Modularized genome transformation system of yarrowia lipolytica, transformation method and application

Abstract

The invention discloses a modular genome transformation system of yarrowia lipolytica, a transformation method and application. Belongs to the technical field of gene engineering. The system provided by the invention adopts a three-level modular design: an L0-level element is a standardized biological component library and comprises a promoter, a coding region, a terminator and other single functional units subjected to sequence domestication; the L0 component is combined into a complete functional module (such as a gene expression cassette or a selection marker) by the L1-level element through GoldenGate assembly; the L1-level element is a homologous recombinant vector targeting specific genome sites (such as KU80 and 26s rDNA), the L1 module is connected in series to a pUC57 skeleton with a homologous arm through a SapI enzyme, the method realizes accurate integration of multiple sites and multiple genes through a CRISPR-Cas9 auxiliary conversion and chemical conversion method, and solves the bottleneck problems of poor site adaptability, difficult iteration, high cost and the like in the prior art.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a modular genome modification system, modification method and application of Yersinia lipophila. Background Technology

[0002] As an unconventional yeast, Yersinia lipolytica has become one of the core chassis cells in the field of synthetic biology for the "carbon source-lipid / high value-added product" transformation due to its natural and efficient lipid accumulation ability (lipid content can reach 30%-70% of cell dry weight), broad-spectrum substrate utilization (it can utilize inexpensive carbon sources such as glycerol, lipids, alkanes and lignocellulose hydrolysates), strong heterologous protein secretion ability, and relatively mature genetic manipulation basis.

[0003] The Yersinia lipolyticis genome contains several identified sites suitable for homologous integration (such as KU70 / KU80, 26S rDNA, zeta, and multiple intergenic regions like YL_Js_P3 and YL_Js_P6). The sequence characteristics of these sites (e.g., GC content, flanking sequence conservation) directly affect homologous recombination efficiency. Currently, the construction of homologous repair fragments (key DNA fragments for gene knock-in / knockout) targeting these sites mainly relies on two types of technologies: In vitro synthetic assembly methods (such as Gibson assembly / GoldenGate assembly): 300-500 bp genomic homologous arms are commercially synthesized and then spliced ​​with a linearized vector using an enzyme ligation reaction. This method can achieve 100% assembly accuracy and is suitable for scenarios requiring strict sequence accuracy, but it is costly and time-consuming.

[0004] PCR-mediated fragment assembly (overlap extension PCR / fusion PCR): By designing primers with overlapping regions, homologous arms are directly spliced ​​to the target fragment after multiple rounds of PCR amplification. This method can complete the construction of fragments <5kb in length within 24 hours, is low-cost, and is suitable for the rapid assembly of non-standardized elements, but has a high amplification error rate.

[0005] However, the aforementioned existing technologies have the following key shortcomings that prevent them from meeting the needs of industrial transformation: Poor site adaptability: For different homologous integration sites, homologous arm sequences or PCR primers need to be redesigned. The lack of a unified adaptation system leads to low cross-site element reuse rate (<10%), and high cost and low efficiency of multi-site modification.

[0006] Site specificity issues: For special structural sites (such as the repetitive sequence of 26S rDNA, the high GC region of YL_Js_P5), the error rate of PCR-mediated amplification is >20%; although the in vitro synthesis method is precise, it requires additional optimization of homologous arm design (such as segmented synthesis), which extends the cycle by 1-2 weeks.

[0007] Iterative upgrades are difficult: when replacing a single element (such as a promoter), it is necessary to resynthesize homologous arms or design primers for the target site, making it impossible to achieve local modular modification, and extending the iteration cycle of multi-site modification by more than 50%.

[0008] Cost and cycle imbalance: When using in vitro synthesis methods to modify multiple sites, the cost of homologous arm synthesis increases exponentially; while PCR methods are low-cost, specific sites require multiple attempts, resulting in high hidden costs. Neither method can simultaneously meet the requirements of "multiple sites, high efficiency, and low cost".

[0009] There are no optimization methods for transformation efficiency: Both methods rely on the homologous recombination efficiency of yeast itself. For low-efficiency sites (such as YL_Js_P3, where the natural recombination efficiency is <5%), no targeted guidance strategy is provided, resulting in a high transformation failure rate, especially in the scenario of simultaneous transformation of multiple sites (failure rate >70%).

[0010] Therefore, how to develop a genome modification system for Yersinia lipophila that is "multi-site adapted, site-specific compatible, easy to iterate, low-cost and capable of optimizing transformation efficiency" to support its large-scale application in industrial scenarios such as multi-pathway synergistic optimization and multi-product co-synthesis is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0011] In view of this, the present invention develops a modular genome modification system, modification method and application for Yersinia lipophila, which solves the problems of poor site adaptability, insufficient compatibility of special sites, difficulty in iteration, imbalance of cost and cycle and low transformation efficiency in the existing technology.

[0012] To solve the above-mentioned technical problems, this application adopts the following technical solution: The primary objective of this application is to provide a modular genome modification system for Yersinia lipophila, the system comprising three levels of modular elements: L0 level elements, L1 level elements, and L2 level elements; the elements are assembled stepwise via standardized restriction enzyme site interfaces; The L0-level element is a homologous recombinant vector composed of a vector backbone and endogenous elements of *Yersinia lipophila*. The vector backbone contains BbsI and BsaI restriction endonuclease recognition sites. The endogenous elements of *Yersinia lipophila* are selected from promoters, target gene coding regions, or terminators. Each endogenous element of *Yersinia lipophila* has a pre-defined BbsI recognition sequence at its end. Furthermore, all BbsI, BsaI, and SapI recognition sites within each endogenous element of *Yersinia lipophila* have been removed by synonymous mutations. The L1 level components include functional module carriers and screening module carriers; The functional module vector is a homologous recombination vector composed of a vector backbone and L0-level elements; the vector backbone contains BsaI and SapI restriction endonuclease recognition sites. The screening module vector is a homologous recombination vector composed of a vector backbone and a screening marker; the vector backbone contains BsaI and SapI restriction endonuclease recognition sites; the screening marker is pre-programmed with a BsaI recognition sequence. The L2-level element is a homologous recombination vector consisting of a vector backbone and homologous arms, or a homologous recombination vector consisting of a vector backbone, homologous arms, and at least one L1-level element; the homologous arms are 300-500 bp DNA sequences designed for specific integration sites in the Yersinia lipolyticis genome; the vector backbone of the L2-level element contains multiple SapI restriction endonuclease recognition sites between the homologous arms for receiving the assembly of L1-level modules.

