Library construction and screening method for integrating multi-copy methanol into yarrowia lipolytica by using key genes

Abstract

The invention discloses an engineering strain for enhancing methanol metabolism capability of yarrowia lipolytica as well as a construction method and application of the engineering strain. The construction method comprises the following steps: by taking a pWLH plasmid as a carrier, integrating a non-homologous end connection (NHEJ) repair gene DL4, PAXX and an XRCC4 expression cassette, constructing a recombinant plasmid pWLH-DL4-PAXX-XRCC4, and transforming yarrowia lipolytica Po1f, so as to obtain an NHEJ enhanced strain PO1f / pWLH-DL4-PAXX-XRCC4; the method comprises the following steps: respectively constructing recombinant plasmids pWLH-Aox-Das-Dak and pWLH-Mdh-Hps-Ph i by using methanol metabolism key genes Aox, Das and Dak as well as Mdh, Hps and Ph i; the Xump and Rump path fragments of the plasmid are connected with a truncated Ura3 tag (Ura41), and a multi-copy methanol utilization gene integration library is constructed through five rounds of iterative transformation and SC-Ura screening; the methanol consumption is quantified through the cell accumulation amount and high performance liquid chromatography (HPLC), and the efficient methanol utilization strain is obtained through screening. According to the constructed engineering strain, by enhancing NHEJ-mediated multi-copy gene integration, the methanol metabolism efficiency of yarrowia lipolytica is remarkably improved, and a new strategy is provided for development of non-natural methylotrophic chassis cells.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a method for constructing a multi-copy methanol utilization key gene integrated Yarrowia lipolytica library mediated by a non-homologous end-joining mechanism, and a method for screening yeasts with strong methanol utilization ability. Background Art

[0002] Methanol, a low-cost, easily storable "liquid carbon source," is an important raw material in biomanufacturing. It can be obtained through low-carbon processes such as electrochemical reduction of carbon dioxide or natural gas reforming, and has significant potential for carbon emission reduction. However, genetic manipulation tools for natural methylotrophic microorganisms (such as some bacteria and yeast) are scarce, and their product spectrum is limited, making it difficult to meet the needs of industrial production of complex compounds. Therefore, modifying commonly used industrial microorganisms (such as Yarrowia lipolytica) to achieve efficient methanol assimilation has become a research hotspot in recent years.

[0003] Yarrowia lipolytica is a GRAS-certified microorganism certified by the U.S. Food and Drug Administration (FDA). It has an efficient acetyl-CoA metabolic pathway, strong robustness, and high lipid accumulation capacity, and has been widely used in the production of organic acids, oils, and secondary metabolites. However, wild-type Yarrowia lipolytica lacks a natural methanol assimilation pathway and cannot directly use methanol as a carbon source. Current studies have mostly attempted to construct synthetic methylotrophic strains by introducing exogenous methanol oxidase (AOX), formaldehyde assimilation pathways (such as the RuMP / XuMP cycle), and formate metabolism-related genes. However, existing technologies mostly rely on plasmid expression systems or single-copy genome integration, which have problems such as genetic instability, high metabolic burden, and insufficient expression intensity, resulting in low methanol utilization efficiency.

[0004] Traditional gene integration techniques are mostly based on homologous recombination (HR), which relies on homology arm design and host genome repair systems (such as the Ku70 protein). This results in low efficiency and difficulty in precisely controlling copy number. Furthermore, the Yarrowia lipolytica genome exhibits significant positional effects, with the expression intensity of exogenous genes varying by more than 20-fold at different integration sites, further limiting the optimization of metabolic pathways.

[0005] Non-homologous end joining (NHEJ) is a DNA repair mechanism that is independent of homologous sequences and can achieve multi-copy gene integration through random insertion. It is particularly suitable for transforming industrial strains that lack efficient homologous recombination. Existing research has shown that by optimizing NHEJ efficiency (e.g., by modulating the activity of host DNA repair proteins) and combining it with multiple rounds of transformation screening, efficient multi-copy gene integration can be achieved in Yarrowia lipolytica, significantly improving the flux of target metabolic pathways.

