Yarrowia lipolytica hyper-mutation system strain based on double-chain specific cytidine deaminase as well as construction method and application of Yarrowia lipolytica hyper-mutation system strain

Abstract

The invention discloses a yarrowia lipolytica hypermutation system strain based on double-chain specific cytidine deaminase as well as a construction method and application of the yarrowia lipolytica hypermutation system strain. The construction method comprises the following steps: integrating a Ura3 gene into a yarrowia lipolytica genome to obtain a stable expression strain HM00; the method comprises the following steps: connecting a PTEFIN regulation and control element, a DddAtox gene, a linker sequence and a MutH * gene to a pWLH plasmid by virtue of a seamless cloning technology, and constructing recombinant plasmids pWLH-DddAtox-linker-MutH * and pWLH-DddAtox-linker-MutH; the method comprises the following steps: transforming recombinant plasmids pWLH-DddAtox-linker-MutH * and pWLH-DddAtox-linker-MutH into a strain HM00, so as to respectively obtain a strain HM22 of a hypermutation system and a strain HM21 of a hypermutation system.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biosynthesis and bioenergy technology, and more particularly to a Yarrowia lipolytica supermutant system strain based on double-stranded specific cytidine deaminase and a construction method and use thereof. BACKGROUND

[0002] Yarrowia lipolytica has significant advantages in food-grade application fields. Its products such as eicosapentaenoic acid (EPA) and citric acid have passed the QPS certification of the European Union EFSA and IDF. Animal experiments have proved that its biomass and metabolic products exhibit good safety in purified or unpurified states. Yarrowia lipolytica has a unique substrate metabolic network: it naturally utilizes oil and fat substances (C12-C18 alkanes, triglycerides), industrial by-products (glycerol) and organic acids (acetic acid). The yeast also exhibits multiple tolerance characteristics, such as tolerance to 12% NaCl, 3% acetic acid, 4% methanol and lignin-derived phenolic compounds. This robustness enables it to directly utilize unpurified industrial waste, such as lignocellulose hydrolysate containing organic acid inhibitors. These characteristics make Yarrowia lipolytica an important industrial microbial strain.

[0003] As a highly potential industrial microorganism, Yarrowia lipolytica exhibits unique application value in many fields. Research on accelerating its evolution is of great significance to meet the needs of industrial production and promote the progress of biotechnology. Traditional methods for accelerating the evolution of Yarrowia lipolytica are as follows: adaptive laboratory evolution is widely used to accelerate the evolution of Yarrowia lipolytica and enhance its industrial phenotype. Traditional mutagenesis and random mutation combined with adaptive evolution strategies are also widely used for strain performance optimization. Traditional Yarrowia lipolytica evolution engineering has shown significant application value in the field of phenotype optimization. However, its mutation rate is still low, and multiple generations of culture and screening are required, which is time-consuming and depends on the rationality of stress design.

[0004] In order to improve the speed of adaptive evolution, the current strategies for artificially increasing the frequency of genomic mutations mainly focus on reducing DNA replication fidelity and interfering with DNA damage repair processes. These artificially modified base mutation frequencies, also known as supermutant systems, have been developed in Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. Supermutant systems have shown excellent results in accelerating strain evolution and enhancing genetic diversity.

[0005] Currently, the main way to accelerate the evolution of Yarrowia lipolytica is still at the traditional evolution level, and there is no report on the development of a supermutant system in Yarrowia lipolytica to improve the mutation rate of the strain. SUMMARY

[0006] The present application aims at overcoming the deficiencies of the prior art, and provides a Yarrowia lipolytica supermutant system strain based on double-strand specific cytidine deaminase.

[0007] The second object of the present application is to provide a construction method of a Yarrowia lipolytica supermutant system strain based on double-strand specific cytidine deaminase.

[0008] The third object of the present application is to provide a use of a Yarrowia lipolytica supermutant system strain based on double-strand specific cytidine deaminase.

[0009] The fourth object of the present application is to provide a second Yarrowia lipolytica supermutant system strain based on double-strand specific cytidine deaminase.

[0010] The fifth object of the present application is to provide a construction method of a second Yarrowia lipolytica supermutant system strain based on double-strand specific cytidine deaminase.

[0011] The sixth object of the present application is to provide a use of a second Yarrowia lipolytica supermutant system strain based on double-strand specific cytidine deaminase.