[0013] As a preferred technical solution, the vector backbone of the L0-level element is the pUAP1 plasmid; the promoter is selected from ylTEF, ylGAPHD, ylPGK, ylLEU2 or ylURA3; the coding region of the target gene is codon-optimized; and the terminator is CYC1.

[0014] As a preferred technical solution, the vector backbone of the L1-level element is a pCK1, pCK2, pCK3, or pCK4 plasmid; wherein pCK1, pCK2, and pCK3 are used to assemble the functional module vector, and pCK4 is used to assemble the screening module vector; the screening marker is the HisG-URA3-HisG gene fragment; the vector backbone of the L2-level element is a pUC57 plasmid; and the homologous arm is a homologous arm sequence targeting the Yersinia lipolytica genome.

[0015] As a preferred technical solution, the specific integration site of the Yersinia lipolytica genome is selected from KU80, YL_Js_P3, YL_Js_P6, YL_Js_P5, YL_Js_P1, YL_Js_P4, YL_Js_P2, zeta, or 26S rDNA sites.

[0016] Another objective of this application is to provide the application of the above-described system in the genome modification of Yersinia lipophila.

[0017] Another object of this application is to provide a method for genome modification of Yersinia lipophila using the above-described system, comprising the following steps: S1. Construction of L0-level elements: The selected endogenous elements of Yersinia lipolytica were cloned into the modified pUAP1 vector, and the key restriction sites inside the endogenous elements of Yersinia lipolytica were domesticated to obtain standardized L0-level element plasmids. S2. Constructing L1-level elements: Using the GoldenGate assembly reaction, the selected L0-level elements containing promoters, L0-level elements containing the coding region of the target gene, and L0-level elements containing terminators are assembled into pCK1, pCK2, or pCK3 vectors to form functional module vectors; or the selection markers are assembled into the pCK4 vector to form selection module vectors. S3. Constructing L2-level components: Using the GoldenGate assembly reaction, the selected L1-level functional modules and screening modules are assembled into the pUC57 vector with specific homologous arms in a predetermined order to form a complete homologous repair vector. S4. Preparation of homologous repair fragment: Using the L2-level vector obtained in S3 as a template, linearized homologous repair fragments were obtained by PCR amplification. These fragments contain homologous arms and all modules that need to be integrated. S5. Yeast Transformation and Screening: The homologous repair fragments obtained in S4 were transformed into Yeastia lipolyticis host cells. Positive transformants were screened using selective culture media, and CRISPR-Cas9-sgRNA plasmids were optionally co-transformed to improve integration efficiency. S6. Verification: Verify whether the target gene is precisely integrated into a specific site in the genome using colony PCR or sequencing.

[0018] The above methods can be used to achieve gene knockout and knock-in: Gene knockout: When there is no functional / selection module inside the L2 level element, PCR amplification of the fragment containing only homologous arms is performed, and the target gene is knocked out after being transformed into yeast. Gene knock-in: Transform L2-level element PCR products containing target functional modules and screening modules into yeast to achieve precise knock-in of single or multiple genes (≤3), such as overexpression of genes for key lipid synthesis enzymes (acetyl-CoA carboxylase).

[0019] As a preferred technical solution, the GoldenGate assembly reaction system for L0-level components in step S1 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pUAP1 empty vector; 0.30 μL of Yersinia lipophila endogenous element fragment containing a BbsI cleavage site; 10×T4 ligase buffer 1.00μL; T4 ligase 0.25 μL; BSA (1 mg / mL) 0.50 μL; 0.25 μL of BBSI (10 U / μL); Add ddH2O to a final volume of 10 μL; As a preferred technical solution, the GoldenGate assembly reaction system of the functional module carrier in step S2 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pCK1(2 / 3) empty vector; pUAP1 promoter 0.30 μL; pUAP1 - Encoding region 0.30 μL; pUAP1 terminator 0.30 μL; 10×T4 ligase buffer 1.00μL; T4 ligase 0.25 μL; BSA (1 mg / mL) 0.50 μL; 0.25 μL of BBSI (10 U / μL); Add ddH2O to a final volume of 10 μL; As a preferred technical solution, the GoldenGate assembly reaction system of the screening module carrier in step S2 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pCK4 empty vector; 0.30 μL of the HisG-URA3-HisG fragment containing the BsaI cleavage site; 10×T4 ligase buffer 1.00μL; T4 ligase 0.25 μL; BSA (1 mg / mL) 0.50 μL; 0.25 μL of BBSI (10 U / μL); Add ddH2O to a final volume of 10 μL; As a preferred technical solution, the GoldenGate assembly reaction system for L2 level components in step S3 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pUC57-homogeneous fragment empty vector; pCK1 - Functional Module 10.30μL; pCK2 - Functional Module 20.30μL; pCK3-Functional Module 30.30μL; pCK4-screening module 0.30μL; 10×rcut smart buffer1.00μL; T4 ligase 0.25 μL; SapI (10 U / μL) 0.50 μL; Add ddH2O to a final volume of 10 μL.

[0020] As a preferred technical solution, the host cell of Yersinia lipophila in step S5 is the Po1f strain.

[0021] As a preferred technical solution, the optional co-transformation plasmid in step S5 is the pYaliA1-hCas9 plasmid, which carries sgRNA designed for the target integration site.

[0022] Another object of this application is to provide: the engineered Yersinia lipophila strain constructed by the above method.

[0023] Another objective of this application is to provide the application of the engineered Yersinia lipolyticis strain constructed by the above method in the production of oils or high-value-added heterologous products.

[0024] Another object of this application is to provide a recombinant plasmid, said recombinant plasmid being pCK4-HisG-URA3-HisG.

[0025] Another object of this application is to provide: the application of the above-mentioned recombinant plasmid, wherein the application is in any of the following directions: (1) Application in constructing a modular genome modification system for Yersinia lipophila; (2) Application in genome modification of Yersinia lipophila; (3) Application in gene knock-in of Yersinia lipophila.

[0026] Another object of this application is to provide a recombinant plasmid, said recombinant plasmid being pUC57-KU80, pUC57-YL_Js_P3, pUC57-YL_Js_P6, pUC57-YL_Js_P5, pUC57-YL_Js_P1, pUC57-YL_Js_P4, or pUC57-YL_Js_P2.

