g / L, leading to significant changes in the strain's growth kinetic parameters and growth stability (OD). 600 The stable value decreased, and the lag phase extended to 5-6 days before reaching the peak of the logarithmic growth phase. Therefore, the evolutionary bacteria were screened on MM medium plates with a concentration of 25 g / L ethanol. The specific process is as follows:

[0053] When the strain reached the 29th generation (using ethanol at a concentration of 30 g / L), MM-Leu-Ura solid plates with an acetic acid concentration of 25 g / L were prepared. The 29th generation strain was diluted to 10⁻⁶ ppm using sterile water. 4 10 5 10 6 107 In order to isolate single colonies, spread them evenly on MM-Leu-Ura plates using a spreader and incubate them in a 30°C constant temperature incubator for a period of time until relatively obvious single colonies grow.

[0054] The 23 largest single colonies were selected from the plate and inoculated into deep-well plates containing 3.2 mL of YPD medium. They were incubated at 30°C and 220 rpm for 36 h as seed culture. Five of these single colonies showed high OD after 24 h of incubation. 600 If the OD value is less than 1.0, the remaining 18 strains are inoculated into 3.2 mL of MM-Leu-Ura medium containing 25 g / L ethanol. The medium is sealed with aluminum foil to reduce ethanol evaporation. The YL strain is used as a control to control the initial OD value. 600 The culture was carried out at 0.3°C, 30°C, and 220 rpm for 60 hours, and the OD of the bacterial culture was measured. 600 By comparing the growth of the evolved strains with the original strains under high concentrations of ethanol, single colonies with high ethanol tolerance were screened and preserved with glycerol.

[0055] Growth of some single colony plates as follows Figure 3 As shown in (b). Measurements revealed that 18 strains grew better than strain YL in 25 g / L ethanol liquid medium. Among them, six strains (1, 2, 3, 9, 12, and 13) showed outstanding performance, with strain 1 exhibiting the highest OD. 600 Reached 5.21 ( Figure 3 The ethanol tolerance was significantly higher than that of strain YL. Therefore, strain 1, which exhibited the highest ethanol tolerance, was selected for subsequent experiments and named Yarrowia lopolytica ETHO1, which was then biopreserved. Yarrowia lopolytica ETHO1 will subsequently be referred to as the evolved strain YL. aleE .

[0056] 2. Evolutionary Bacteria YL aleE Performance evaluation

[0057] Will YL aleE YL strain according to initial OD 600 An ethanol concentration of 0.3 g / L was added to a 250 mL Erlenmeyer flask containing 30 mL of YP medium, and the ethanol concentration was controlled at 20 g / L. Three parallel controls were set up for each group, along with a blank control (no bacteria, BLANK group). The culture was carried out at 30℃ and 220 rpm, and samples were taken every 12 h to measure the OD of the bacterial culture. 600 The ethanol content of the samples was determined by HPLC, and the ethanol consumption of each engineered strain was compared at the same time point to analyze the ethanol utilization capacity of the strains.

[0058] Simultaneously, the oil content of samples (three controls for each strain) after culturing for 24 h, 48 h, 72 h, 96 h, and 120 h was determined and compared using the phosphate-vanillin (SPV) colorimetric method.

[0059] Result: From Figure 4 It can be known that the evolutionary bacterium YL aleE The ethanol utilization capacity of the strain YL was significantly higher than that of the wild-type strain, with an ethanol consumption of 13.97 g / L within 72 hours, while the ethanol consumption of the YL strain within 72 hours was only 2.17 g / L. The evolution of strain YL at various time points during fermentation... aleE The oil yield of the evolved strain YL was higher than that of the YL strain, especially at 24 h and 48 h after the start of fermentation. aleE The oil production of this strain is about twice that of the YL strain.

[0060] Example 2: Validation of the effectiveness of the heterologous ethanol metabolism pathway in wild-type Yersinia lipolytica

[0061] 1. Gene cloning and construction of recombinant plasmids

[0062] (1) Amplification of the target gene

[0063] The nucleotide sequence of the target gene was obtained from the NCBI website and then sent to Sangon Biotech (Shanghai) Co., Ltd. for the synthesis of the target gene.

