Method for stable integration of exogenous DNA in pachysolen tannophilus, iterative gene editing method and application thereof

Abstract

The present application belongs to the field of microbial metabolic engineering and industrial microorganism technology, and particularly relates to a method for stably integrating exogenous DNA into Pseudozyma roseolata, an iterative gene editing method and application thereof. The preservation number of the strain MYL-2 is CCTCC M 20252968, and the strain can efficiently accumulate oil under nitrogen-limited culture conditions, the main fatty acid components in the cells are C16-C18 fatty acids, and the strain has application potential as a microbial oil production strain. The present application provides a method for stably integrating exogenous DNA into Pseudozyma roseolata, and constructs a recombinant expression vector pNFH-loxp-RAD51 and a Cre recombination vector containing a loxP site, and for the first time, precise removal and repeated use of a screening marker mediated by a Cre-LoxP system are realized in the strain, thereby laying a foundation for multi-round gene editing and efficient engineering modification of the strain.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering and industrial microbial technology, and specifically relates to a method for stably integrating exogenous DNA and a gene editing method. Background Technology

[0002] Microbial oils are a type of oil resource obtained through microbial fermentation and can serve as important raw materials in the fields of daily chemicals, food, pharmaceuticals, and bioenergy. Compared to traditional animal and vegetable oils, the production process of microbial oils has advantages such as not being limited by land resources and climate conditions, having diverse forms of carbon source utilization, and having their fatty acid composition that can be controlled through metabolic engineering, thus possessing considerable potential for industrial applications.

[0003] Cyclosporium pinkis ( Sporidiobolus pararoseus Belonging to the Rhodotorula group of Basidiomycetes, *Rhodotorula* is a non-traditional yeast strain with both biosafety and multifunctional application potential. Besides showing application potential in food processing and biocontrol, it also possesses a natural ability to accumulate lipids. For example, patent 202110825523.X discloses a strain of *Rhodotorula* ZQHL, characterized by high lipid yield and high glucose conversion rate. *Rhodotorula* has unique value in the development of functional lipids, and its environmental adaptability and substrate utilization range are broad, making it suitable for industrial fermentation needs. However, compared to model oil-producing yeasts such as *Yarrowia lipolytica*, the genetic background of *Rhodotorula* is unclear, its genetic transformation efficiency is low, and it lacks a universal and standardized genetic manipulation system, thus limiting its application in metabolic engineering.

[0004] The Cre-LoxP system is a gene recombination system composed of Cre recombinase (Causes recombination, a DNA endonuclease derived from bacteriophage P1) and the loxP recognition sequence (locus of X-over P1, a 34 bp specific palindromic DNA sequence). This system boasts advantages such as high sequence recognition specificity, independence from host cofactors, high editing efficiency, and low off-target rate, and is widely used for site-directed gene mutation and site-directed integration of exogenous genes. Cre-loxp-mediated gene deletion is highly efficient and precise, and has been applied in various microorganisms. However, a Cre-LoxP-mediated genetic manipulation method for *Cladosporium rosenbergii* has not yet been established, and its application in the metabolic engineering of this strain remains unexplored.

[0005] Therefore, establishing an efficient genetic transformation method suitable for *Synthia spp.* and constructing a gene editing system that enables the recovery of screening markers to meet the needs of multiple rounds of engineering modification of this strain is not only of great research significance for exploring the application potential of this strain in the synthesis of functional oils, but also provides a new path for the green and efficient development of the oil industry. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a method for the stable integration of exogenous DNA by *Syngonium pulmonale*, an iterative gene editing method, and their applications.

[0007] The technical solution of this invention is implemented as follows:

[0008] In a first aspect, the present invention provides a strain of *Sterilosporium rosenbergii* MYL-2, which is classified and named as follows: Sporidiobolus pararoseus The accession number is CCTCC M 20252968, the depositary institution is the China Center for Type Culture Collection, the deposit date is December 19, 2025, and the deposit address is Wuhan University, Wuhan, China.

[0009] The strain MYL-2 exhibits significant lipid accumulation capacity under nitrogen-limited culture conditions. Its main intracellular fatty acid components are C16-C18 fatty acids, with DCW and C16-C18 fatty acid titers of 10.18±0.46 g / L and 5.26±0.68 g / L, respectively, demonstrating its potential as a microbial lipid production strain.

