An expression vector of yarrowia lipolytica and application thereof

Abstract

The application discloses a Yarrowia lipolytica expression vector and application thereof, and particularly discloses a nucleic acid molecule C-M6, wherein the DNA sequence of the nucleic acid molecule C-M6 is SEQ ID NO:23; further disclosed is a vector containing the nucleic acid molecule, wherein the vector comprises the following elements: a centromere, an autonomous replication sequence, a resistance gene, a promoter, a screening marker gene and a terminator, and the centromere is the nucleic acid molecule in claim 1. The nucleic acid molecule provided by the application is used as the centromere in a yeast vector, and the obtained yeast vector has higher stability compared with a conventional vector carrying an ARS and a vector carrying a wild-type centromere sequence, and can obtain higher protein expression yield for expressing a target protein.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a Yersinia lipophila expression vector and its application. Background Technology

[0002] The development of molecular tools for gene expression and regulation is crucial for metabolic engineering and synthetic biology research, enabling precise control over target genes to optimize metabolic pathways, study microbial metabolic mechanisms, or synthesize new compounds. In metabolic engineering, various vectors have been developed and applied to regulate the expression levels of key enzymes, introduce heterologous pathways and precisely control their expression, and dynamically regulate toxic genes or pathways (such as the methanol utilization pathway) to address toxicity issues. In synthetic biology, convenient free vectors are often used to modularly design gene circuits containing different components or to construct molecular tools such as metabolite-responsive sensors to achieve feedback control.

[0003] For yeast hosts, gene overexpression primarily relies on two pathways: genome integration and free vector construction. Although genome integration technology has been widely used for metabolic pathway modification and stable expression of recombinant proteins, its inherent heterogeneity and low transformation efficiency remain significant technical bottlenecks. In contrast, free vectors are easier to modify and engineer, offering high transformation efficiency and ease of operation, and are commonly used for transient expression using site-specific recombinases, CRISPR / Cas9, and other gene editing tools. In the model yeast strain *Saccharomyces cerevisiae* (Saccharomyces cerevisiae),... Saccharomyces cerevisiae In the field of [unclear - possibly related to yeast], researchers have developed low-copy plasmids based on centromere (CEN) / autonomously replicating sequences (ARS) and high-copy plasmids based on 2 μ replicons. Naturally occurring multi-copy 2 μ plasmids exist only in yeasts, existing as self-replicating circular DNA, and their free state is maintained through a synergistic mechanism of segregation and copy number control. However, due to the lack of natural 2 μ plasmids in many non-traditional yeasts, such high-copy expression tools cannot be effectively used. Furthermore, the autonomously replicating sequence (ARS) serves as a chromosome replication initiation point, enabling plasmids to replicate autonomously; for example, it originates from Kluyveromyces [unclear - possibly related to yeast]. Kluyveromyces lactis The 452 bp ARS element can function in various yeasts, including *Saccharomyces* and *Pichia pastoris*. However, studies have shown that plasmid systems based on CEN / ARS still suffer from insufficient stability, and this technological bottleneck has not yet been overcome.

[0004] Yarrowia lipolytica ( Yarrowia lipolyticaAs an unconventional oil-producing yeast, *Yersinia lipophila* is widely distributed in natural ecological environments such as soil, seawater, and oil-contaminated environments. Its powerful ability to degrade lipids and proteins has attracted considerable attention in the industrial field. Researchers have developed various genetic tools based on genetic and molecular biology research to achieve the transformation and integration of exogenous genes in *Yersinia lipophila*. However, no naturally occurring free plasmids have yet been identified in this yeast. Currently, a series of CEN / ARS-based functional vectors constructed in *Yersinia lipophila* have only been used for subcellular localization and promoter strength analysis, and are not suitable for stable heterologous expression. Therefore, the application prospects of this yeast as a cell factory are hindered by the lack of stable, high-copy-value free plasmids. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a nucleic acid molecule that serves as the centromere in a yeast vector. The yeast vector constructed from this molecule exhibits higher stability and higher yield of the target protein compared to traditional vectors carrying ARS sequences and vectors carrying wild-type centromere sequences.

[0006] The present invention also proposes a carrier.

[0007] The present invention also proposes a recombinant yeast comprising the above-mentioned carrier.

[0008] The present invention also proposes a reagent kit.

[0009] The present invention also proposes a method for producing proteins in cells.

[0010] The present invention also proposes an application.