[0012] The technical solution of the present application is summarized as follows:

[0013] The construction method of the Yarrowia lipolytica supermutant system strain based on double-strand specific cytidine deaminase comprises the following steps:

[0014] (1) stably express the Ura3 gene into the Yarrowia lipolytica genome to obtain the strain HM00; the Ura3 gene is an abbreviation of the lactosyl-5'-phosphoric acid decarboxylase gene endogenous to Yarrowia lipolytica PO1f;

[0015] The nucleotide sequence of the Ura3 gene is shown in SEQ ID NO. 1;

[0016] (2) link the P TEFIN fragment and the DddA tox gene, the linker sequence, and the MutH* gene to construct the fragment P TEFIN -DddA tox -linker-MutH* into the plasmid pWLH to construct the recombinant plasmid pWLH-DddA tox -linker-MutH*; the DddA tox gene is an abbreviation of the double-strand DNA cytidine deaminase protein coding gene from Burkholderia cenocepacia, which is codon-optimized for Yarrowia lipolytica PO1f; the P TEFINFragment is the abbreviation of strong endogenous promoter of Yarrowia lipolytica PO1f; MutH* gene is the abbreviation of site-specific endonuclease MutH protein coding gene of Escherichia coli, which is codon-optimized for Yarrowia lipolytica PO1f and mutated two amino acids (E91A; F94A);

[0017] The nucleotide sequence of the DddA tox gene is shown as SEQ ID NO. 2;

[0018] The nucleotide sequence of the pWLH plasmid is shown as SEQ ID NO. 3;

[0019] The P TEFIN The nucleotide sequence of the fragment is shown as SEQ ID NO. 4;

[0020] The nucleotide sequence of the MutH* gene is shown as SEQ ID NO. 5;

[0021] (3) Transform the recombinant plasmid pWLH-DddA tox -linker-MutH* into the strain HM00 to obtain a Yarrowia lipolytica supermutagenesis system strain HM22 based on double-strand specific cytidine deaminase;

[0022] The Yarrowia lipolytica supermutagenesis system strain HM22 based on double-strand specific cytidine deaminase constructed by the above construction method.

[0023] The use of the Yarrowia lipolytica supermutagenesis system strain HM22 based on double-strand specific cytidine deaminase.

[0024] Another construction method of a Yarrowia lipolytica supermutagenesis system strain based on double-strand specific cytidine deaminase, comprising the following steps:

[0025] (1) Integrate the Ura3 gene into the Yarrowia lipolytica genome for stable expression to obtain the strain HM00; the Ura3 gene is the abbreviation of the orotidine-5'-phosphate decarboxylase gene endogenous to Yarrowia lipolytica PO1f;

[0026] The nucleotide sequence of the Ura3 gene is shown as SEQ ID NO. 1;

[0027] (2) Construct the fragment P TEFIN Fragment and DddA tox gene, linker sequence, MutH gene, and link the constructed fragment P TEFIN -DddA tox -linker-MutH into the plasmid pWLH to construct the recombinant plasmid pWLH-DddA tox-linker-MutH; the DddA tox The gene is a double-stranded DNA cytosine deaminase protein coding gene of Burkholderia cenocepacia origin optimized according to the Yarrowia lipolytica PO1f codon; the P TEFIN The fragment is a strong promoter endogenous to Yarrowia lipolytica PO1f; the MutH gene is a site-specific endonuclease MutH protein coding gene of Escherichia coli origin optimized according to the Yarrowia lipolytica PO1f codon;

[0028] The DddA tox The nucleotide sequence of the DddA gene is shown as SEQ ID NO. 2;

[0029] The nucleotide sequence of the pWLH plasmid is shown as SEQ ID NO. 3;

[0030] The P TEFIN The nucleotide sequence of the P fragment is shown as SEQ ID NO. 4;

[0031] The nucleotide sequence of the MutH gene is shown as SEQ ID NO. 5;

[0032] (3) Transform the recombinant plasmid pWLH-DddA tox -linker-MutH into strain HM00 to obtain Yarrowia lipolytica supermutant system strain HM21 based on double-stranded specific cytosine deaminase.