[0027] Another object of this application is to provide that the application is in any of the following directions: (1) Application in constructing a modular genome modification system for Yersinia lipophila; (2) Application in genome modification of Yersinia lipophila; (3) Application in gene knock-in of Yersinia lipophila; (4) Application in gene knockout of Yersinia lipophila.

[0028] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Extremely high site adaptability and component reuse rate: By separating the homologous arm and functional module, the component reuse rate is close to 100%. Cross-site modification does not require redesigning components, which completely solves the problem of repeated design and synthesis in multi-site modification. It adapts to 9 typical sites and other potential sites, significantly reducing costs and cycle time.

[0029] (2) Excellent site-specific compatibility: The “domestication” step of L0-level elements removes internal restriction sites, ensuring that even special sites (such as the 26S rDNA high GC region and zeta repeat sequence region) can be assembled efficiently and accurately.

[0030] (3) Seamless iteration and rapid upgrade: hierarchical module splitting + independent design of site homology arms. If metabolic pathways need to be optimized, only a single L0 level component needs to be replaced (such as replacing a weak promoter with a strong promoter), and then standardized L1 and L2 level assembly can be performed again. There is no need to change the homology arms or other modules. The site iteration cycle is shortened to 1-2 days, realizing true "plug and play" and rapid iteration, and shortening the iteration cycle.

[0031] (4) Efficient balance between cost and cycle: The strategy of “synthesizing a small number of standardized elements + efficient enzymatic assembly” avoids the huge cost of synthesizing long fragments for each site. Once the L0 and L2 libraries are built, the newly constructed assembly can be completed in 3-5 days. The cost is mainly concentrated in enzyme preparations, which is far lower than that of commercial synthesis.

[0032] (5) Standardization and scalability: The entire system is built on the GoldenGate assembly standard, with unified interfaces and fixed operating procedures, making it easy to promote and apply in the laboratory. Application scenarios of the system in this application: Gene knockout: When there is no functional / screening module inside the L2 level element, PCR amplification only contains fragments with homologous arms, and after transformation into yeast, the target gene is knocked out; Gene knock-in: The PCR product of the L2 level element containing the target functional module is transformed into yeast to achieve precise knock-in of single genes / multiple genes (≤3), with strong application flexibility. Attached Figure Description

[0033] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0034] Figure 1 for: Figure 1 a represents the original vector map of pUAP1; Figure 1 b is a schematic diagram of an L0-level component with pUAP1 as the skeleton.

[0035] Figure 2 for: Figure 2 a represents the original vector pattern of pCK1 / pCK2 / pCK3; Figure 2 b is a schematic diagram of the L1 level functional module with pCK1 / pCK2 / pCK3 as the skeleton.

[0036] Figure 3 for: Figure 3 a represents the original pCK4 vector map; Figure 3 b is a schematic diagram of the L1 level screening module with pCK4 as the skeleton.

[0037] Figure 4 for: Figure 4 a is the original vector map of the pUC57-homogeneous arm; Figure 4 The diagram shows a L2-level element (recombination sequence) with pUC57-homogeneous arm as the backbone.

[0038] Figure 5 The results show the overexpression pathway construction of the MVA pathway in Yersinia lipophila. The wild-type band is approximately 1000 bp, and the MVA pathway integrated band is approximately 7800 bp. Here, wt represents the wild-type blank control, M represents the marker, i.e., the commercially available control band, and A, B, C, D, and E represent single colonies picked from the plate for verification. The band sizes of A, B, and D are wild-type, and the band sizes of C and E are the corresponding bands after successful integration.

[0039] Figure 6 The results are as follows: YALI1_A09939g knockout results. The lanes from left to right are: marker, wild-type control, experimental strain A, experimental strain B, experimental strain C, and experimental strain D. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The Po1f- strain (ATCC MYA-2613) used in the embodiments of the present invention was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0042] Example 1 A modular genome editing system for Yersinia lipophila, the construction process of which is as follows: S1. Construction of L0 level elements: (pUAP1 empty vector + lipolysinus endogenous elements) (1) Vector backbone: pUAP1 (source: https: / / doi.org / 10.1111 / nph.13532, which describes the properties and construction method of the plasmid) was used as the empty vector. This empty vector has a BbsⅠ recognition site (GAAGAC) added to the red fluorescent protein fragment. The pUAP1 backbone has a BsaI recognition site (GGTCTC) in the direction of the red fluorescent protein fragment. (2) Endogenous elements of Yersinia lipophila: selected from promoters, target gene coding regions or terminators; The promoter, target gene coding region, or both ends of the promoter mentioned above each have a pre-defined sequence, the composition of which is as follows: Two protective bases (gc) - BbsI recognition site (GAAGAC) - two spacer bases (tc) - BbsI cleavage site (CTCA) - BsaI pre-cleavage site.

[0043] When the intrinsic element is selected from the promoter, its BsaI pre-cleavage site is GGAG / AATG; When the intrinsic element is selected from the target coding region, its BsaI precut site is AATG / AGCC; When the intrinsic element is selected from the terminator, its BsaI pre-cleavage site is AGCC / CGCT; Taking an internal element as an example of a promoter: the preset sequence at both ends can be as shown in SEQ ID NO.1; or as shown in SEQ ID NO.2.

[0044] Promoters: DNA sequences on the genome of Yersinia lipolyticis that bind to RNA polymerase and initiate mRNA synthesis, such as ylTEF, ylGAPHD, ylPGK, ylLEU2, and ylURA3. Transcriptional activity covers 0.7%-100% TEF intensity. They can be obtained by PCR or by artificial synthesis.

[0045] Target gene coding region: (The region in the DNA structure that can transcribe messenger RNA (mRNA) and guide protein synthesis. It is generally obtained by PCR, but can also be obtained by artificial synthesis).

[0046] CYC terminator: (A DNA sequence that gives RNA polymerase a transcription termination signal, usually obtained by PCR, but can also be obtained by artificial synthesis).

[0047] (3) Domestication of endogenous elements in Yersinia lipophila: By inputting the sequence into the snapgene (7.1.2) software, the BbsI / BsaI / SapI sites inside the element are removed by nonsense mutation (such as site-directed PCR mutation) (e.g., replacing redundant enzyme digestion sequences in the coding region).

[0048] The sequences of the domesticated endogenous elements (excluding the pre-defined sequence, which was obtained by PCR after being added to primers) are as follows: ylTEF nucleotide sequence: SEQ ID NO.3.

[0049] ylGAPHD nucleotide sequence: SEQ ID NO.4.