[0006] Currently, research on engineering Yarrowia lipolytica to utilize methanol is mostly focused on the modification of genes related to the methanol metabolism pathway. There has been no report on enhancing the methanol utilization of Yarrowia lipolytica by increasing the copy number of key genes for methanol utilization by enhancing non-homologous end joining in Yarrowia lipolytica. Summary of the Invention

[0007] The primary purpose of the present invention is to provide a library construction and screening method for integrating multiple copies of key methanol utilization genes into Yarrowia lipolytica to overcome the disadvantage of low methanol metabolism efficiency in the prior art.

[0008] The second object of the present invention is to provide a method for constructing an engineered strain that enhances the repair efficiency of non-homologous end joining (NHEJ) in Yarrowia lipolytica.

[0009] The third object of the present invention is to provide a method for constructing a recombinant plasmid of key genes in the methanol metabolism pathway.

[0010] The fourth object of the present invention is to provide a highly efficient methanol-utilizing Yarrowia lipolytica strain obtained by the above method and its application in industrial biocatalysis.

[0011] The technical solution of the present invention is summarized as follows:

[0012] A method for constructing a multi-copy methanol utilization key gene library using the NHEJ repair efficiency of Yarrowia lipolytica includes the following steps:

[0013] The method for constructing an engineered strain for enhancing the NHEJ repair efficiency of Yarrowia lipolytica comprises the following steps:

[0014] (1) Using the pWLH plasmid as a template, primers pWLH-octt-F and pWLH-clone-R were used to amplify the vector fragment. Using the LD01-DL4, LD02-PAXX, and LD03-XRCC4 plasmids as templates, the DL4, PAXX, and XRCC4 expression cassettes were amplified, respectively. The vector and expression cassette fragments were assembled by Gibson ligation to obtain the recombinant plasmid pWLH-DL4-PAXX-XRCC4.

[0015] The nucleotide sequence of the DL4 gene is shown in SEQ ID NO.1;

[0016] The nucleotide sequence of the PAXX gene is shown in SEQ ID NO.2;

[0017] The nucleotide sequence of the XRCC4 gene is shown in SEQ ID NO.3;

[0018] (2) The recombinant plasmid pWLH-DL4-PAXX-XRCC4 was transformed into Yarrowia lipolytica Po1 f to obtain the engineered strain PO1 f / pWLH-DL4-PAXX-XRCC4.

[0019] The method for constructing a recombinant plasmid for the methanol metabolism pathway comprises the following steps:

[0020] (1) Construction of plasmid pWLH-Aox-Das-Dak:

[0021] The vector pWLH2 was amplified using pWLH as a template; the Aox, Das, and Dak expression cassettes were amplified using CYRYLME001, CYRYLME002, and CYRYLME004 plasmids as templates, respectively, and the recombinant plasmid pWLH-Aox-Das-Dak was obtained by Gibson ligation.

[0022] The nucleotide sequence of the Aox gene is shown in SEQ ID NO.4;

[0023] The nucleotide sequence of the Das gene is shown in SEQ ID NO.5;

[0024] The nucleotide sequence of the Dak gene is shown in SEQ ID NO.6;

[0025] (2) Construction of plasmid pWLH-Mdh-Hps-Phi:

[0026] The vector was amplified using pWLH as a template; the Mdh, Hps, and Phi expression cassettes were amplified using CYRYLME008, CYRYLME009, and CYRYLME010 plasmids as templates, respectively, and the recombinant plasmid pWLH-Mdh-Hps-Phi was obtained by Gibson ligation.

[0027] The nucleotide sequence of the Mdh gene is shown in SEQ ID NO.7;

[0028] The nucleotide sequence of the Hps gene is shown in SEQ ID NO.8;

[0029] The nucleotide sequence of the Phi gene is shown in SEQ ID NO.9;

[0030] The construction and screening method of a multi-copy methanol utilization key gene integration library includes the following steps:

[0031] (1) Preparation of multiple rounds of iterative fragments:

[0032] Using pWLH-Aox-Das-Dak and pWLH-Mdh-Hps-Phi as templates, the Xump and Rump pathway fragments were amplified, respectively. A screening tag fragment was amplified using a truncated Ura3 promoter tag (Ura41) as a template. The Xump and Rump fragments were ligated to the Ura41 tag to obtain the Xump-Ura41 and Rump-Ura41 integration fragments, respectively.