[0033] Advantages of the present application:

[0034] The recombinant plasmid pWLH-DddA tox -linker-MutH* and the recombinant plasmid pWLH-DddA tox -linker-MutH, the recombinant plasmid is transformed into Yarrowia lipolytica in vivo to construct Yarrowia lipolytica supermutant system strain HM22 and HM21 based on double-stranded specific cytosine deaminase. This method of constructing Yarrowia lipolytica supermutant system through nicking-double-strand deamination without unzipping is simple in operation, and can obtain Yarrowia lipolytica chassis strains with high mutation rate. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Principle of nicking-double-strand deamination for supermutant system.

[0036] Figure 2 Principle of characterizing mutation rate through Ura3 gene mutation for supermutant system.

[0037] Figure 3 For recombinant plasmid pWLH-DddA tox -linker-MutH* and pWLH-DddA tox The schematic diagram of construction of -linker-MutH.

[0038] Figure 4 Flow chart of fluctuation test of Yarrowia lipolytica hypermutation system strains HM21 and HM22 based on double-strand specific cytidine deaminase.

[0039] Figure 5 The apparent mutation frequency of Yarrowia lipolytica hypermutation system strains HM21 and HM22 based on double-strand specific cytidine deaminase. DETAILED DESCRIPTION

[0040] The original strain Yarrowia lipolytica PO1f was purchased from Thermofisher (https: / / www.thermofisher.com / cn / zh / home / brands / invitrogen.html) in September 2019.

[0041] P TEFIN Fragment: from Yarrowia lipolytica PO1f.

[0042] The nucleotide sequence of the pWLH plasmid is shown in SEQ ID NO. 4 (Fang LX, Chen YR, He QX, et al. Mining novel gene targets for improving tolerance to furfural and acetic acid in Yarrowia lipolytica using whole-genome CRISPRi library [J]. Bioresource Technology, 2024, 403: 130764).

[0043] In order to obtain Yarrowia lipolytica strains with better industrial phenotypes, traditional evolution methods face multiple limitations in this host. The hypermutation system can accelerate the mutation process, greatly shorten the strain evolution time, and faster obtain excellent phenotypes. Deepen the correlation between cell genes and phenotypes. To a greater extent, the potential of cells is exerted.

[0044] The application will be further described below in conjunction with specific examples.

[0045] Example 1

[0046] The construction of the supermutant strain HM22 of *Yarrowia lipophila* based on double-stranded specific cytidine deaminase includes the following steps:

[0047] (1) The Ura3 gene was artificially synthesized, integrated into the genome of Yersinia lipolytica by transforming competent cells, and stably expressed. The strain HM00, which stably expressed the Ura3 gene, was obtained by screening in SC-Ura auxotrophic medium. The Ura3 gene is an abbreviation for the gene encoding orotidine-5'-phosphate decarboxylase endogenous in Yersinia lipolytica PO1f.

[0048] The nucleotide sequence of the Ura3 gene is shown in SEQ ID NO.1;

[0049] (2) Fragment P from Yersinia lipophila PO1f was obtained from the NCBI database. TEFIN Primers PTEFIN-F and PTEFIN-R were designed and synthesized. Using the Yersinia lipophila PO1f genome as a template, PCR amplification was performed using primers PTEFIN-F and PTEFIN-R to obtain fragment P. TEFIN Artificial synthesis of DddA tox Genes and MutH* genes; P through seamless cloning TEFIN Fragment, DddA tox Gene, fragment P constructed by linking MutH* genes TEFIN -DddA tox -linker-MutH* was incorporated into plasmid pWLH to construct the recombinant plasmid pWLH-DddA tox -linker-MutH*, such as Figure 3 As shown; the recombinant plasmid was transformed into competent E. coli cells for replication and passage; the DddA... tox The gene is an abbreviation for the double-stranded DNA cytidine deaminase protein encoding gene derived from Burkholderia cenocepacia, optimized according to the PO1f codon of Yersinia lipolyticis; the P TEFIN The fragment is an abbreviation for the endogenous strong promoter of Yersinia lipophila PO1f; the MutH* gene is an abbreviation for the site-specific endonuclease MutH protein encoding gene from Escherichia coli, optimized according to the codon of Yersinia lipophila PO1f and mutated by two amino acids (E91A; F94A);

[0050] The DddA tox The nucleotide sequence of the gene is shown in SEQ ID NO.2;

[0051] The nucleotide sequence of the pWLH plasmid is shown in SEQ ID NO.3;