[0010] Secondly, a method for stably integrating exogenous DNA into *Cyclophorus paniculatus* includes the following steps:

[0011] (1) Take the bacterial culture of *Syngonium rosenbergii*, wash and centrifuge, resuspend in pre-cooled LiAc solution, and the precipitate obtained by centrifugation is the competent cells;

[0012] (2) Add PEG 3350, LiAc solution, salmon sperm DNA and exogenous DNA to competent cells in sequence, mix well and incubate with shaking, then add DMSO for heat shock and collect the cells;

[0013] (3) The bacterial cells were resuspended in YPD liquid culture and incubated upside down at 30℃ for 48–72 h for recovery culture. They were then plated onto YPD solid medium containing selection markers for screening. Colony PCR was used for verification to obtain positive transformants that stably integrated exogenous DNA. The transformation efficiency was approximately 1.34 × 10⁻⁶. 2 CFU / μg DNA.

[0014] Preferably, in step (1) above, the *Sporidiobolus pararoseus* is *Sporidiobolus pararoseus* MYL-2, with the taxonomic name *Sporidiobolus pararoseus*, accession number CCTCC M 20252968, deposited at the China Center for Type Culture Collection on December 19, 2025, and deposited at Wuhan University, Wuhan, China; the OD of the bacterial culture... 600 The concentration of the LiAc solution is 0.8–1.0 mM; the concentration of the LiAc solution is 100 mM–200 mM.

[0015] Preferably, in step (2) above, the mass concentration of PEG 3350 is 40%–50%, the concentration of LiAc solution is 1 M, and the concentration of salmon sperm DNA is 1–5 mg / mL; the shaking incubation conditions are 28–30℃ and 200–220 r / min for 30–60 min; and the heat shock conditions are 36–42℃ for 25–35 min.

[0016] Preferably, in step (3) above, the screening marker is Hygr or Zeocin, with the minimum inhibitory concentration of Hygr being 60 μg / mL and the minimum inhibitory concentration of Zeocin being 25 μg / mL.

[0017] Thirdly, an iterative gene editing method, with the following steps:

[0018] 1) Using the above-mentioned genetic transformation method, the recombinant vector containing Cre recombinase was introduced into an engineered strain containing the loxP site. The transformation product was plated on YPD medium containing Zeocin and positive transformants were screened.

[0019] 2) Induce the expression of Cre recombinase in positive transformants (the recombinant vector containing Cre recombinase is used as a selection marker to drive the transient expression of Cre recombinase in the engineered strain), thereby knocking out the target gene and precisely removing the selection marker gene to obtain the gene-edited engineered strain;

[0020] 3) Using the engineered strain from step 2) as the recipient, repeat steps 1) to 2) to complete the reuse of the screening marker gene, obtain engineered strains for multi-target gene editing, and realize iterative gene editing.

[0021] Preferably, in step 1), the genome of the engineered strain integrates an expression cassette containing a promoter, a RAD51 coding gene, a target gene, and a terminator. The loxP recognition sites are located on both sides of the selection marker and are oriented in the same direction. The selection marker gene is the resistance gene Hygr, with an optimized sequence as shown in SEQ ID NO.1. The recombinant vector containing Cre recombinase contains a promoter, a terminator, a resistance gene Zeocin, a reporter gene eGFP, and a replication element pan-ARS. The reporter gene eGFP is used to verify the transformation efficiency of the recombinant vector using fluorescence detection. The replication element pan-ARS can achieve autonomous replication in various yeast strains.

[0022] Preferably, the promoter is TPI, TEF or GPD, and the terminator is CYC1 or XPR2; the gene sequence encoding Cre recombinase is shown in SEQ ID NO.2; the optimized sequence of Zeocin resistance gene is shown in SEQ ID NO.3; the optimized sequence of reporter gene eGFP is shown in SEQ ID NO.4; and the sequence of replication element pan-ARS is shown in SEQ ID NO.5.

[0023] Preferably, the engineered strains in step 2) are inoculated onto a medium containing Zeocin, a medium containing Hygr, and a medium without antibiotics, respectively. Colonies that can grow on the medium containing Zeocin and the medium without antibiotics, cannot grow on the medium containing Hygr, and show an eGFP fluorescent signal are selected and identified as engineered strains with precisely excised screening marker genes.

[0024] Fourthly, the application of the aforementioned genetic transformation methods or iterative gene editing methods in the targeted construction of engineered microbial lipid-producing strains.