[0050] ylPGK nucleotide sequence: SEQ ID NO.5.

[0051] ylLEU2 nucleotide sequence: SEQ ID NO.6.

[0052] ylURA3 nucleotide sequence: SEQ ID NO.7.

[0053] CYC1 nucleotide sequence: SEQ ID NO.8.

[0054] (4) Assembly of L0 level components GoldenGate L0-level devices are based on pUAP1 and incorporate various primary elements (such as promoters, coding genes, and terminators). They are constructed using the golden gate method. The reaction system is shown in Table 1, and the reaction procedure is shown in Table 2. Figure 1 a represents the original vector map of pUAP1; Figure 1 b is a schematic diagram of an L0-level component with pUAP1 as the skeleton.

[0055] Table 1 L0-level component reaction system Table 2 GoldenGate Assembly Procedure For the validation of L0-level elements (using PCR or sequencing by a sequencing company), the primers in Table 3 are common primers on the pUAP1 backbone. All L0-level elements can be validated using these primers. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4.

[0056] Table 3 PCR reaction system Table 4 PCR reaction procedure S2. Construct L1 level components: I. Functional Module Carrier: (pCK1 / pCK2 / pCK3 empty carrier + functional module) (1) Vector backbone: pCK1, pCK2 or pCK3 empty plasmids are used as the vector backbone (source: https: / / doi:10.1021 / acssynbio.9b00511, which describes the properties and construction methods of plasmids).

[0057] The empty vectors pCK1, pCK2, and pCK3 all have a BsaI recognition site (GGTCTC) on the outward side of the lacZ (blue-white screening) fragment, and the sticky ends produced after cleavage are GGAG / CGCT; the pCK backbone has a SapI recognition site (GCTCTTC) on the direction of the lacZ (blue-white screening) fragment, and the sticky ends produced after cleavage are 3bp (pCK1: ATG / GCA, pCK2: GCA / TAC, pCK3: GCA / CAG).

[0058] (2) Functional modules constructed by L0 level components: The functional modules constructed by L0 level components consist of a promoter, an encoding area, and a terminator (composed of three L0 level components).

[0059] (3) Assembly of the functional module carrier GoldenGate: pCK1, pCK2, and pCK3 were used to assemble functional module carriers with L0-level components, respectively, using the Goldengate method. The specific reaction systems and assembly procedures are shown in Tables 5 and 2. Figure 2 a represents the original vector pattern of pCK1 / pCK2 / pCK3; Figure 2 b is a schematic diagram of the L1 level functional module with pCK1 / pCK2 / pCK3 as the skeleton; Table 5 L1 Level Component Reaction System II. Screening module carrier: (pCK4 empty carrier + hisG-URA3-hisG screening marker) (1) Vector skeleton: pCK4 empty plasmid was used as the vector skeleton (source: https: / / doi:10.1021 / acssynbio.9b00511, which describes the properties and construction method of the plasmid).

[0060] The pCK4 empty vector has a BsaI recognition site (GGTCTC) on the outward side of the lacZ (blue-white screening) fragment, and the sticky ends produced after cleavage are GGAG / CGCT; on the pCK backbone, there is a SapI recognition site (GCTCTTC) on the direction of the lacZ (blue-white screening) fragment, and the sticky ends produced after cleavage are 3bp (pCK4: CAG / GGT).

[0061] (2) Selection marker: The selection marker is hisG-URA3-hisG (synthesized from the neutralizing gene, the sequence of which is shown in SEQ ID NO.5).

[0062] (3) Assembly of the GoldenGate screening module carrier: The pCK4 and the screening marker hisG-URA3-hisG were assembled into a screening module carrier using the Golden Gate method. The specific reaction system and assembly procedure are shown in Table 6 and Table 2. Figure 3 a represents the original pCK4 vector map; Figure 3 b is a schematic diagram of the L1 level screening module with pCK4 as the skeleton.

[0063] Table 6. pCK4-hisG-URA3-hisG Reaction System The validation of L1-level elements (using PCR or sequencing by a sequencing company) can be performed on both the functional and screening modules. The primer sequences used are the common sequences on the pCK1-4 backbone. The PCR reaction system is shown in Table 7, and the PCR reaction program is shown in Table 8.

[0064] Table 7 PCR Reaction System Table 8 PCR reaction procedure S3. Construct L2 level components: (1) Vector backbone: pUC57 was used as an empty plasmid (source: https: / / doi.org / 10.1038 / nbt.2375, which describes the properties and construction method of the plasmid) and modified in the laboratory to serve as the vector backbone. The specific modification process is as follows: Homologous arms: Yeast lipophila homologous arms (UP / DOWN, 300-500bp in length), containing lacZ (blue-white screening) fragments between homologous arms, with SapI recognition sites (GCTCTTC) added outwards to the lacZ (blue-white screening) fragments, and sticky ends generated after cleavage are ATG / GGT.

[0065] Amplification of each fragment required for homologous arm construction: The homologous arms of *Yersinia lipophila* (UP) (AT ratio ~70%, length 300-500bp), the homologous arms of *Yersinia lipophila* (DOWN) (AT ratio ~70%, length 300-500bp), the lacZ (blue-white screening) fragment, and the pUC57 backbone were amplified. A 17-25bp homologous region from adjacent fragments was added to the primer design for gebison ligation. The primer design is shown in Table 9.

[0066] Table 9 Primers used for constructing the pUC57-homologous arm The nucleotide sequences of the amplified fragments are as follows: KU80-UP nucleotide sequence: SEQ ID NO.53; KU80-DOWN nucleotide sequence: SEQ ID NO.54; YL_Js_P1-UP nucleotide sequence: SEQ ID NO.55; YL_Js_P1-DOWN nucleotide sequence: SEQ ID NO.56; YL_Js_P2 UP nucleotide sequence: SEQ ID NO.57; YL_Js_P2 DOWN nucleotide sequence: SEQ ID NO.58; YL_Js_P3-UP nucleotide sequence: SEQ ID NO.59; YL_Js_P3-DOWN nucleotide sequence: SEQ ID NO.60; YL_Js_P4-UP nucleotide sequence: SEQ ID NO.61; YL_Js_P4-DOWN nucleotide sequence: SEQ ID NO.62; YL_Js_P5 UP nucleotide sequence: SEQ ID NO.63; YL_Js_P5 DOWN nucleotide sequence: SEQ ID NO.64; YL_Js_P6-UP nucleotide sequence: SEQ ID NO.65; YL_Js_P6-DOWN nucleotide sequence: SEQ ID NO.66; 26S rDNA-UP nucleotide sequence: SEQ ID NO. 67; 26S rDNA-DOWN nucleotide sequence: SEQ ID NO. 68; ZETA-UP nucleotide sequence: SEQ ID NO.69; ZETA-DOWM nucleotide sequence: SEQ ID NO.70; pUC57 backbone nucleotide sequence: SEQ ID NO.71; lacZ nucleotide sequence: SEQ ID NO.72; The pUC57 backbone, lacZ fragment, and purified Yersinia lipolytica homologous arm (UP / DOWN) were mixed at a certain molar ratio and ligated using Gbison ligase. The reaction system and reaction procedure are shown in Table 10 and Table 11. A recombinant vector backbone containing Yersinia lipolytica homologous arm and lacZ selection marker was constructed.