[0052] The P TEFIN The nucleotide sequence of the fragment is shown in SEQ ID NO.4;

[0053] The nucleotide sequence of the MutH* gene is shown in SEQ ID NO.5;

[0054] The nucleotide sequence of the PTEFIN-F primer is shown in SEQ ID NO. 6;

[0055] The nucleotide sequence of the PTEFIN-R primer is shown in SEQ ID NO.7;

[0056] (3) The recombinant plasmid pWLH-DddA tox -linker-MutH* was transformed into competent cells of Yersinia lipolyticis HM00, and transformants were screened on SC-Leu auxotrophic medium to obtain Yersinia lipolyticis hypermutant strain HM22 based on double-stranded specific cytidine deaminase.

[0057] Example 2

[0058] The construction of the supermutant strain HM21 of Yersinia lipophila based on double-stranded specific cytidine deaminase includes the following steps:

[0059] (1) Same as step (1) in Example 1

[0060] (2) Fragment P from Yersinia lipophila PO1f was obtained from the NCBI database. TEFIN Primers PTEFIN-F and PTEFIN-R were designed and synthesized. Using the Yersinia lipophila PO1f genome as a template, PCR amplification was performed using primers PTEFIN-F and PTEFIN-R to obtain fragment P. TEFIN Artificial synthesis of DddA tox Genes and MutH genes; P through seamless cloning TEFIN Fragment, DddA tox Gene, fragment P constructed by linking the MutH gene. TEFIN -DddA tox -linker-MutH was constructed into plasmid pWLH to construct recombinant plasmid pWLH-DddA tox -linker-MutH, such as Figure 3 As shown; the recombinant plasmid was transformed into competent E. coli cells for replication and passage; the DddA... tox The gene is an abbreviation for the double-stranded DNA cytidine deaminase protein encoding gene derived from Burkholderia cenocepacia, optimized according to the PO1f codon of Yersinia lipolyticis; the P TEFINFragment is the abbreviation of strong endogenous promoter of Yarrowia lipolytica PO1f; MutH* gene is the abbreviation of site-specific endonuclease MutH protein coding gene of Escherichia coli, which is codon-optimized for Yarrowia lipolytica PO1f and mutated by three bases;

[0061] The DddA tox The nucleotide sequence of the gene is shown as SEQ ID NO. 2;

[0062] The nucleotide sequence of the pWLH plasmid is shown as SEQ ID NO. 3;

[0063] The P TEFIN The nucleotide sequence of the fragment is shown as SEQ ID NO. 4;

[0064] The nucleotide sequence of the PTEFIN-F primer is shown as SEQ ID NO. 6;

[0065] The nucleotide sequence of the PTEFIN-R primer is shown as SEQ ID NO. 7;

[0066] The nucleotide sequence of the MutH gene is shown as SEQ ID NO. 8;

[0067] (3) The recombinant plasmid pWLH-DddA tox -linker-MutH was transformed into the competent cells of Yarrowia lipolytica HM00, the transformants were screened on SC-Leu auxotrophic medium, and the Yarrowia lipolytica hypermutation system strain HM21 based on double-strand-specific cytidine deaminase was obtained.

[0068] Example 3: Fluctuation test of Yarrowia lipolytica hypermutation system strain HM22 based on double-strand-specific cytidine deaminase, strain HM21, and strain HM00

[0069] 1. Strain activation and preservation

[0070] The preserved HM22 glycerol bacteria, HM21 glycerol bacteria, and HM00 strain glycerol bacteria were taken out from the -80℃ refrigerator, streaked on SC-Ura-Leu plates, and after the colonies grew, 3 biological replicates were set for each strain, and the three colonies were inoculated into 2 ml SC-Ura-Leu medium. After 20 h of culture at 30℃ and 220 rpm in a shaking tube, the strain was activated. The shaking tube was mixed, 700 μL of bacterial solution was taken from it and added to 300 μL of glycerol, and the EP tube was stored at -80℃.

[0071] The SC medium is prepared as follows: 20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 2 g / L amino acid dropout powder, 20 mg / L histidine, 20 mg / L tryptophan, 100 mg / L leucine (Leu) and 20 mg / L uracil (Ura) are added according to the nutritional marker of the strain, dissolved in ddH2O and made up to volume, and the pH is adjusted to 6.0 with saturated sodium hydroxide solution, and autoclaved at 115°C for 15 min. If a solid medium is prepared, the pH is adjusted to 6.5 with saturated sodium hydroxide solution, and 20 g / L agar powder is added.