[0025] Preferably, the chassis strain used for the above-mentioned directional construction is *Cyclophorus pseudopinnatifida* MYL-2, with the accession number CCTCC M 20252968.

[0026] The present invention has the following beneficial effects:

[0027] 1. This invention establishes a method for the stable integration of exogenous DNA into *Cladosporium rosenbergii* MYL-2, including competent cell preparation, construction of a PEG-LiAc-DNA transformation system, heat shock treatment, and resuscitation screening steps. This method enables stable integration of exogenous DNA into MYL-2 cells and improves integration efficiency. The strain MYL-2, with accession number CCTCC M 20252968, exhibits significant lipid accumulation capacity under nitrogen-limited culture conditions. Its cells have a high proportion of C16-C18 fatty acids in their dry weight, demonstrating potential as a microbial lipid-producing strain. Furthermore, this invention systematically determined and established an resistance screening system suitable for this strain, with a minimum inhibitory concentration (MIC) of 60 μg / mL for Hygr and 25 μg / mL for Zeocin. These screening conditions can be used for efficient screening and stable inheritance of engineered strains.

[0028] 2. This invention also establishes an iterative gene editing method, achieving for the first time the effective application of the Cre-LoxP system in *Cyclospora rosenbergii*. A Cre-LoxP gene recombination system suitable for *Cyclospora rosenbergii* MYL-2 was constructed, including a recombinant expression vector containing loxP sites. The loxP recognition sites are located on both sides of the selection marker and are oriented in the same direction, using the Hygr resistance gene as the selection marker. The Cre recombinase expression vector uses the Zeocin resistance gene as the selection marker to drive the transient expression of Cre recombinase in the engineered strain. When the Cre recombinase expression vector is introduced into the engineered strain containing loxP sites, the Cre recombinase can specifically recognize and mediate the recombination reaction between the oriented loxP sites, thereby achieving precise excision of the selection marker gene (Hygr) and repeated use of the selection marker in subsequent rounds of gene editing. This method does not rely on host-specific recombination factors and has advantages such as high recombination efficiency, simple operation process, and strong applicability. This application provides a reliable technical foundation for the targeted design, modification, and industrial development of microbial lipid-synthesizing strains. Attached Figure Description

[0029] 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.

[0030] Figure 1 Phylogenetic tree of *Syndrome rosenbergii* MYL-2.

[0031] Figure 2Morphological characteristics of *Syndrome rosenbergii* MYL-2; where A shows the colony morphology of strain MYL-2 on YPD medium, and B shows the morphology observed under a microscope.

[0032] Figure 3 The values ​​represent the dry weight (DCW) and fatty acid titers of *Syngonium rosenbergii* MYL-2 cells after fermentation in NL-1 medium; where A represents the C16-C18 fatty acid content, and B represents the DCW and C16-C18 fatty acid titers.

[0033] Figure 4 The results show the minimum inhibitory concentrations (MICs) of hygromycin (Hygr) and bleomycin (Zeocin) screening markers for *Cladosporium rosenbergii* MYL-2. A represents the MIC of MYL-2 against the Hygr screening marker, and B represents the MIC of MYL-2 against the Zeocin screening marker.

[0034] Figure 5 The MYL-2 / RAD51 transformant was grown on a YPD screening plate with 60 μg / mL Hygr added.

[0035] Figure 6 The agarose gel electrophoresis results of the MYL-2 / RAD51 engineered bacteria were verified by colony PCR; where A is the agarose gel electrophoresis result of the MYL-2 / RAD51 engineered bacteria verified using primers RAD51-YZ-F / TCYC1-R (900 bp), and B is the agarose gel electrophoresis result of the MYL-2 / RAD51 engineered bacteria verified using primers NFH-Hygr-5F-YH / NFH-Hygr-3R-YH (1026 bp).

[0036] Figure 7 To verify the precise excision and recovery of the Hygr selection marker by Cre recombinase; where A represents the growth of colonies on antibiotic-free YPD plates, and B represents the growth of colonies on YPD plates containing 60 μg / mL Hygr. Detailed Implementation

[0037] 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.

[0038] 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.

[0039] This invention provides a *Cyclophorus rosenbergii* strain MYL-2 and its standardized genetic manipulation method. By establishing the Cre-LoxP system in this strain, precise excision and reuse of selection markers are achieved. This method can be used for multiple rounds of genetic modification of *Cyclophorus rosenbergii*, providing a technical foundation for related metabolic engineering research and the construction of microbial lipid-engineered strains.