[0064] Specific primers were designed based on the synthesized gene sequences adh2 (amino acid sequence as shown in SEQ ID NO.7) and ada (amino acid sequence as shown in SEQ ID NO.8):

[0065] ada: upstream primer leu-ada-F (SEQ ID NO.1) and downstream primer FBAin-T7-R (SEQ ID NO.2);

[0066] Promoter FBAin (nucleotide sequence shown in SEQ ID NO.9): upstream primer T7-FBAin-F (SEQ ID NO.3) and downstream primer adh2-FBAin-R (SEQ ID NO.4);

[0067] adh2: upstream primer FBAin-adh2-F (SEQ ID NO.5) and downstream primer xpr2-TD-adh2-R (SEQ ID NO.6).

[0068] PCR amplification was performed to obtain the target gene fragment (the reaction system and procedure are shown in Tables 3-4).

[0069] Table 3 PCR amplification system

[0070]

[0071] Table 4 PCR Amplification Program

[0072]

[0073] After the target gene is amplified, the purified target fragment is obtained by gel extraction.

[0074] (2) Construction and transformation of recombinant plasmids

[0075] The pINA1269 plasmid was digested with the restriction enzyme SpeI and used as a backbone. Homologous recombination was then performed with the adh2, FBAin, and ada gene fragments obtained in (1). 1 μL of the plasmid backbone, 3 μL of the target fragment, and 3 μL of recombinase buffer were incubated at 50℃ for 30 min to obtain the recombinant plasmid pINA1269-adh2-ada. The recombinant plasmid map is shown below. Figure 5 (a) The recombinant plasmid was then transformed into E. coli DH5α competent cells using conventional methods.

[0076] (3) Colony PCR identification and sequencing

[0077] After transformation, single colonies of *E. coli* were picked from the plates and colony PCR was performed using leu-Seq-F (SEQ ID NO.10) and xpr2-Seq-R (SEQ ID NO.11) as primers. The colony PCR reaction system (20 μL) and reaction procedure are shown in Tables 5-6.

[0078] Table 5 PCR amplification reaction system

[0079]

[0080] Table 6 PCR Amplification Reaction Procedure

[0081]

[0082] After colony PCR, 5 μL of PCR product was subjected to nucleic acid gel electrophoresis, and the results were observed using a UV gel imaging system. PCR products with the correct band size were sent for sequencing, and the bacterial culture of positive clones was inoculated into LB liquid medium (containing ampicillin) and cultured at 37°C with shaking at 220 rpm for 12 hours.

[0083] PCR electrophoresis results are shown below Figure 5In (b), the recombined size of the three fragments adh2, FBAin, and ada is around 3500 bp, and the fragments in lanes 1, 4, and 8 are the correct size, indicating that the target gene has been successfully integrated into competent E. coli cells.

[0084] (4) Plasmid extraction

[0085] Recombinant plasmids were extracted using the Tiangen Plasmid Mini-Prep Kit. Refer to the kit's instruction manual for detailed steps.

[0086] 2. Recombinant plasmids were transfected into competent evolved lipophilic yeast cells.

[0087] The extracted recombinant plasmid was transfected into competent YL strain cells using the Zymogen Frozen EZ Yeast Transformation Kit II. Refer to the kit instructions for specific steps.

[0088] The transformed strain was verified by PCR amplification to obtain a positive strain, Po1f-Δku70-1269-adh2-ada, whose pINA1269-adh2-ada plasmid was successfully transformed into yeast cells.

[0089] The fragment size obtained by amplifying the experimental bacteria using primers leu-Seq-F (SEQ ID NO.10) and leu-dn-R (SEQ ID NO.12) should be approximately 5500 bp. If a positive band is amplified, it indicates successful gene introduction. Yeast validation results are as follows... Figure 6 As shown, the single colony verification results for lanes 1 and 7 are a single band with the correct size.

[0090] After passing the above yeast verification, the strain was preserved with glycerol.