[0033] The nucleotide sequence of the Ura41 fragment is shown in SEQ ID NO.10;

[0034] (2) Construction of multi-round iterative integration library:

[0035] The engineered strain PO1f / pWLH-DL4-PAXX-XRCC4 was transformed sequentially for five rounds, with the Xump-Ura41 and Rump-Ura41 fragments transformed in each round. The fragments were then plated on SC-Ura plates for screening to obtain the first to fifth round iterative strain libraries.

[0036] (3) Screening of efficient methanol utilization strains:

[0037] Each round of strains was inoculated into a fermentation medium containing methanol, and the strains with significantly improved methanol utilization ability were screened by measuring cell accumulation (the difference between OD600 at 48 h and OD600 at 0 h) and quantifying methanol consumption by high-performance liquid chromatography (HPLC).

[0038] The engineered strain PO1f / pWLH-DL4-PAXX-XRCC4 and its iterative library strain constructed by the above method.

[0039] The application of the above-mentioned engineered strain in methanol metabolism, biomanufacturing and as a non-natural methylotrophic chassis cell. Advantages of the present invention:

[0040] By enhancing NHEJ repair efficiency and achieving random integration of multiple copies of methanol utilization genes, the methanol metabolism capacity of Yarrowia lipolytica was significantly improved. Combining multiple rounds of iterative integration with a high-throughput screening strategy, a highly efficient strain was rapidly obtained, with simple operation and high library diversity. This represents the first time that a non-native methylotrophic metabolic pathway has been established in Yarrowia lipolytica, expanding its potential for industrial biotechnology applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0042] Figure 1Constructing a methanol metabolism pathway map for Yarrowia lipolytica.

[0043] Figure 2 Schematic diagram of the construction of the recombinant plasmid pWLH-DL4-PAXX-XRCC4.

[0044] Figure 3 Schematic diagram of the construction of the methanol metabolism pathway gene recombinant plasmids Xump and Rump.

[0045] Figure 4 Schematic diagram of the multi-round iterative integration of heterologous genes and the accumulation of fermentation cells in 10 mL EP tubes of Yarrowia lipolytica strains X001-X060 and R001-R060 carrying multiple copies of key genes for methanol utilization.

[0046] Figure 5 is the methanol consumption of the dominant strain. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0048] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0050] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] The original strain Yarrowia lipolytica PO1f was purchased from Thermofisher (China, https: / / www.thermofisher.com / cn / zh / home / brands / invitrogen.html) in September 2019.

[0053] DL4 gene: Yarrowia lipolytica PO1 f;

[0054] PAXX gene: Yarrowia lipolytica PO1 f;

[0055] XRCC4 gene: Yarrowia lipolytica PO1 f.

[0056] The synthesis of products within microorganisms is often regulated by complex genetic and metabolic networks within the cell. Therefore, identifying and effectively regulating the expression of endogenous potential genes within the cell not only fully taps the potential for cell product synthesis, but also accelerates the construction of high-yield engineered E. coli strains and deepens the understanding of the cell product synthesis and regulatory mechanisms.

[0057] The present invention will be further described below with reference to specific embodiments.

[0058] Example 1

[0059] The construction of a strain with enhanced efficiency of the non-homologous end joining repair mechanism of Yarrowia lipolytica comprises the following steps:

[0060] (1) Using pWLH as a template, primers pWLH-octt-F and pWLH-clone-R were used to amplify the vector pWLH1; using LD01-DL4 plasmid as a template, primers pEXP1-WLH-F and xpr2t-clone-R were used to amplify the DL4 expression cassette; using LD02-PAXX plasmid as a template, primers TEFin-xpr2t-F and l ip2t-clone-R were used to amplify the PAXX expression cassette; using LD03-XRCC4 plasmid as a template, primers were used to amplify the XRCC4 expression cassette. The obtained vector and expression cassette fragments were ligated by Giboson to obtain the recombinant plasmid pWLH-DL4-PAXX-XRCC4, as shown in FIG. Figure 2As shown; the DL4 gene is the DNA ligase 4 gene; the PAXX and XRCC4 are the repair factor genes of DNA ligase 4.