[0067] Table 10 Gbison Reaction System Table 11 Gbison Reaction Procedure When the integration site is KU80, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arm (UP / DOWN) is as follows: pUC57-KU80: SEQ ID NO.73; When the integration site is YL_Js_P3, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arm (UP / DOWN) is as follows: pUC57-YL_Js_P3: SEQ ID NO.74; When the integration site is YL_Js_P6, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arms (UP / DOWN) is as follows: pUC57-YL_Js_P6: SEQ ID NO.75; When the integration site is YL_Js_P5, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arm (UP / DOWN) is as follows: pUC57-YL_Js_P5: SEQ ID NO.76; When the integration site is YL_Js_P1, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arm (UP / DOWN) is as follows: pUC57-YL_Js_P1: SEQ ID NO.77; When the integration site is YL_Js_P4, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arms (UP / DOWN) is as follows: pUC57-YL_Js_P4: SEQ ID NO.78; When the integration site is YL_Js_P2, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arm (UP / DOWN) is as follows: pUC57-YL_Js_P2: SEQ ID NO.79; When the integration site is zeta, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipophila homologous arm (UP / DOWN) is as follows: SEQ ID NO.80; When the integration site is 26S rDNA, the nucleotide sequence of the recombinant plasmid constructed using the pUC57 backbone, lacZ fragment, and purified Yersinia lipolyticis homologous arm (UP / DOWN) is as follows: SEQ ID NO.81; The recombinant vector backbone was transformed into competent E. coli cells, and blue-positive clones were selected by blue-white screening. The plasmid was extracted and sequenced to verify that the homologous arms and lacZ fragment were correctly inserted and the sequence was error-free.

[0068] (2) Assembly of L2 level component GoldenGate: The vector backbone containing the homologous arm of *Yersinia lipolytica* and the lacZ selection marker prepared above was assembled with three functional modules and one selection module in the L1 level element using the Golden Gate method: functional module 1 (from pCK1) - functional module 2 (from pCK2) - functional module 3 (from pCK3) - selection module (from pCK4). The reaction system and assembly procedure are shown in Tables 12 and 2. The L2 level element construction process and the schematic diagram of the final structure are shown in Table 2. Figure 4 As shown in b.

[0069] Table 12 L2 Level Component Reaction System Using the above methods, a modular genome modification system of Yersinia lipolyticis containing L0-level, L1-level, and L2-level elements was successfully constructed.

[0070] S4. Construct pYaliA1-hCas9 containing the corresponding site n20 sequence (optional) Vector backbone: pYaliA1-hCas9 was used as the original plasmid (purchased externally; the sequence map can be found in Addgene ID #87687). This plasmid was modified in the laboratory to serve as the vector backbone, with only the n20 sequence at the corresponding site modified. Circular PCR was used, and the specific modification process is as follows: Table 13 Circular PCR primers used for pYaliA1-hCas9 site construction Table 14 PCR amplification reaction system Table 15 PCR Amplification Reaction Procedure The nucleotide sequences of the amplified fragments are as follows: pYaliA1-hCas9-KU80: SEQ ID NO.100; pYaliA1-hCas9-YL_Js_P1: SEQ ID NO.101; pYaliA1-hCas9-YL_Js_P2: SEQ ID NO.102; pYaliA1-hCas9-YL_Js_P3: SEQ ID NO.103; pYaliA1-hCas9-YL_Js_P4: SEQ ID NO.104; pYaliA1-hCas9-YL_Js_P5: SEQ ID NO.105; pYaliA1-hCas9-YL_Js_P6: SEQ ID NO.106; pYaliA1-hCas9-26srDNA: SEQ ID NO.107; pYaliA1-hCas9-ZETA: SEQ ID NO. 108.

[0071] After removing the template by treating the above PCR product with DpnⅠ, it can be directly transformed into E. coli competent cells to obtain the corresponding plasmid.

[0072] Example 2 The overexpression pathway of the MVA (mevaleric acid pathway) in *Yarrowia lipolytica* was constructed by overexpressing three genes—ERG10 (acetoacetyl-CoA thiolase), ERG13 (hydroxymethylglutaryl-CoA synthase), and tHMG1 (truncated 3-hydroxy-3-methylglutaryl-CoA reductase)—at the KU80 site in the *Yarrowia lipolytica* genome. The specific process is as follows: S1. Construction of L0 level elements: (pUAP1 empty vector + lipolysinus endogenous elements) L0 level elements include pUAP1-ylTEF (promoter endogenous element), pUAP1-CYC1 (terminator endogenous element), pUAP1-ERG10, pUAP1-ERG13, and pUAP1-tHMG1 (three target gene coding region endogenous elements). (1) Using the genome of lipophila as a template, PCR amplification was performed using the primer sets in the table below to obtain fragments containing cleavage sites and to recover the fragments. The specific primer set sequences are shown in Table 16, the amplification reaction system is shown in Table 17, and the reaction procedure is shown in Table 18.

[0073] Table 16 Primers used for overexpression of the lipolysis MVA pathway Table 17 PCR Amplification Reaction System Table 18 PCR Amplification Reaction Procedure The amplified ylTEF, CYC1, ERG10, ERG13, and tHMG1 sequences are as follows: ylTEF: SEQ ID NO. 3; CYC1: SEQ ID NO. 8; ERG10: SEQ ID NO.119; ERG13: SEQ ID NO.120; tHMG1: SEQ ID NO.121.

[0074] (2) Assembly of L0 level components GoldenGate Following the reaction system in Table 1 and the assembly procedure in Table 2 of Example 1, five L0-level elements were assembled: pUAP1-ylTEF (promoter endogenous element), pUAP1-CYC1 (terminator endogenous element), pUAP1-ERG10, pUAP1-ERG13, and pUAP1-tHMG1 (three target gene coding region endogenous elements).