[0091] 3. Screening of single colonies with high ethanol tolerance

[0092] (1) Preliminary screening and seed culture preparation of yeast ethanol metabolism engineered strains

[0093] Eight vigorous single colonies of Po1f-Δku70-1269-adh2-ada were selected from the plate and cultured in 24-well plates. 3.2 mL of YPD medium was added to each well. After overnight incubation, three of the best-growing single colonies were selected and inoculated into new 24-well plates. The ethanol concentration was controlled at 20 g / L. The inoculation was based on the OD value of the bacterial culture. 600 Two strains of the best-tolerance single colonies (adh2-ada-2 and adh2-ada-3, respectively) were selected for preservation and inoculated into 25 mL Erlenmeyer flasks containing 5 mL YPD medium to prepare seed culture.

[0094] (2) Inoculation and sampling testing

[0095] Single colonies of the selected *Yersinia lipolytica* strains, adh2-ada-2 and adh2-ada-3, were inoculated into 24-well plates. A *Yersinia lipolytica* strain inoculated with the original plasmid (pINA1269) was used as the control group (polf-Δku70-1269 group). Ethanol concentrations of 10, 15, 20, 25, 30, and 35 g / L were set up, with three replicates for each concentration. Samples were taken at 24, 48, 72, 120, 144, and 168 h after the start of culture to detect the OD500 of the strains. 600 The changes.

[0096] Figure 7 The results in (a)-(f) showed that under higher ethanol concentrations, the strains introduced with heterologous ethanol utilization genes exhibited superior growth phenotypes compared to the control group. The experimental results indicated that under high ethanol stress (≥20 g / L), the biomass accumulation of the engineered strains introduced with the adh2 and ada genes was significantly higher than that of the wild-type control group. However, in low ethanol environments (≤20 g / L), there was no significant difference in growth curves between the engineered strains and the control strains. It is speculated that the intrinsic ethanol metabolism genes are sufficient to support the growth of the strains under lower ethanol conditions, thus explaining the lack of significant difference between the experimental and control groups. This experiment preliminarily demonstrates the effectiveness of the heterologous ethanol utilization genes adh2 and ada.

[0097] Example 3: Optimization of lipid synthesis pathway in Yersinia lipophila ETHO1 by overexpressing the DGA gene

[0098] (1) Gene cloning and construction of recombinant plasmids

[0099] ① Primer design and target gene amplification

[0100] Based on the sequence information of the DGA1 (nucleotide sequence as shown in SEQ ID NO.25) and DGA2 (nucleotide sequence as shown in SEQ ID NO.27) genes, primers were designed to construct the Cas9-A08-DGA1 and Cas9-AXP-DGA2 plasmids.

[0101] Using the YL strain genome as a template, the promoter TEFin fragment was amplified using uHAA08uHATEFin-F (SEQ ID NO.13) and DGA1-TEFin-R (SEQ ID NO.14) as primers;

[0102] Using the YL strain genome as a template, the DGA1 fragment was amplified using TEFin-DGA1-F (SEQ ID NO.15) and T7-DGA1-R (SEQ ID NO.16) primers;

[0103] Using the DGA1 fragment obtained after amplification and enzyme digestion as a template, TEFin-DGA1-F, L-T7-DGA1-R (SEQ ID NO.17) and A08DGA-T7-R (SEQ ID NO.18) were used as primers to amplify and extend the DGA1 fragment;

[0104] Using the YL strain genome as a template, the promoter GPD fragment (nucleotide sequence shown in SEQ ID NO.26) was amplified using axp-GPD-F (SEQ ID NO.19) and DGA2-gpd-R (SEQ ID NO.20) primers.

[0105] Using the YL strain genome as a template, the DGA2 fragment was amplified using GPDp-DGA2-F (SEQ ID NO.21) and xpr2-DGA2-R (SEQ ID NO.22) primers;

[0106] Using pINA1269 plasmid as a template and DGA2-XPR2-F (SEQ ID NO.23) and axp-XPR2-R (SEQ ID NO.24) as primers, the terminator xpr2 fragment (nucleotide sequence shown in SEQ ID NO.28) was amplified.