[0011] According to a first aspect of the present invention, a nucleic acid molecule is provided, the DNA sequence of which is SEQ ID NO:23.

[0012] According to a second aspect of the present invention, a carrier is provided that comprises the nucleic acid molecule described in the first aspect of the present invention.

[0013] In some embodiments of the present invention, the vector includes the following elements: centromere, autonomously replicating sequence, resistance gene, promoter, selection marker gene, and terminator, wherein the centromere is the nucleic acid molecule described in the first aspect of the present invention.

[0014] In some embodiments of the present invention, the vector comprises, from upstream to downstream, the following components in sequence: centromere, autonomous replication sequence, resistance gene promoter, resistance gene, replication initiation site, promoter, selection marker gene, and terminator.

[0015] In some embodiments of the present invention, the autonomous replication sequence includes an ori1001 fragment or an ARS fragment.

[0016] In some embodiments of the present invention, the resistance gene includes the AmpR gene or the Kmr gene.

[0017] In some embodiments of the present invention, the screening marker gene includes the URA3 gene.

[0018] In some embodiments of the present invention, the promoter includes any one of TEFin, TEF, and TEF1.

[0019] In some embodiments of the present invention, the terminator includes any one of CYC1 and ADH1.

[0020] According to a third aspect of the present invention, a recombinant yeast is provided, the recombinant yeast comprising the vector described in the second aspect of the present invention.

[0021] In some embodiments of the present invention, the host strain of the recombinant yeast includes *Yersinia lipophila* (…). Yarrowia lipolytica ).

[0022] According to a fourth aspect of the present invention, a kit is provided, the kit comprising at least one of (1) to (2): (1) The nucleic acid molecule described in the first aspect of the present invention; (2) The carrier described in the second aspect of the present invention.

[0023] According to a fifth aspect of the invention, a method for producing proteins in cells is provided, the method comprising the step of contacting the cells with at least one of (1) to (3): (1) The nucleic acid molecule described in the first aspect of the present invention; (2) The carrier described in the second aspect of the present invention; (3) The reagent kit described in the fourth aspect of the present invention.

[0024] According to a sixth aspect of the present invention, the application of the nucleic acid molecule described in the first aspect of the present invention, the vector described in the second aspect of the present invention, or the kit described in the fourth aspect of the present invention in constructing a stable lipophilic Yeast vector is proposed.

[0025] The present invention has at least the following beneficial effects: This invention uses Yersinia lipophila ( Yarrowia lipolyticaUsing strain Po1f as the expression host, the endogenous centromere sequence of yeast was amplified using molecular biology techniques and methods. Random mutations of the centromere sequence were then performed using error-prone PCR to obtain a series of mutant products from C-M1 to C-M12. Subsequently, the autonomously replicating sequence ori1001 element amplified from the genome was combined with the aforementioned centromere sequence and selection markers. Ura3 Using the pUC19 plasmid containing the E. coli replication system as a backbone, a series of vectors, pCE-M1 to pCE-M12, were constructed, and their stability was determined. After comparison with traditional replication plasmids, the effective mutant C-M6, which improves stability, was selected. The recombinant vectors constructed using C-M6 exhibit higher stability and higher target protein yield compared to traditional vectors. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the pCE vector construction in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the pARS1 vector construction in Embodiment 1 of the present invention; Figure 3 This is a graph showing the plasmid stability detection results in Example 1 of the present invention; Figure 4 This is a graph showing the results of plasmid passage stability determination in Example 1 of the present invention; Figure 5 This is a graph showing the 1,8-cineole yield detection results in Example 1 of the present invention. Detailed Implementation

[0027] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0028] In the following examples, unless otherwise specified, all instruments and reagents are commercially available through conventional purchasing channels.