[0061] The nucleotide sequence of the DL4 gene is shown in SEQ ID NO.1;

[0062] The nucleotide sequence of the PAXX gene is shown in SEQ ID NO.2;

[0063] The nucleotide sequence of the XRCC4 gene is shown in SEQ ID NO.3;

[0064] (2) The recombinant plasmid pWLH-DL4-PAXX-XRCC4 was transformed into Yarrowia lipolytica Po1 f to obtain the engineered strain PO1 f / pWLH-DL4-PAXX-XRCC4.

[0065] Example 2

[0066] The construction of a plasmid for the methanol metabolism pathway includes the following steps:

[0067] (1) Using pWLH as a template, primers were used to amplify the vector pWLH2; using CYRYLME001 plasmid as a template, primers were used to amplify the Aox expression cassette; using CYRYLME002 plasmid as a template, primers were used to amplify the Das expression cassette; using CYRYLME004 plasmid as a template, primers were used to amplify the Dak expression cassette. The obtained vector and expression cassette fragments were ligated by Giboson to obtain the recombinant plasmid pWLH-Aox-Das-Dak, as shown in FIG. Figure 3 As shown; the Aox gene is a Pichia-derived alcohol oxidase gene; the Das is a Pichia-derived dihydroxyacetone synthase gene; the Dak is a dihydroxyacetone kinase gene;

[0068] The nucleotide sequence of the Aox gene is shown in SEQ ID NO.4;

[0069] The nucleotide sequence of the Das gene is shown in SEQ ID NO.5;

[0070] The nucleotide sequence of the Dak gene is shown in SEQ ID NO.6;

[0071] (2) Using pWLH as a template, primers were used to amplify the vector; using CYRYLME008 plasmid as a template, primers were used to amplify the Mdh expression cassette; using CYRYLME009 plasmid as a template, primers were used to amplify the Hps expression cassette; using CYRYLME010 plasmid as a template, primers were used to amplify the Phi expression cassette. The obtained vector and expression cassette fragments were ligated by Giboson to obtain the recombinant plasmid pWLH-Mdh-Hps-Phi, as shown in FIG. Figure 3 As shown; the Mdh gene is a methanol dehydrogenase gene; the Hps gene is a 3-hexulose 6-phosphate synthase gene; the Phi gene is a 6-phosphate 3-hexanone isomerase gene;

[0072] The nucleotide sequence of the Mdh gene is shown in SEQ ID NO.7;

[0073] The nucleotide sequence of the Hps gene is shown in SEQ ID NO.8;

[0074] The nucleotide sequence of the Phi gene is shown in SEQ ID NO.9;

[0075] Example 3: Construction and screening of a multi-copy methanol utilization key gene library mediated by non-homologous end joining mechanism

[0076] 1. Acquisition of multiple rounds of iterative fragments

[0077] Using pWLH-Aox-Das-Dak as a template, primers were used to amplify the Xump pathway fragment; using pWLH-Mdh-Hps-Phi as a template, primers were used to amplify the Rump pathway fragment; using as a template, primers were used to amplify the truncated Ura3 promoter tag fragment; the obtained Xump and Rump fragments were respectively connected in vitro with Ura41 with a truncated Ura3 promoter tag to obtain the Xump-Ura41 fragment and the Rump-Ura41 fragment.

[0078] The nucleotide sequence of the Ura41 gene is shown in SEQ ID NO.10

[0079] 2. Library Construction

[0080] (1) The PO1f / pWLH-DL4-PAXX-XRCC4 strain obtained in Example 1 was transferred to 3 mL of SD-Leu liquid culture medium and cultured at 30°C and 200 rpm for 18 h. A certain amount of the PO1f / pWLH-DL4-PAXX-XRCC4 strain was transferred to a high-temperature sterilized 1.5 mL centrifuge tube, centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. 1 mL of high-temperature sterilized deionized water was added to the centrifuge tube, mixed thoroughly, and centrifuged at 4000 rpm for 5 min. The supernatant was discarded. The fragments obtained in step 1 were respectively transferred to the PO1f / pWLH-DL4-PAXX-XRCC4 strain. After recovery at 30°C and 200 rpm for 3 h, 300 μL was spread on an SC-Ura solid plate to obtain the first round of Xump and Rump iterative strains. The remaining bacteria were transferred to 3 mL of SD-Leu liquid culture medium for culture.