[0107] The Cas9-A08 plasmid was constructed using the same method as the Cas9-AXP plasmid. The plasmid pCRISPRyl-URA3 was digested with restriction endonucleases SacII and NotI. The YL strain genome was amplified using primer pairs CU-SacII-uHA-A08-F (SEQ ID NO.29) / dHA-spei-uHAA08-R (SEQ ID NO.30) and UHA-A08DHA-F (SEQ ID NO.31) / SgRNA-dhaa08-R (SEQ ID NO.32) to obtain the upper and lower homologous arms of the A08 knock-in site. Primers dHA-A08-sgRNA-F (SEQ ID NO.33) / A08sgRNA-R (SEQ ID NO.34) and A08sgRNA-F (SEQ ID NO.35) / CU-NotI-sgTer-R (SEQ ID NO.35) were used to amplify the genome of the YL strain. (ID NO. 36) Amplified plasmid Cas9-AXP to obtain the sgRNA expression module of A08. The above fragments were then gel-cleaved and homologously recombinated with the enzyme-digested plasmid to obtain the Cas9-A08 plasmid.

[0108] ② Construction and transformation of recombinant plasmids

[0109] The Cas9-A08 and Cas9-AXP plasmids were digested and recovered using SpeI enzyme, and homologous recombination was performed with the above fragments, respectively. The specific procedure was as follows: 1 μL of the digested and recovered plasmid, 3 μL of the target gene fragment, and 4 μL of homologous recombination enzyme were added, and the mixture was incubated at 50℃ for 30 min to perform homologous recombination, thereby obtaining two recombinant plasmids, Cas9-A08-DGA1 and Cas9-AXP-DGA2 (see figures of the two recombinant plasmids). Figure 8 (a) and (b)).

[0110] Two recombinant plasmids were transformed into *E. coli* DH5α competent cells, and positive clones were screened by plating on LB agar plates containing ampicillin. Single colonies of *E. coli* were picked from the plates and colony PCR was performed using primers uHA-A08-seqF (SEQ ID NO.37) / dHA-A08-seqR (SEQ ID NO.38) and uHA-AXP-seqF (SEQ ID NO.39) / dHA-AXP-seqR (SEQ ID NO.40) for verification. Clones with the correct band size were sent for sequencing. Positive clones were inoculated into LB liquid medium (containing ampicillin) and incubated at 37°C with shaking for 12 hours. Recombinant plasmids were then extracted using a plasmid miniprep kit. The colony PCR verification system and procedure were the same as in Example 2.

[0111] (2) Yeast cell transformation of recombinant plasmids

[0112] The transformation process was the same as described in Example 2. This resulted in the acquisition of strains ETHO1-cas9-A08::DGA1, which overexpresses the DGA1 gene, and ETHO1-cas9-AXP::DGA2, which overexpresses the DGA2 gene.

[0113] The transformed strains were subjected to PCR amplification for verification to obtain positive strains with successful DGA gene knock-in into the genome. Since genes DGA1 and DGA2 contain upper and lower homologous arm sequences, primers were designed in the peripheral regions of these homologous arms. Primers used to verify successful overexpression of the DGA1 gene were Gene-dHAA08-seq-R (SEQ ID NO.41) and Gene-uHAA08seq-F (SEQ ID NO.42), and primers used to verify successful overexpression of the DGA2 gene were Gene-dHAAXP-seq-R (SEQ ID NO.43) and Gene-uHAAXPseq-F (SEQ ID NO.44).

[0114] Results: For the validation of the DGA1 gene, the band size of the control strain was between 1000-2000 bp, and the band size of strain 1 was between 3000-5000 bp, which is consistent with the theoretical value. Therefore, it is determined that the DGA1 gene has been successfully knocked into the A08 site of the genome. Figure 9 (a)); For the validation of the DGA2 gene, the band size of the control strain in the amplification results was between 1000-2000 bp, and the band size of strains 1-8 was between 3000-5000 bp, which is consistent with the theoretical value. Therefore, it is determined that the DGA2 gene has been successfully knocked into the genomic AXP site (a). Figure 9 (b)

[0115] After DGA gene integration, the strain was cultured multiple times in a selective medium to remove the free tool plasmid. The successfully validated yeast strain was then repeatedly streaked onto YPD plates containing 5-FOA (2.0 g / L). Strains that grew on these plates were identified as strains with the Ura selection marker lost. Further streaking on YNB-Leu plates (where growth was not observed) was necessary to accurately confirm the loss of the tool plasmid. Once the tool plasmid was successfully removed, the strain could then proceed to the next gene knockout and integration operation.