[0040] The culture medium formula used in this application is as follows:

[0041] Isolation medium (Bangladesh Red medium): 5.0 g peptone, 1.0 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 10.0 g glucose, 0.1 g chloramphenicol, 0.033 g Bengal Red, 20.0 g agar, add distilled water to a final volume of 1000 mL, sterilize at 121℃ for 20 min and set aside.

[0042] Enrichment medium (YPD liquid medium): yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, sterilized at 121℃ for 20 min for later use.

[0043] Nitrogen-limiting medium NL-1: 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 dihydrate 0.22 g / L, trace elements 10 mL / L, pH 6.0; the trace elements consist of the following: zinc sulfate heptahydrate 1 mg / 100 mL, ferrous sulfate heptahydrate 5.5 mg / 100 mL, copper sulfate pentahydrate 0.76 mg / 100 mL, and manganese sulfate monohydrate 0.76 mg / 100 mL.

[0044] Example 1: Isolation, purification and identification of *Sterilostomyces pinki* MYL-2

[0045] 1. Sample collection

[0046] Sample source: Soil samples collected in Ulanhot, Inner Mongolia in November 2024.

[0047] 2. Strains Isolation and Purification

[0048] (1) Sample preparation: Weigh 2-4 g of soil sample and place it in a centrifuge tube containing 10 mL of sterile water. Vortex for 30 s to mix. Under aseptic conditions, serially dilute to 1×10⁻⁶. -3 Select 1×10 -1 1×10 -2 1×10 -3Three gradients were prepared, with 100 μL of each diluted solution spread onto Bengal Red agar plates, and three replicates were set for each gradient.

[0049] (2) Culture and purification: Place the plate after plating in a constant temperature incubator at 28~30℃ for 1~3 days; pick out the single colony with typical morphology on the plate, inoculate it into 5 mL of YPD liquid medium, and culture at 28~30℃ and 220 rpm for 1~2 days with shaking. Repeat the streak purification 3 times to ensure the purity of the strain.

[0050] (3) Preservation of bacterial strains: Take 700 μL of purified bacterial solution and mix it with 300 μL of 50% (v / v) sterile glycerol, put it into a preservation tube and label it, and freeze it in an ultra-low temperature freezer at -80℃ for later use.

[0051] 3. Strain identification

[0052] (1) PCR amplification of the ITS gene:

[0053] ① Template preparation: Take 10 μL of purified bacterial culture and add 50 μL of Lysis Buffer. Incubate at 80℃ for 15 min (to break the fungal cell wall). After a short centrifugation for 10 s, take the supernatant as the PCR amplification template.

[0054] ②PCR system (20 μL): 2×Magic Green Taq SuperMix 10 μL, template 1 μL, primer ITS1 (10 μM) 1 μL, primer ITS4 (10 μM) 1 μL, ddH2O 7 μL;

[0055] ③ Primer sequences: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), ITS4 (5'-TCCTCCGCTTATTGATATGC-3');

[0056] ④ Amplification conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 55℃ annealing for 10 s, 72℃ extension for 42 s, for a total of 30 cycles; 72℃ final extension for 10 min, with an expected amplified fragment length of 500~750 bp.

[0057] (2) Electrophoresis detection and sequencing: Prepare 1% agarose gel (containing nucleic acid dye), take 3~5 μL of PCR product for electrophoresis (120 V, 10~20 min), and observe the clarity of the bands using a gel imaging system; select products with single and clear bands and send them to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing to obtain the ITS gene sequence (SEQ ID NO.6). Use BLAST analysis tool to compare with the NCBI database to establish a phylogenetic tree ( Figure 1The ITS gene sequence of strain MYL-2 has the highest homology with that of S. pararoseus, and they cluster together in the same branch in the phylogenetic tree.

[0058] (3) Morphological identification: The colonies are orange-red or magenta, with a smooth, creamy, viscous surface, raised and irregular edges, and some colonies are regularly round. Figure 2 A); Under the microscope, the cells are oval-shaped, uniform in size, dispersed and do not aggregate, and have an intact structure. Figure 2 B). Based on the combined morphological and molecular biological identification results, strain MYL-2 was confirmed as *S. pararoseus*.