[0075] (3) Transformation and screening The five L0-level elements obtained above were introduced into E. coli DH5α competent cells, and single clones were obtained through resistance selection. The specific process is as follows: Transformation: Take 5 μL of ligation product, add 100 μL of competent cells in an ice bath, mix gently, and incubate on ice for 30 min; Heat shock at 42℃ for 45 seconds (to create pores in the cell membrane and allow plasmids to enter the cell), then immediately place on ice for 2 minutes; add 900 μL LLB liquid culture medium (antibiotic-free), and culture at 37℃ with shaking at 200 rpm for 1 hour (to allow the bacteria to recover and express the plasmid resistance gene).

[0076] Screening for single clones: Take 100 μL of culture and spread it evenly on an LB solid plate containing the corresponding antibiotic (pUAP1 contains the chloramphenicol resistance gene); incubate upside down at 37°C for 12-16 h, and single clones will form on the plate.

[0077] (4) Verification The L0-level components were validated using PCR. The reaction system and procedure were the same as those for the L0-level components in Example 1 (Tables 3 and 4). The L0-level components that were validated correctly were then used for subsequent operations.

[0078] S2. Construct L1 level components: L1 level components include pCK1-TEFin-ERG10-tCYC1, pCK2-TEFin-ERG13-tCYC1, pCK3-TEFin-tHMG1-tCYC1 (functional module carriers) and pCK4-hisG-URA3-hisG (screening module carriers).

[0079] I. Functional Module Carrier: (pCK1 / pCK2 / pCK3 empty carrier + functional module) The L0-level elements constructed in S1 were connected into a complete plasmid using the Golden Gate method. The reaction system and assembly procedure are shown in Table 19 and Table 2, respectively.

[0080] Table 19 L1 Level Component Reaction System II. Screening module carrier: (pCK4 empty carrier + hisG-URA3-hisG screening marker) The pCK4 skeleton was fitted with the hisG-URA3-hisG fragment in the Golden Gate manner. (1) Amplification of hisG-URA3-hisG fragment Using the Yersinia lipolyticis genome as a template, the hisG-URA3-hisG fragment was amplified using the primers in Table 20. The specific reaction system and reaction procedure are shown in Tables 21 and 22.

[0081] Table 20 Primers used for the hisG-URA3-hisG fragment Table 21 PCR amplification reaction system Table 22 PCR Amplification Reaction Procedure hisG-URA3-hisG nucleotide sequence: SEQ ID NO.124.

[0082] (2) The pCK4 backbone was ligated to the hisG-URA3-hisG fragment obtained in step (1) using the Golden Gate method to form a complete plasmid. The reaction system and assembly procedure are shown in Table 23 and Table 2, respectively.

[0083] Table 23 pCK4-hisG-URA3-hisG Reaction System The nucleotide sequence of pCK4-hisG-URA3-hisG is as follows: SEQ ID NO.125.

[0084] Verification of L1 level components The L1-level components were validated using PCR. The reaction system and procedure were the same as those for the L1-level components in Example 1 (Tables 7 and 8). The L0-level components that were validated correctly were then used for subsequent operations.

[0085] S3. Construct L2 level components: The L2-level components are assembled using the golden gate method with the pUC57-homologous arm carrier backbone containing the homologous arm sequence and lacZ (blue-white screening) sequence, along with three functional modules and one screening module: TEFin-ERG10-tCYC1 (from pCK1)-TEFin-ERG13-tCYC1 (from pCK2)-TEFin-tHMG1-tCYC1 (from pCK3)-hisG-URA3-hisG (from pCK4). The reaction system and assembly procedure are shown in Table 24 and Table 2, respectively.

[0086] Table 24 L2 Level Component Reaction System S4. Preparation of homologous repair fragments: Using the constructed L2-level element plasmid as a template, linear homologous repair fragments were obtained by PCR amplification using specific primers and recovered using a kit. The specific homologous fragment PCR reaction system and reaction procedure are shown in Tables 25 and 26, respectively.

[0087] Table 25 PCR amplification reaction system Table 26 PCR Amplification Reaction Procedure S5. Yeast Transformation and Screening: (1) Host strain: Yarrowia lipolytica Po1f strain.

[0088] (2) Transformation method: a. Host pretreatment: Streak the Po1f strain onto YPD plates (or resuspend in 100 μL of sterile water and then plate) and incubate at 30°C for 24 h; resuspend the colonies in 1 mL of sterile water, centrifuge at 3000 g for 5 min, wash twice, and finally collect 5 × 10⁻⁶ colonies. 7 One cell (OD600=5) was prepared for use; b. Preparation of transformation mixture: Add 500 ng of L2-stage vector PCR product to the cell pellet, then add the transformation mixture (PEG 87.5 μL + LiAc 5.0 μL + ssDNA 2.5 μL + DTT 5.0 μL), and gently mix. c. Transformation and screening: Incubate at 39℃ for 60 min, centrifuge at 3000g for 5 min, discard the supernatant; resuspend in 500 μL LYPD medium, incubate at 30℃ with shaking for 2 h (recovery culture); centrifuge again, resuspend in 100 μL sterile water, inoculate into SD-URA3 selective medium, incubate at 30℃ for 48 h, and screen for positive transformants; S6. PCR verification of Yersinia lipophila colonies: Single colonies were picked from the transformation plate and transferred to 300 µL LYPD medium. The culture was carried out overnight for approximately 12-16 hours until the bacterial cells became slightly turbid. The genome was then crudely extracted for PCR verification. The specific procedure is as follows: (1) Pipette 150 µL of bacterial culture into a new 1.5 mL EP tube, centrifuge at 12000 g for 3 min, discard the supernatant, and store the remainder in a 4℃ refrigerator for later use; (2) Add 100 µL of cell lysis buffer containing 200 mM lithium acetate and 1% SDS, vortex to mix, and incubate at 70 °C for 5 minutes; (3) After incubation, add 300 µL of anhydrous ethanol, vortex for 15 s, centrifuge at 12000 g for 3 min, and discard the supernatant (a white precipitate will appear at the bottom of the EP tube). (4) Add 300 µL of 70% anhydrous ethanol, wash, centrifuge at 12000 g for 3 min, discard the supernatant, and use a pipette tip to remove the liquid; (5) Place in a metal bath at 60 °C for 5 min, evaporate the ethanol, add 100 µL ddH2O, gently blow and mix, and centrifuge at 12000 g for 30 s; (6) Take 1 µL of supernatant as a template for PCR. The reaction system and reaction procedure for PCR amplification are shown in Table 27 and Table 28.