[0116] After obtaining two strains overexpressing DGA1 and DGA2 respectively, DGA2 gene overexpression was performed on the DGA1 overexpressing strain ETHO1-cas9-A08::DGA1. Specifically, the recombinant plasmid Cas9-AXP-DGA2 was transformed into the constructed ETHO1-cas9-A08::DGA1 strain using conventional methods. This resulted in the strain ETHO1-cas9-A08::DGA1-AXP::DGA2 (hereinafter referred to as YL), which simultaneously overexpresses both DGA1 and DGA2 genes. aleE -1 strain), and the colonies were verified by PCR.

[0117] Using Gene-dHAAXP-seq-R and Gene-uHAAXPseq-F as primers, the amplification results showed that the control strain had a band size between 1000-2000 bp, while the band sizes of strains 1, 2, 4, 5, 6, and 8 were between 3000-5000 bp, consistent with theoretical values. Therefore, it is determined that the DGA2 gene has been successfully knocked into the genomic AXP site. Figure 9 (c) indicates that the corresponding strains are all target strains that have been successfully constructed and simultaneously overexpress DGA1 and DGA2.

[0118] To verify the correct strain, the Ura selection marker needs to be discarded, and the bacterial culture should be spread on a YPD-5FOA plate for reverse selection. The specific steps are the same as the experimental setup described above.

[0119] Example 4: Integration of strains YL-1-1 and YL aleE Construction and performance evaluation of -2

[0120] 1. Construction of the integrated strain YL-1-1

[0121] By overexpressing the DGA1 and DGA2 genes in strain YL, and introducing the heterologous ethanol utilization pathway genes adh2 and ada using plasmid 1269, an unevolved engineered strain YL-1-1 was constructed. The plasmid construction and yeast transformation process were the same as described in Examples 2 and 3.

[0122] 2. Integrated strain YL aleE -2 construction

[0123] Previously, an ethanol utilization pathway was introduced into a wild-type *Yarrowia lipophila* strain using plasmid 1269. Cultured at high ethanol concentrations, the results showed that the strain with the introduced heterologous ethanol utilization-related gene exhibited a superior growth phenotype compared to the control group, preliminarily demonstrating the effectiveness of the ethanol utilization gene module adh2-ada. Therefore, the aim was to introduce a heterologous ethanol utilization pathway into an evolved strain and to add it to the yeast genome for stable inheritance and expression. Specifically, plasmid D17-adh2-ada was constructed and transformed into the strain ETHO1-cas9-A08::DGA1-AXP::DGA2, which simultaneously overexpresses DGA1 and DGA2 genes as described in Example 3. Based on this, a strain YL was constructed that heterologously expresses the ethanol utilization gene and overexpresses the key DGA lipid synthesis gene. aleE -2. The conversion process is still carried out using the Zymogen Frozen EZ Yeast Transformation Kit II, and the specific implementation steps are the same as those set in Example 2.

[0124] The specific construction method of the D17-adh2-ada plasmid is as follows: Using the pINA1269-adh2-ada plasmid constructed in Example 2 as a template, the adh2-ada expression module was amplified using d17-1269uas-F (SEQ ID NO.45) and D17-1269XPR2-R (SEQ ID NO.46) as primers, and ligated to the Cas9-D17 plasmid backbone. Colony PCR verification was performed using uHA-D17-seqF (SEQ ID NO.47) and dHA-D17seqR (SEQ ID NO.48). After successful verification, the plasmid was transformed into ETHO1-cas9-A08::DGA1-AXP::DGA2. The transformation process was still completed using the Zymogen Frozen EZ Yeast Transformation Kit II, and yeast verification was performed using uHA-D17-seqF (SEQ ID NO.47) and Gene-D17-seqR (SEQ ID NO.49).

[0125] 3. Evaluation of the integrated strain's ethanol utilization efficiency, lipid synthesis, and single-cell protein accumulation performance.