[0081] (2) The remaining strains from step (1) were cultured for h, and the second round of Xump and Rump iterative strains were obtained according to the method of step 1. The remaining bacteria were transferred to 3 mL SD-Leu liquid culture medium for culture.

[0082] (3) The remaining strains from step (2) were cultured for h, and the third round of Xump and Rump iterative strains were obtained according to the method of step 1. The remaining bacteria were transferred to 3 mL SD-Leu liquid culture medium for culture.

[0083] (4) The remaining strains from step (3) were cultured for h, and the fourth round of Xump and Rump iterative strains were obtained according to the method of step 1. The remaining bacteria were transferred to 3 mL SD-Leu liquid culture medium for culture.

[0084] (5) Cultivate the remaining strains from step (4) for h, and obtain the fifth round of Xump and Rump iterative strains according to the method of step 1.

[0085] 3. Screening of strains with strong methanol utilization ability

[0086] The formula of the fermentation medium is: 7.6 g / L YNB, 1.31 g / L SCM, sterilized at 121° C. for 20 min, and after the medium is cooled to room temperature, 2% (v / v) membrane-sterilized methanol is added.

[0087] Randomly select single colonies from the first to fifth rounds of plates obtained in step 2 and inoculate them into 3 mL of YPD liquid medium. Incubate at 30°C, 200 rpm for 18 hours. Transfer a certain amount of the culture broth to a 1.5 mL autoclaved centrifuge tube and centrifuge at 4000 rpm for 5 minutes. Discard the supernatant. Add 1 mL of autoclaved deionized water to the centrifuge tube, mix thoroughly, centrifuge at 4000 rpm for 5 minutes, and discard the supernatant. Transfer all colonies to a 10 mL EP tube containing 3 mL of fermentation broth. Remove 1 mL of the fermentation broth and measure the initial OD600. Incubate the remaining culture broth at 30°C, 200 rpm for 48 hours. A certain amount of the culture broth after 48 hours of fermentation was measured at 48 hours OD600. Strains were screened for methanol capacity based on the cell accumulation (48 hour OD600 minus 0 hour OD600).

[0088] 4. Determination of methanol consumption

[0089] Take 0.5mL of the 0h fermentation broth and 48h fermentation broth obtained in step 3 of this embodiment respectively, centrifuge at 12000rpm for 5min, collect 300μL supernatant, and filter with 0.22μm organic filter membrane. The filtrate is analyzed using high performance liquid chromatography (Ultimate 3000 system, Dionex), and the Aminex HPX-87H ion exclusion column is maintained at 30°C. 20μL sample is used for analysis, the eluent is 5mM H2SO4, the flow rate is 0.6mL / min, and it lasts for 45min. The peak time of methanol is qualitatively determined by the peak time of the methanol standard with reference to the peak time of the methanol standard, and the peak area of ​​methanol with different concentrations is quantitatively determined. The methanol consumption is the methanol of the 0h sample minus the methanol of the 48h sample.

[0090] 4. Results

[0091] like Figure 4 As shown, a library of multi-copy integrated methanol utilization key genes mediated by non-homologous end joining mechanism was used to screen strains with different methanol utilization abilities, and methanol consumption was quantitatively determined by methanol liquid phase analysis. This indicates that the library constructed by this method can randomly insert different copy numbers of methanol utilization key genes into Yarrowia lipolytica, thereby enhancing the methanol utilization ability of Yarrowia lipolytica and constructing a non-natural methylotrophic Yarrowia lipolytica.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. A method for constructing an engineered strain that enhances the efficiency of non-homologous end joining (NHEJ) repair in Yarrowia lipolytica, characterized by The steps include: (1) Using the pWLH plasmid as a template, primers pWLH-octt-F and pWLH-clone-R were used to amplify the vector fragment; using the LD01-DL4, LD02-PAXX, and LD03-XRCC4 plasmids as templates, primers pEXP1-WLH-F / xpr2t-clone-R and TEFin-xpr2t-F / l ip2t-clone-R were used to amplify the DL4, PAXX, and XRCC4 expression cassettes, respectively; the vector and expression cassette fragments were assembled by Gibson ligation to obtain the recombinant plasmid pWLH-DL4-PAXX-XRCC4; The nucleotide sequence of the DL4 gene is shown in SEQ ID NO.1; The nucleotide sequence of the PAXX gene is shown in SEQ ID NO.2; The nucleotide sequence of the XRCC4 gene is shown in SEQ ID NO.3; (2) The recombinant plasmid pWLH-DL4-PAXX-XRCC4 was transformed into Yarrowia lipolytica Po1f to obtain the engineered strain PO1f / pWLH-DL4-PAXX-XRCC4.