[0072] 2. Fermentation in a well plate

[0073] The mixed bacterial solution is taken out from the bacterial solution, 20 μL of which is added to an EP tube containing 980 μL of SC-leu medium, and diluted 50 times. The 1000 μL gun is adjusted to 500 μL to suck the bacterial solution in the first EP tube, 100 μL of which is taken out and added to a second EP tube containing 900 μL of SC-leu medium, and diluted 10 times. The bacterial solution in the second EP tube is sucked as above, 100 μL of which is taken out and added to a third EP tube containing 900 μL of SC-leu medium, and diluted 10 times. The bacterial solution in the third EP tube is sucked as above, 100 μL of which is taken out and added to a first enzyme-labeled plate containing 100 μL of SC-Leu medium, and diluted 2 times, and repeated eight times to obtain eight technical parallels. (The enzyme-labeled plate uses the middle six rows of 6*8, and 100 μL of SC-Leu is added in advance, and the remaining cells are added with 200 μL of SC-leu in advance) Shake well, a total of 10000 times dilution, and incubate in a 28°C incubator for 48 h.

[0074] 3. Mutant plating

[0075] A 100 μL range of the gun is used to resuspend the bacterial solution. 60 μL of the bacterial solution is taken from each of the eight technical parallels and added to a fourth EP tube, and 10 μL, 50 μL and 250 μL of the bacterial solution is taken from the fourth EP tube and plated on SC+FOA (light shielding) solid plates, and placed in a 28°C incubator for light shielding culture for 2-3 days. The colonies grown on the plates are counted to calculate the mutation frequency.

[0076] 4. Dilution plating

[0077] Resuspend the mixed bacterial culture in EP tube #4 by aspiration. Add 10 μL of bacterial culture from EP tube #4 to EP tube #5 (pre-added with 990 μL of SC-Leu liquid medium) and dilute 100-fold. Resuspend the mixed bacterial culture in EP tube #5 by aspiration. Add 100 μL of bacterial culture from EP tube #5 to EP tube #6 (pre-added with 900 μL of SC-Leu liquid medium) and dilute 10-fold. Repeat this process for a total of 1000-fold dilutions. Plate 10 μL of bacterial culture from EP tube #6 onto an SC agar plate. Resuspend the mixed bacterial culture in EP tube #6 by aspiration. Add 100 μL of bacterial culture from EP tube #6 to EP tube #7 (pre-added with 900 μL of SC-Leu liquid medium) and dilute 10-fold. Repeat this process for a total of 10000-fold dilutions. Plate 10 μL of bacterial culture from EP tube #7 onto an SC agar plate. Incubate at 28°C for 2-3 days. The mutation frequency was calculated by counting the colonies grown on the plate.

[0078] 5. Mutation frequency calculation

[0079] Based on the fact that 5-FOA can be converted into 5-fluorouracil (5-FU) by the Ura3 gene product of *Yarrowia lipolytica* genome, leading to cell death, it can be concluded that the Ura3 gene of strains forming colonies on SC+FOA solid plates has been inactivated by deamination mutation via a hypermutation system. Colonies from 10 μL, 50 μL, and 250 μL bacterial suspensions were counted to determine the number of mutated colonies per unit volume of suspension during the fluctuation experiment. The number of colonies grown after 1000-fold and 10000-fold dilutions on SC plates represents the total number of colonies per unit volume. Therefore, through... Figure 4 From the formula, we can calculate the observed mutation rate.

[0080] 6. Results

[0081] The mutation frequency was calculated by plate counting. Figure 5 It can be seen that the mutation rate of the *Yersinia lipophila* supermutant strain HM22 based on double-stranded specific cytidine deaminase is 2.328*10^6. -3 The mutation rate of the supermutant strain HM21 of *Yarrowia lipolyticis* was 8.12*10^6. -2 The mutation rate of Yersinia lipolyticis strain HM00 is 3.33*10. -9 The HM22 strain increased the PO1f mutation rate of *Yarrowia lipolyticis* to 6.99 × 10⁻⁶ compared to the original strain HM00. 5 The HM21 strain increased the mutation rate of Yersinia lipolyticis PO1f to 2.44 × 10⁻⁶ times that of the original strain HM00. 7 times.