[0126] Will YL aleE YL aleE -1、YL aleE -2. YL and YL-1-1 strains were selected according to their initial OD values. 600 An ethanol concentration of 0.3 g / L was inoculated into a 250 mL Erlenmeyer flask containing 30 mL of MM-Leu-Ura medium. The ethanol concentration was controlled at 20 g / L. The flask was incubated at 30 °C and 220 rpm. Samples were taken every 24 h to determine the OD of the bacterial culture. 600 The ethanol content of the samples was determined by HPLC, and the ethanol consumption of each engineered strain was compared at the same time points (0, 24, 48, 72, 96, and 120 h) to analyze the ethanol utilization capacity of the strains.

[0127] The oil content of five strains (three controls for each strain) was determined and compared after culturing for 24h, 48h, 72h, 96h, and 120h using the phosphate-vanillin (SPV) colorimetric method.

[0128] Simultaneously, the Kjeldahl method was used to determine and compare the oil content of five strains (three controls for each strain) after culturing for 24 h, 48 h, 72 h, 96 h, and 120 h. The protein content in yeast was determined using the national standard Kjeldahl method (GB 5009.5-2016 National Food Safety Standard - Determination of Protein in Food). Total nitrogen in the samples was also determined, and the protein content was obtained using a conversion factor of 6.25. The specific operating procedure is as follows: After sampling, the culture medium was washed, followed by freeze-drying or low-temperature drying (<50℃) to constant weight, and then grinding. 0.1 g of sample was accurately weighed into a digestion tube. Approximately 5 g of mixed catalyst was then added to the digestion tube. 15-20 mL of concentrated sulfuric acid was slowly added along the wall, gently shaken to ensure the sample was completely wetted with acid. The digestion tube was placed on a digestion oven and heated at a low temperature (approximately 200℃) to prevent excessive foaming and sample overflow. After the foam decreased, the temperature was gradually increased to 380-420℃. Continue digesting at a gentle boil until the solution turns a completely clear blue-green color (indicating that the organic matter has been completely decomposed), and continue digesting for at least 1 hour. The total digestion time is usually 2-4 hours.

[0129] Next, transfer all or part (quantitatively) of the cooled digestion solution into the reaction chamber of the distillation apparatus. Rinse the digestion tube several times with a small amount of water, and add the washings to the reaction chamber. Prepare the receiving flask by adding 20 mL of boric acid absorption solution and 2-4 drops of mixed indicator (the solution should turn purple-red). Slowly add excess sodium hydroxide solution (approximately 40-50 mL) to the reaction chamber to make the solution strongly alkaline (a brown precipitate or darkening of the liquid). Immediately begin distillation, ensuring the end of the receiving tube is submerged below the boric acid solution. Distill for approximately 10-15 minutes, collecting about 150 mL of distillate. Lower the receiving flask, rinse the end of the tube with a small amount of water, and stop distillation. Titrate the distillate with a standardized hydrochloric acid solution; the endpoint is reached when the color changes from blue-green to pale purple-red. Record the volume of acid consumed. Then perform calculations.

[0130] Result: From Figure 10 According to AB, strain YL aleE YL aleE -1、YL aleE The ethanol tolerance and utilization capacity of strain -2 is significantly higher than that of non-evolved strains, and it can utilize all of the ethanol in about 72 hours after fermentation.

[0131] Depend on Figure 10 According to C, at 24 h and 120 h after the start of fermentation, the integrated strain YL aleEThe lipid synthesis capacity of strain -2 was stronger than that of the incompletely modified strain, and the lipid content remained higher than that of the non-evolved strain at all other time points. However, after 24 hours, the intracellular lipid content of all strains decreased with increasing time, presumably due to the decomposition and metabolism of intracellular lipids during cell growth and division. The preliminary results of this invention have confirmed the effectiveness of this modification strategy, successfully establishing an ethanol-to-lipid synthesis pathway in *Yarrowia lipophila*.