[0059] 4. Fermentation of *Cladosporium rosenbergii* MYL-2 cells in nitrogen-limiting medium NL-1 and determination of cell dry weight and fatty acid titer

[0060] (1) Fermentation of *Cyclophorus rosiflora* MYL-2 on nitrogen-limited NL-1 medium

[0061] After activating and culturing *Cyclophorus rosenbergii* MYL-2 in YPD liquid medium for 16–24 h (30℃, 220 rpm), 1 mL of the bacterial culture was centrifuged at 12000 × g for 1 min to remove the supernatant. The culture was then resuspended in nitrogen-limiting medium and added to nitrogen-limiting medium for fermentation to achieve initial OD. 600 The concentration of the spores was 0.06–0.1. Fermentation was carried out at 30℃ and 220 rpm for 5 days to obtain the fermentation broth of *Syngonium rosenbergii* MYL-2. The fermentation broth was centrifuged, and the cells were collected and freeze-dried. The dry cell weight (DCW) and fatty acid titer were then measured to determine the biomass, fatty acid composition, and lipid accumulation capacity of MYL-2.

[0062] (2) Detection of DCW and fatty acid titer

[0063] DCW is obtained by weighing the freeze-dried bacterial cells.

[0064] Fatty acid titers were obtained as follows: 1–2 mg of lyophilized bacterial cells were weighed, and glycerol heptadecanate was added as an internal standard. 500 μL of 0.5 N sodium hydroxide-methanol solution was added, and the mixture was vortexed at 1200 × g for 2 h. After vortexing, 40 μL of 98% concentrated sulfuric acid was added to neutralize the sample, and the pipette tip was immersed below the sample surface and slowly added dropwise. 400 μL of n-hexane was added to each sample, and the mixture was vortexed at 1200 × g for 10 min to extract fatty acid methyl esters. The mixture was centrifuged at 12000 × g for 2 min, and the n-hexane phase was collected for gas chromatography analysis. Gas chromatography analysis conditions: An Agilent 8890 GC system equipped with a flame ionization detector and an HP-INNOWAX capillary column (30 m × 0.25 mm × 0.25 µm) was used for GC analysis. The initial temperature was 210 °C (held for 1 min), and the temperature was increased to 220 °C at a rate of 2 °C / min, and held at 220 °C for 3 min. The carrier gas was nitrogen, the flow rate was 1 mL / min, and the detector temperature was 280℃.

[0065] The above experiments show that after fermentation in NL-1 medium, the proportion of C16-C18 fatty acids in the DCW of *Synthia spp.* MYL-2 is 51.53%, indicating it is an oil-producing yeast. Figure 3 A), DCW and C16-C18 fatty acid titers were 10.18±0.46 g / L and 5.26±0.68 g / L, respectively. Figure 3 B).

[0066] Example 2: Establishment of screening markers for *Syndrome rosenbergii* MYL-2, lithium acetate chemical transformation and screening of positive transformants

[0067] 1. Determination of the minimum inhibitory concentration (MIC) of the screening marker

[0068] (1) Determination of the minimum inhibitory concentration (MIC) of hygromycin (Hygr): After sterilizing YPD medium and cooling it to 50-55℃, Hygr was added under aseptic conditions to prepare resistance plates with different concentration gradients (three replicates for each concentration), with YPD plates without Hygr as the control. Wild-type MYL-2 were cultured to the logarithmic growth phase (OD200). 600 =0.1±0.02), 100 μL of bacterial suspension was spread on plates of each concentration and incubated at 30℃ for 48 h. The lowest Hygr concentration corresponding to "no visible colony growth on the plate (or only small colonies with a diameter <0.5 mm and a single plate colony count ≤1)" was taken as the MIC. The MIC of MYL-2 against Hygr was measured to be 60 μg / mL. Figure 4 A).

[0069] (2) The MIC of bleomycin (Zeocin) was determined according to the method described above for Hygr. The MIC of MYL-2 against Zeocin was determined to be 25 μg / mL by screening with Zeocin concentration gradient plates. Figure 4 B).

[0070] 2. Chemical transformation of lithium acetate and screening of positive transformants

[0071] (1) Culture of strains

[0072] The MYL-2 glycerol-preserved strain was streaked onto YPD solid medium for resuscitation and activation. A single colony was picked and inoculated into 5 mL of YPD liquid medium, and cultured overnight at 30°C and 220 r / min with shaking to obtain a seed culture. The next day, the seed culture was transferred to 50 mL of YPD liquid medium, and the initial OD was adjusted. 600 The concentration was reduced to 0.2, and the mixture was incubated at 30℃ and 200 r / min for 4–5 h until the OD value was reached. 600 It reaches 0.8 to 1.0.