2. The Yarrowia lipolytica engineered strain PO1 f / pWLH-DL4-PAXX-XRCC4 with enhanced NHEJ repair efficiency constructed by the method of claim 1.

3. Use of the engineered strain according to claim 2 in improving the efficiency of multi-copy gene integration.

4. A method for constructing a recombinant plasmid for the methanol metabolism pathway, characterized by: The steps include: (1) Construction of plasmid pWLH-Aox-Das-Dak: The vector pWLH2 was amplified using pWLH as a template; the Aox, Das, and Dak expression cassettes were amplified using CYRYLME001, CYRYLME002, and CYRYLME004 plasmids as templates, respectively, and the recombinant plasmid pWLH-Aox-Das-Dak was obtained by Gibson ligation. The nucleotide sequence of the Aox gene is shown in SEQ ID NO.4; The nucleotide sequence of the Das gene is shown in SEQ ID NO.5; The nucleotide sequence of the Dak gene is shown in SEQ ID NO.6; (2) Construction of plasmid pWLH-Mdh-Hps-Phi: The vector was amplified using pWLH as a template; the Mdh, Hps, and Phi expression cassettes were amplified using CYRYLME008, CYRYLME009, and CYRYLME010 plasmids as templates, respectively, and the recombinant plasmid pWLH-Mdh-Hps-Phi was obtained by Gibson ligation. The nucleotide sequence of the Mdh gene is shown in SEQ ID NO.7; The nucleotide sequence of the Hps gene is shown in SEQ ID NO.8; The nucleotide sequence of the Phi gene is shown in SEQ ID NO.

9.

5. The methanol metabolic pathway recombinant plasmids pWLH-Aox-Das-Dak and pWLH-Mdh-Hps-Phi constructed by the method of claim 4.

6. Use of the recombinant plasmid according to claim 5 in constructing a non-natural methylotrophic Yarrowia lipolytica.

7. Construction and screening method of multi-copy methanol utilization key gene integration library, characterized by The steps include: (1) Preparation of multiple rounds of iterative fragments: Using pWLH-Aox-Das-Dak and pWLH-Mdh-Hps-Phi as templates, the Xump and Rump pathway fragments were amplified, respectively. The screening tag fragment was amplified using the truncated Ura3 promoter tag (Ura41) as a template. The Xump and Rump fragments were ligated to the Ura41 tag to obtain the Xump-Ura41 and Rump-Ura41 integration fragments, respectively. The nucleotide sequence of the Ura41 fragment is shown in SEQ ID NO.10; (2) Construction of multi-round iterative integration library: The engineered strain PO1f / pWLH-DL4-PAXX-XRCC4 according to claim 2 is sequentially transformed for five rounds, with Xump-Ura41 and Rump-Ura41 fragments transformed in each round, and the fragments are spread on SC-Ura plates for screening to obtain the first to fifth round iterative strain libraries; (3) Screening of efficient methanol utilization strains: Each round of strains was inoculated into a fermentation medium containing methanol, and the strains with significantly improved methanol utilization ability were screened by measuring cell accumulation (the difference between OD600 at 48h and OD600 at 0h) and quantifying methanol consumption by high-performance liquid chromatography (HPLC).

8. A multi-copy methanol utilization key gene integration library strain constructed by the method of claim 7.

9. Use of the strain according to claim 8 in methanol metabolism, biomanufacturing and as a non-natural methylotrophic chassis cell.

10. The screening method according to claim 7, characterized in that The fermentation medium formula is: 7.6 g / L YNB, 1.31 g / L SCM, and after sterilization, 2% by volume of methanol is added; the methanol consumption is detected by HPLC, and the chromatographic conditions are: Aminex HPX-87H ion exclusion column, 5 mM H2SO4 eluent, flow rate 0.6 mL / min, and detection time 45 minutes.

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