[0132] like Figure 10 As shown in Figure D, strains YL and YL1-1 did not meet the requirements for cell dry weight in protein measurement, therefore no valid data on protein accumulation were available. However, YL... aleE Series of strains (YL) aleE YL aleE -1 and YL aleE -2) They exhibited high and stable protein synthesis capabilities, especially in the later stages (72-120 h), where the protein-to-dry-weight ratio remained at a high level, among which YL aleE The protein content of single cells at -2% accounted for 73.8% of the cell dry weight. The presumed reason is that the introduction of the ada and adh2 genes effectively accumulated acetyl-CoA and synthesized lipids (24-48 h), but in the later stages of cell fermentation, triacylglycerols were degraded to synthesize fatty acids. Increased fatty acid synthesis helps promote the synthesis of membrane proteins and cellular synthetic proteins, thereby increasing the protein content of single cells. Simultaneously, it is possible that the modification provided more energy and reducing power (through acetyl-CoA, ATP, and NADPH), driving cellular protein synthesis.

[0133] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A yeast strain that utilizes ethanol to produce oil and single-cell proteins, characterized in that, The yeast engineered strain is based on Yersinia lipolytica ETHO1 as the starting strain. The DGA1 and DGA2 genes are cloned into a plasmid to obtain a recombinant overexpression plasmid, which is then transformed into Yersinia lipolytica ETHO1 to obtain Yersinia lipolytica engineered strain. The adh2 and ada genes are cloned into a plasmid to obtain a recombinant plasmid, which is then transformed into the Yersinia lipolytica engineered strain to obtain the oil-producing and single-cell protein-producing yeast engineered strain. The Yarrowia lopolytica ETHO1 was deposited at the China Center for Type Culture Collection (CCTCC) on September 8, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M20251984.

2. The engineered yeast strain according to claim 1, characterized in that, The nucleotide sequence of the DGA1 gene is shown in SEQ ID NO.25; the nucleotide sequence of the DGA2 gene is shown in SEQ ID NO.

27.

3. The engineered yeast strain according to claim 1, characterized in that, The amino acid sequence of the adh2 gene is shown in SEQ ID NO.7; the amino acid sequence of the ada gene is shown in SEQ ID NO.

8.

4. A method for constructing an engineered yeast strain as described in any one of claims 1-3, characterized in that, The method includes the following steps: using Yersinia lipolytica ETHO1 as the starting strain, cloning the DGA1 and DGA2 genes into a plasmid to obtain a recombinant overexpression plasmid, and transforming it into Yersinia lipolytica ETHO1 to obtain an engineered Yersinia lipolytica strain; cloning the adh2 and ada genes into a plasmid to obtain a recombinant plasmid, and transforming the recombinant plasmid into the engineered Yersinia lipolytica strain to obtain the oil-producing and single-cell protein-producing yeast engineered strain.

5. The construction method according to claim 4, characterized in that, The method for constructing the engineered *Yersinia lipophila* strain is as follows: constructing a recombinant overexpression plasmid overexpressing the DGA1 gene and a recombinant overexpression plasmid overexpressing the DGA2 gene; transforming the recombinant overexpression plasmid overexpressing the DGA1 gene into the strain to obtain recombinant bacteria; and then transforming the recombinant overexpression plasmid overexpressing the DGA2 gene into the recombinant bacteria to obtain the engineered *Yersinia lipophila* strain.

6. The construction method according to claim 5, characterized in that, The recombinant overexpression plasmid for overexpressing the DGA1 gene uses the Cas9-A08 plasmid as its base; the recombinant overexpression plasmid for overexpressing the DGA2 gene uses the Cas9-AXP plasmid as its base.

7. A method for producing oil and single-cell protein using ethanol, characterized in that, The method includes the steps of fermenting and culturing the yeast engineered strain according to any one of claims 1-3 in a culture medium with ethanol as the carbon source to obtain cells containing oil and single-cell protein, and then separating and purifying the oil and single-cell protein.

8. The method according to claim 7, characterized in that, The fermentation culture method includes the following steps: the yeast engineered strain is cultured in a medium with ethanol as the sole carbon source for 120 h.

9. The use of the yeast engineered strain according to any one of claims 1-3 in the production of oils and single-cell proteins using ethanol as a carbon source.

Images