[0073] (2) Preparation of competent cells

[0074] Take 50 mL of mid-logarithmic bacterial culture, centrifuge at 4℃, 4000×g for 5 min, and discard the supernatant; resuspend the precipitate with 50 mL of pre-cooled sterile ddH2O and wash twice (centrifuge at 4℃, 4000×g for 5 min each time, and discard the supernatant); then resuspend the bacterial cells with 20 mL of pre-cooled 150 mM LiAc solution, centrifuge at 4℃, 4000×g for 5 min, and discard the supernatant; resuspend the final precipitate in 150 mM LiAc solution, dispense 1 mL into each of 5 tubes, centrifuge at 8000×g for 1 min to collect the precipitate, and obtain competent cells, which should be placed on ice for later use.

[0075] (3) Construction of transformation system

[0076] Take one tube of competent cells and add 240 μL of 50% (w / v) PEG 3350, 54 μL of 1 M LiAc, 50 μL of 2 mg / mL salmon sperm DNA, and approximately 500 ng of linearized recombinant plasmid in sequence. Use ddH2O to bring the volume to a final volume of 360 μL and mix gently to avoid generating air bubbles.

[0077] (4) Transformation and screening

[0078] The mixture was incubated at 30℃ and 200 r / min with shaking for 30 min; 34 μL of DMSO was added, gently mixed, and then heat-shocked at 37℃ for 30 min; subsequently, it was centrifuged at 4℃ and 4000×g for 5 min, and the supernatant was discarded; the precipitate was resuspended in 1 mL of YPD liquid medium and washed once (centrifuged at 4℃ and 6000×g for 3 min, and the supernatant was discarded), and then resuspended in 2 mL of YPD liquid medium, and cultured at 30℃ and 220 r / min with shaking for 3 h; the resuscitation solution was spread onto YPD solid screening plates containing the corresponding antibiotics and incubated upside down at 30℃ for 48–72 h until single colonies appeared; under these conditions, the transformation efficiency was measured to be approximately 1.34 × 10⁻⁶. 2 CFU / μg DNA.

[0079] (5) Verification of positive clones

[0080] Single colonies were picked from screening plates and positive clones were verified by colony PCR. The PCR products were detected by agarose gel electrophoresis, and clones whose amplified fragment size was consistent with the expectation were identified as positive candidate strains.

[0081] Example 3: Construction of the recombinant vector for *Cytomyces pinki* MYL-2 and Cre-LoxP system-mediated label reuse

[0082] 1. Strains and vectors

[0083] The host strain was *S. pararoseus* MYL-2. The vector backbone used was the *Yarrowia lipolytica* pYLXP plasmid; the screening drugs were hygromycin (Hygr) and bleomycin (Zeocin).

[0084] 2. Construction of recombinant expression vectors

[0085] Table 1. Primer sequences and applications for constructing the recombinant expression vector of *Cyclophorus rosenbergii* MYL-2.

[0086]

[0087]

[0088] (1) Construction of vector pNFH-loxp-RAD51:

[0089] ① Functional element amplification: Using the MYL-2 genome as a template, the TPI promoter (primer NFH-loxp-pTPI-F / NFH-pTPI-3R-YH-2) and the TEF promoter (primer NFH-N3-ov-loxp-F / NFH-pTEF1-R) were amplified; using the Saccharomyces cerevisiae genome as a template, the RAD51 coding region (primer NFH-RAD51-ov-P) was amplified. TEF The XPR2 terminator (primer XPR2_term-5F / NFH-loxp-Hygr-R) and the CYC1 terminator (primer Tcyc1-F / Tcyc1-R) were amplified using the *Yarrowia lipolytica* genome as a template. The loxP site standard recognition sequence 5'-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3' was selected. The Hygr resistance gene was optimized according to the MYL-2 codon preference and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (SEQ ID NO.1), and the amplification primers were NFH-Hygr-5F-YH / NFH-Hygr-3R-YH. All primers were designed using Geneious Primer and a 20 bp homologous arm was introduced at the 5' end (Table 1). The PCR products were purified after confirmation by 1% agarose gel electrophoresis.