Claims

1. A method for constructing a Yarrowia lipolytica hypermutation system based on double-strand specific cytosine deaminase, characterized in that Comprising the following steps: (1) stably express Ura3 gene into Yarrowia lipolytica genome to obtain strain HM00; the Ura3 gene is an abbreviation of whey-5'-phosphoric acid decarboxylase gene endogenous to Yarrowia lipolytica PO1f; The nucleotide sequence of the Ura3 gene is shown as SEQ ID NO. 1; (2) the P TEFIN fragment and the DddA tox gene, a linker sequence, and a MutH* gene TEFIN -DddA tox -linker-MutH* into the plasmid pWLH, resulting in the recombinant plasmid pWLH-DddA tox -linker-MutH*; said DddA tox gene is an abbreviated version of the Yarrowia lipolytica POlf codon-optimized double-stranded DNA cytosine deaminase protein-encoding gene of Burkholderia cenocepacia origin; said P TEFIN fragment is an abbreviated version of a strong promoter endogenous to Yarrowia lipolytica POlf; and said MutH* gene is an abbreviated version of the Yarrowia lipolytica POlf codon-optimized and mutated for two amino acids (E91A; F94A) MutH site-specific endonuclease protein-encoding gene of Escherichia coli origin. The DddA tox The nucleotide sequence of the gene is shown as SEQ ID NO. 2; The nucleotide sequence of the pWLH plasmid is shown as SEQ ID NO. 3; The P TEFIN The nucleotide sequence of the fragment is shown as SEQ ID NO. 4; The nucleotide sequence of the MutH* gene is shown as SEQ ID NO. 5; (3) Transforming the recombinant plasmid pWLH-DddA tox -linker-MutH* into strain HM00 to obtain Yarrowia lipolytica hypermutation system strain HM22 based on double-strand specific cytidine deaminase.

2. Yarrowia lipolytica double-chain-specific cytidine deaminase-based ultramutagenesis system strain HM22 constructed by the construction method of claim 1.

3. Use of Yarrowia lipolytica double-chain-specific cytidine deaminase-based ultramutagenesis system strain HM22 of claim 2.

4. A method for constructing a Yarrowia lipolytica hypermutation system based on double-strand specific cytidine deaminase, characterized in that Comprising the following steps: (1) stably express Ura3 gene into Yarrowia lipolytica genome to obtain strain HM00; the Ura3 gene is an abbreviation of whey-5'-phosphoric acid decarboxylase gene endogenous to Yarrowia lipolytica PO1f; The nucleotide sequence of the Ura3 gene is shown as SEQ ID NO. 1; (2) P TEFIN Fragments and DddA tox Gene, linker sequence, fragment P constructed by linking the MutH gene TEFIN -DddA tox -linker-MutH was constructed into plasmid pWLH to construct recombinant plasmid pWLH-DddA tox -linker-MutH; the DddA tox The gene is an abbreviation for the double-stranded DNA cytidine deaminase protein encoding gene derived from Burkholderia cenocepacia, optimized according to the PO1f codon of Yersinia lipolyticis; the P TEFIN The fragment is an abbreviation for the endogenous strong promoter of Yersinia lipophila PO1f; the MutH gene is an abbreviation for the gene encoding the site-specific endonuclease MutH protein from Escherichia coli, optimized according to the codon of Yersinia lipophila PO1f. The DddA tox The nucleotide sequence of the gene is shown as SEQ ID NO. 2; The nucleotide sequence of the pWLH plasmid is shown as SEQ ID NO. 3; The P TEFIN The nucleotide sequence of the fragment is shown as SEQ ID NO. 4; The nucleotide sequence of the MutH gene is shown as SEQ ID NO. 8; (3) transform recombinant plasmid pWLH-Trex2-PmCDA1-linker-MutH into strain HM00 to obtain Yarrowia lipolytica double-chain-specific cytidine deaminase-based ultramutagenesis system strain HM21.

5. Yarrowia lipolytica double-chain-specific cytidine deaminase-based ultramutagenesis system strain HM21 constructed by the construction method of claim 4.

6. Yarrowia lipolytica double-chain-specific cytidine deaminase-based ultramutagenesis system strain HM21 of claim 5.

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