[0089] Table 27 PCR Reaction System Table 28 PCR Reaction Procedure Results Analysis: The experimental results are as follows: Figure 5 The wild-type band before integration of the MVA pathway overexpression pathway was approximately 1000 bp, and the band after integration was approximately 7800 bp, indicating that the integration experiment was successful.

[0090] Example 3 Knockout of YALI1_A09939g genome of Yersinia lipolyticis Since this embodiment only requires knocking out the YALI1_A09939g gene, it does not require L0 or L1 level elements. Only L2 level elements containing specific homologous arms need to be constructed for gene knockout. The specific process is as follows: S1. Construct the pUC57-YALI1_A09939g plasmid (see schematic diagram for plasmid reference). Figure 4 (pUC57-homologous arm in a) (1) Using the genome of Yersinia lipophila as a template, PCR amplification of each fragment was performed using the primer set in Table 29, and each amplified fragment was recovered.

[0091] Table 29 Primers used to construct pUC57-YALI1_A09939g plasmid YALI1_A09939g-UP nucleotide sequence: SEQ ID NO.134; YALI1_A09939g-DOWN nucleotide sequence: SEQ ID NO.135; LacZ: SEQ ID NO.72; pUC57: SEQ ID NO.71.

[0092] (2) The pUC57 backbone and lacZ fragment obtained in step (1) were mixed with the purified Yersinia lipolyticis pUC57-YALI1_A09939 homologous arm (UP / DOWN) at a certain molar ratio, and ligation was performed using Gbison ligase to construct a recombinant plasmid (pUC57-YALI1_A09939) containing the Yersinia lipolyticis homologous arm and the lacZ selection marker. The Gbison operation method is as follows: Table 30 Gbison Reaction System Table 31 Gbison Reaction Procedure pUC57-YALI1_A09939 nucleotide sequence: SEQ ID NO.136.

[0093] (3) The recombinant plasmid was transformed into competent Escherichia coli cells. Blue positive clones were selected by blue-white screening. The plasmid was extracted and sequenced to verify that the homologous arm and lacZ fragment were correctly inserted and the sequence was correct. The pUC57-homologous fragment empty vector was obtained.

[0094] S2. Yeast Transformation and Screening (1) Host strain: Yarrowia lipolytica Po1f strain.

[0095] (2) Transformation method: a. Host pretreatment: Streak the Po1f strain onto YPD plates (or resuspend in 100 μL of sterile water and then plate) and incubate at 30°C for 24 h; resuspend the colonies in 1 mL of sterile water, centrifuge at 3000 g for 5 min, wash twice, and finally collect 5 × 10⁻⁶ colonies. 7 One cell (OD600=5) was prepared for use; b. Preparation of transformation mixture: Add 500 ng of L2-stage vector PCR product to the cell pellet, then add the transformation mixture (PEG 87.5 μL + LiAc 5.0 μL + ssDNA 2.5 μL + DTT 5.0 μL), and gently mix. c. Transformation and screening: Incubate at 39℃ for 60 min, centrifuge at 3000g for 5 min, discard the supernatant; resuspend in 500 μL LYPD medium, incubate at 30℃ with shaking for 2 h (recovery culture); centrifuge again, resuspend in 100 μL sterile water, inoculate into SD-URA3 selective medium, incubate at 30℃ for 48 h, and screen for positive transformants.

[0096] S3. PCR verification of Yersinia lipophila colonies Single colonies were picked from the transformation plate and transferred to 300 µL LYPD medium. The culture was carried out overnight for approximately 12-16 hours until the bacterial cells became slightly turbid. The genome was then crudely extracted for PCR verification. The specific procedure is as follows: (1) Pipette 150 µL of bacterial culture into a new 1.5 mL EP tube, centrifuge at 12000 g for 3 min, discard the supernatant, and store the remainder in a 4℃ refrigerator for later use.

[0097] (2) Add 100 µL of cell lysis buffer containing 200 mM lithium acetate and 1% SDS, vortex to mix, and incubate at 70 °C for 5 minutes.

[0098] (3) After incubation, add 300 µL of anhydrous ethanol, vortex for 15 s, centrifuge at 12000 g for 3 min, and discard the supernatant (a white precipitate will appear at the bottom of the EP tube).

[0099] (4) Add 300 µL of 70% anhydrous ethanol, wash, centrifuge at 12000 g for 3 min, discard the supernatant, and use a pipette tip to remove the liquid.

[0100] (5) Place in a metal bath at 60 °C for 5 min, evaporate the ethanol, add 100 µL ddH2O, gently blow and mix, and centrifuge at 12000 g for 30 s.

[0101] (6) Take 1 µL of supernatant as a template for PCR. The reaction system and reaction procedure for PCR amplification are shown in Table 32 and Table 33.

[0102] Table 32 PCR Reaction System Table 33 PCR Reaction Procedure Results Analysis: The experimental results are as follows: Figure 6Before the knockout, YALI1_A09939g had a band of 2328 bp, which was between the 2000-3000 standard bands in the electrophoresis gel. After the knockout, two bands appeared, with ~1000 and ~500 bp respectively. The experimental strain lacked >1000 bp compared with the wild-type control, indicating that the knockout experiment was successful.

[0103] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0104] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A modular genome modification system for Yersinia lipophila, characterized in that, The system comprises three levels of modular components: L0 level components, L1 level components, and L2 level components; these components are assembled step-by-step via standardized enzyme cleavage site interfaces. The L0-level element is a homologous recombinant vector composed of a vector backbone and endogenous elements of *Yersinia lipophila*. The vector backbone contains BbsI and BsaI restriction endonuclease recognition sites. The endogenous elements of *Yersinia lipophila* are selected from promoters, target gene coding regions, or terminators. Each endogenous element of *Yersinia lipophila* has a pre-defined BbsI recognition sequence at its end. Furthermore, all BbsI, BsaI, and SapI recognition sites within each endogenous element of *Yersinia lipophila* have been removed by synonymous mutations. The L1 level components include functional module carriers and screening module carriers; The functional module vector is a homologous recombination vector composed of a vector backbone and L0-level elements; the vector backbone contains BsaI and SapI restriction endonuclease recognition sites. The screening module vector is a homologous recombination vector composed of a vector backbone and a screening marker; the vector backbone contains BsaI and SapI restriction endonuclease recognition sites; the screening marker is pre-programmed with a BsaI recognition sequence. The L2-level element is a homologous recombination vector consisting of a vector backbone and homologous arms, or a homologous recombination vector consisting of a vector backbone, homologous arms, and at least one L1-level element; the homologous arms are 300-500 bp DNA sequences designed for specific integration sites in the Yersinia lipolyticis genome; the vector backbone of the L2-level element contains multiple SapI restriction endonuclease recognition sites between the homologous arms for receiving the assembly of L1-level modules.