[0090] ② Seamless Cloning and Verification: The purified DNA elements were mixed with the pYLXP plasmid backbone in a specific ratio and assembled using Gibson (50℃, 30 min) to obtain a circular recombinant plasmid. The assembly product was transformed into E. coli K1 competent cells, and screening was performed using ampicillin (100 μg / mL). Initial screening was conducted using colony PCR (primers RAD51-YZ-F / YLXP-YZ-1-3R). Positive clones were confirmed by sequencing and named pNFH-loxp-RAD51 (full length 9842 bp). Using pNFH-loxp-RAD51 as a template, NFH-loxp-P was amplified by PCR. TPI -Hygr-T XPR2 -loxp-P TEF -RAD51-T CYC1 A linear fragment (full length 5022 bp; primers NFH-UP-F / Tcyc1-R) was used to transform the MYL-2 strain using a lithium acetate chemical transformation method (same as in Example 2). Transformants were picked on YPD selection plates containing 60 μg / mL Hygr. Figure 5 The transformation efficiency was measured to be 48.2 CFU / μg DNA, and colony PCR was used for identification. Colony PCR was performed using the primer combination RAD51-YZ-F / T. CYC1-R and NFH-Hygr-5F-YH / NFH-Hygr-3R-YH; electrophoresis results are as follows Figure 6 As shown, clones whose amplified fragment size is consistent with the expected size are identified as positive candidate strains, and the MYL-2 / RAD51 engineered strain is obtained accordingly.

[0091] (2) Carrier pNFH-P TPI -Zeocin-T XPR2 -P TEF -Cre-T XPR2 -P GPD -eGFP-T XPR2 - PanARS Construction:

[0092] ① Acquisition of functional elements: Using a vector containing the Cre gene as a template, the coding region of the Cre gene was amplified by PCR (SEQ ID NO. 2; primer NFH-Cre-5F / NFH-Cre-3R); using the MYL-2 genome as a template, the TPI promoter (primer NFHpTPI-Hygr-5F / YLXP-NFHpTPI-3R), TEF promoter (primer NFH-TEF-Cre-F / NFH-pTEF-3R), and GPD promoter (primer NFH-pUC-eGFP-F / NFH-pGPD-3R-YH) were amplified by PCR; using the Yersinia lipolytica genome as a template, the XPR2 terminator (primer XPR2_term-5F / NFH-TEF-Cre-ov-eGFP-R) and the Zeocin resistance gene (SEQ ID NO. 3; primer NFH-Zeocin-5F / NFH-Zeocin-3R) and the eGFP gene (SEQ ID NO. 3) were amplified by PCR. NO.4; primers NFH-GFP-5F-YH-2 / NFH-GFP-3R-YH were optimized based on MYL-2 codon preference and chemically synthesized by a biotechnology company; the pan-species autonomously replicating sequence pan-ARS (SEQ ID NO.5) was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0093] ② Seamless cloning and verification: Following the aforementioned Gibson assembly and E. coli transformation screening process, the recombinant plasmid was sequenced to verify the element sequence, insertion direction, and absence of mutations, resulting in a recombinant plasmid (full length 9287 bp).

[0094] 3. Recycling of Hygr filter markers mediated by the Cre-LoxP system

[0095] (1) Yeast transformation: The Cre recombinant vector was introduced into the MYL-2 / RAD51 engineered strain using the lithium acetate chemical transformation method (same as in Example 2); the transformation product was plated on a YPD screening plate containing 25 μg / mL Zeocin and cultured at 28℃ for 48 h.

[0096] (2) Recovery and validation of Hygr selection marker: Single colonies were picked and streaked onto YPD plates containing 25 μg / mL Zeocin, 60 μg / mL Hygr, and antibiotic-free YPD plates, respectively. After colony growth, single colonies that could grow on Zeocin and antibiotic-free plates but not on Hygr plates were selected, and the enhanced green fluorescent protein (eGFP) fluorescence signal was observed under a microscope. Colonies with strong fluorescence signals were selected and inoculated into YPD liquid medium containing 25 μg / mL Zeocin and cultured with shaking at 30℃ and 220 r / min for 24 h. The above samples were inoculated onto antibiotic-free YPD plates and YPD plates containing 60 μg / mL Hygr, respectively; strains that could grow on antibiotic-free plates but not on Hygr plates were considered to have successfully recovered the Hygr selection marker. Figure 7 The above-mentioned strains were inoculated into antibiotic-free YPD liquid medium and cultured with shaking for 48 h to induce the cells to lose the Cre recombinant plasmid under non-selective pressure conditions.

[0097] 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 method for stably integrating exogenous DNA into *Cyclophorus pseudovae*, characterized in that, The steps are as follows: (1) Take the bacterial culture of *Syngonium rosenbergii*, wash and centrifuge, resuspend in pre-cooled LiAc solution, and the precipitate obtained by centrifugation is the competent cells; (2) Add PEG 3350, LiAc solution, salmon sperm DNA and exogenous DNA to competent cells in sequence, mix well and incubate with shaking, then add DMSO for heat shock and collect the cells; (3) The bacterial cells were resuspended in YPD liquid medium for resuscitation and culture, and then spread on YPD solid medium containing selection markers for screening. Colony PCR was used for verification to obtain positive transformants that stably integrated exogenous DNA.

2. The method according to claim 1, characterized in that: In step (1), the *Cyclophorus rosenbergii* is *Cyclophorus rosenbergii* MYL-2, which is classified as follows: Sporidiobolus pararoseus The accession number is CCTCC M20252968, the depositary institution is the China Center for Type Culture Collection, the deposit date is December 19, 2025, and the deposit address is Wuhan University, Wuhan, China; OD of the bacterial culture. 600 The concentration of the LiAc solution is 0.8–1.0 mM; the concentration of the LiAc solution is 100 mM–150 mM.

3. The method according to claim 2, characterized in that: In step (2), the mass concentration of PEG 3350 is 40%–50%, the concentration of LiAc solution is 1 M, and the concentration of salmon sperm DNA is 1–5 mg / mL; the shaking incubation conditions are 28–30℃ and 200–220 r / min for 30–60 min; and the heat shock conditions are 36–42℃ for 25–35 min.

4. The method according to claim 3, characterized in that: In step (3), the screening markers are Hygr or Zeocin, with the minimum inhibitory concentration of Hygr being 60 μg / mL and the minimum inhibitory concentration of Zeocin being 25 μg / mL.

5. An iterative gene editing method, characterized in that, The steps are as follows: 1) Using the method described in claim 4, a recombinant vector containing Cre recombinase is introduced into an engineered strain containing a loxP site, and the transformation product is plated on YPD medium containing Zeocin and positive transformants are obtained by screening. 2) Induce the expression of Cre recombinase in positive transformants to achieve the knockout of the target gene, and at the same time precisely excise the screening marker gene to obtain the gene-edited engineered strain; 3) Using the engineered strain from step 2) as the recipient, repeat steps 1) to 2) to complete the reuse of the screening marker gene, obtain engineered strains for multi-target gene editing, and realize iterative gene editing.

6. The iterative gene editing method according to claim 5, characterized in that: In step 1), the genome of the engineered strain integrates an expression cassette containing a promoter, a RAD51 coding gene, a target gene, and a terminator. The loxP recognition site is located on both sides of the selection marker and is oriented in the same direction. The selection marker gene is the resistance gene Hygr, with an optimized sequence as shown in SEQ ID NO.

1. The recombinant vector containing Cre recombinase contains a promoter, a terminator, the resistance gene Zeocin, the gene eGFP, and the replication element pan-ARS.

7. The iterative gene editing method according to claim 6, characterized in that: The promoter is TPI, TEF, or GPD, and the terminator is CYC1 or XPR2; the gene sequence encoding Cre recombinase is shown in SEQ ID NO.2; The optimized sequence of the resistance gene Zeocin is shown in SEQ ID NO.3, the optimized sequence of the gene eGFP is shown in SEQ ID NO.4, and the sequence of the replication element pan-ARS is shown in SEQ ID NO.

5.

8. The iterative gene editing method according to claim 7, characterized in that: The engineered strains from step 2) were inoculated onto culture media containing Zeocin, culture media containing Hygr, and culture media without antibiotics, respectively. Colonies that could grow on culture media containing Zeocin and culture media without antibiotics, could not grow on culture media containing Hygr, and showed an eGFP fluorescent signal were selected as engineered strains with precisely excised screening marker genes.

9. The application of the method according to any one of claims 1 to 4 or the iterative gene editing method according to any one of claims 5 to 8 in the targeted construction of microbial lipid-producing engineered strains.

10. The application according to claim 9, characterized in that: The chassis strain used for the directional construction was *Syndrome rosenbergii* MYL-2, with accession number CCTCC M 20252968.

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