2. The system according to claim 1, characterized in that, The vector backbone of the L0-level element is the pUAP1 plasmid; the promoter is selected from ylTEF, ylGAPHD, ylPGK, ylLEU2, or ylURA3; the coding region of the target gene is codon-optimized; and the terminator is CYC1. The vector backbone of the L1 level element is pCK1, pCK2, pCK3 or pCK4 plasmid; wherein pCK1, pCK2, pCK3 are used to assemble the functional module vector, and pCK4 is used to assemble the screening module vector; the screening marker is the HisG-URA3-HisG gene fragment; the vector backbone of the L2 level element is pUC57 plasmid. The specific integration sites in the Yersinia lipolyticis genome are selected from KU80, YL_Js_P3, YL_Js_P6, YL_Js_P5, YL_Js_P1, YL_Js_P4, YL_Js_P2, zeta, or 26S rDNA sites.

3. The application of the system according to any one of claims 1-2 in the genome modification of Yersinia lipophila.

4. A method for genome modification of Yersinia lipophila using the system described in any one of claims 1-2, characterized in that, Includes the following steps: S1. Construction of L0-level elements: The selected endogenous elements of Yersinia lipolytica were cloned into the modified pUAP1 vector, and the key restriction sites inside the endogenous elements of Yersinia lipolytica were domesticated to obtain standardized L0-level element plasmids. S2. Constructing L1-level elements: Using the GoldenGate assembly reaction, the selected L0-level elements containing promoters, L0-level elements containing the coding region of the target gene, and L0-level elements containing terminators are assembled into pCK1, pCK2, or pCK3 vectors to form functional module vectors; or the selection markers are assembled into the pCK4 vector to form selection module vectors. S3. Constructing L2-level components: Using the GoldenGate assembly reaction, the selected L1-level functional modules and screening modules are assembled into the pUC57 vector with specific homologous arms in a predetermined order to form a complete homologous repair vector. S4. Preparation of homologous repair fragment: Using the L2-level vector obtained in S3 as a template, linearized homologous repair fragments were obtained by PCR amplification. These fragments contain homologous arms and all the module vectors that need to be integrated. S5. Yeast Transformation and Screening: The homologous repair fragments obtained in S4 were transformed into Yeastia lipolyticis host cells. Positive transformants were screened using selective culture media, and CRISPR-Cas9-sgRNA plasmids were optionally co-transformed to improve integration efficiency. S6. Verification: Verify whether the target gene is precisely integrated into a specific site in the genome using colony PCR or sequencing.

5. The method according to claim 4, characterized in that, The GoldenGate assembly reaction system for L0 level components in step S1 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pUAP1 empty vector; 0.30 μL of Yersinia lipophila endogenous element fragment containing a BbsI cleavage site; 10×T4 ligase buffer 1.00μL; T4 ligase 0.25 μL; BSA (1 mg / mL) 0.50 μL; 0.25 μL of BBSI (10 U / μL); Add ddH2O to a final volume of 10 μL; The GoldenGate assembly reaction system for the functional module carrier in step S2 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pCK1(2 / 3) empty vector; pUAP1 promoter 0.30 μL; pUAP1 - Encoding region 0.30 μL; pUAP1 terminator 0.30 μL; 10×T4 ligase buffer 1.00μL; T4 ligase 0.25 μL; BSA (1 mg / mL) 0.50 μL; 0.25 μL of BBSI (10 U / μL); Add ddH2O to a final volume of 10 μL; The GoldenGate assembly reaction system for the screening module carrier in step S2 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pCK4 empty vector; 0.30 μL of the HisG-URA3-HisG fragment containing the BsaI cleavage site; 10×T4 ligase buffer 1.00μL; T4 ligase 0.25 μL; BSA (1 mg / mL) 0.50 μL; 0.25 μL of BBSI (10 U / μL); Add ddH2O to a final volume of 10 μL; The GoldenGate assembly reaction system for L2 level components in step S3 is as follows: The total volume of the goldengate system was 10 μL. 0.30 μL of pUC57-homogeneous fragment empty vector; pCK1 - Functional Module 10.30μL; pCK2 - Functional Module 20.30μL; pCK3-Functional Module 30.30μL; pCK4-screening module 0.30μL; 10×rcut smart buffer1.00μL; T4 ligase 0.25 μL; SapI (10 U / μL) 0.50 μL; Add ddH2O to a final volume of 10 μL; In step S5, the Yeast lipolyticis host cell is the Po1f strain; the optional co-transformation plasmid is the pYaliA1-hCas9 plasmid, which carries an sgRNA designed for the target integration site.

6. The engineered Yersinia lipophila strain constructed by the method described in any one of claims 4-5.

7. The use of the engineered Yersinia lipophila strain according to claim 6 in the production of oils or high-value-added heterologous products.

8. A recombinant plasmid, characterized in that, The recombinant plasmid is pCK4-HisG-URA3-HisG.

9. The application of the recombinant plasmid according to claim 8, characterized in that, The application is in any of the following directions: (1) Application in constructing a modular genome modification system for Yersinia lipophila; (2) Application in genome modification of Yersinia lipophila; (3) Application in gene knock-in of Yersinia lipophila.

10. A recombinant plasmid, characterized in that, The recombinant plasmids are pUC57-KU80, pUC57-YL_Js_P3, pUC57-YL_Js_P6, pUC57-YL_Js_P5, pUC57-YL_Js_P1, pUC57-YL_Js_P4, or pUC57-YL_Js_P2.

11. The application of the recombinant plasmid according to claim 10, characterized in that, The application is in any of the following directions: (1) Application in constructing a modular genome modification system for Yersinia lipophila; (2) Application in genome modification of Yersinia lipophila; (3) Application in gene knock-in of Yersinia lipophila; (4) Application in gene knockout of Yersinia lipophila.

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