[0029] Example 1 This embodiment uses *Yarrowia lipolytica* strain Po1f as the expression system. The endogenous centromere sequence of this yeast was amplified using molecular cloning technology, and the element was randomly mutagenized using error-prone PCR, successfully obtaining a series of mutants from C-M1 to C-M12. Simultaneously, the autonomous replication sequences ori1001 and ARS1 were isolated from the genome and modularly assembled with the mutated centromere sequence and the Ura3 selection marker, respectively. Using the pUC19 plasmid (containing an *E. coli* replicon) as the vector backbone, a series of recombinant plasmids, pCE, pCE-M1 to pCE-M12, and pARS1, were constructed. Finally, elements that enhanced plasmid stability compared to the traditional replicon ARS1 were screened. Specific experimental methods and results are as follows: 1. Obtaining key elements such as centromere sequence and ori1001 First, the genome of *Yersinia lipolyticis* strain Po1f was obtained using a genome extraction kit (TIANGEN, catalog number: DP307). Then, various elements required were amplified using this genome and other existing plasmids as templates. The autonomous replication sequences *ori1001*, *ARS1*, and *CEN* were amplified using the genome as templates with three primer pairs: *ori1001-F / R*, *ARS1-F / R*, and *CEN-F / R*. The TEFin promoter was amplified using the pPT plasmid (purchased from Anhui General Technology Co., Ltd.) with the pTEFin-F / R primer pair. The selection marker gene was obtained using the pSH65 plasmid (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) with the Ura3-F / R primer pair. Ura3 Simultaneously, using the genome of Yersinia lipophila Po1f strain as a template, the CYC1 terminator was amplified using the tCYC1-F / R primer pair.

[0030] To obtain mutant centromere elements for screening highly stable vectors, the CEN-F / R primer pair was used for amplification via error-prone PCR. PCR was performed under four different reagent ratios (four different concentrations of Mut Enhancer) to obtain 12 mutant products C-M1 to C-M12 with different mutation rates. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0031] Table 1 PCR reaction system

[0032] Table 2 PCR reaction conditions

[0033] The primer sequences used in the above steps and the corresponding sequence information of the key elements obtained by amplification are as follows: Primer ori1001-F: 5'-CATACTACTGTATATTCAAGCAAGTATATCCGTGGGTGCG-3' (SEQ NOID: 1); Primer ori1001-R: 5'-TATGTCTGATAAAAGGATGTAACATAGGCAAGCTGCTCGT-3' (SEQ NOID: 2); Primer ARS1-F: 5'-CATACTACTGTATATTCAAGCAAGTATATCCGTGGGTGCG-3' (SEQ NO ID: 1); Primer ARS1-R: 5'-CTCATTTACAATTTCATCTTTCATCAAATTTAGGGATGCC-3' (SEQ NO ID: 3); Primer CEN-F: 5'-CTTTCATCAAATTTAGGGATGCCATCAACT-3' (SEQ NO ID: 4); Primer CEN-R: 5'-GCTTTTCGTAGATAATGGAATACAAATGGA-3' (SEQ NO ID: 5); ori1001 fragment: 5'-CATACTACTGTATATTCAAGCAAGTATATCCGTGGGTGCGGGTGATTTGGATCTAAGGTTCGTACTCAACACTCACGAGCAGCTTGCCTATGTTACATCCTTTTATCAGACATA-3' (SEQ NO ID: 6); Centromeric sequence fragment: 5’-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAA.CATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTAT.CCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:8); Primer pTEFin-F: 5’-AGAGACCGGGTTGGCGGCGTATTTGTGTCCCAAAAAACAG-3’ (SEQ NOID:9); Primer pTEFin-R: 5’-CTGCGGTTAGTACTGCAAAAAGTGCTGGTCGGATGACGTG-3’ (SEQ NOID:10); TEFin promoter: 5’-AGAGACCGGGTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGAATCATTCAAAATGGTGAGTTTCAGAGGCAGCAGCAATTGCCACGGGCTTTGAGCACACGGCCGGGTGTGGTCCCATTCCCATCGACACAAGACGCCACGTCATCCGACCAGCACTTTTTGCAGTACTAACCGCAG-3’ (SEQ NO ID:11); Primer Ura3-F: 5’-TTTTTGCAGTACTAACCGCAGATGCCCTCCTACGAAGCTC-3’ (SEQ NO ID:12); Primer Ura3-R: 5’-GACATAACTAATTACATGACTAACAGTTAATCTTCTGGTA-3’ (SEQ NO ID:13); Ura3 fragment: 5’-ATGTCGAAAGCTACATATAAGGAACGTGCTGCTACTCATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAAGCAAACAAACTTGTGTGCTTCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAATTTGTTTACTAAAAACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAGGCATTATCCGCCAAGTACAATTTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGGTAATACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGCAGACATTACGAATGCACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAAGCAGGCGGCAGAAGAAGTAACAAAGGAACCTAGAGGCCTTTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTATCTACTGGAGAATATACTAAGGGTACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAAAGAGACATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAGATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCTCTACAGGATCTGACATTATTATTGTTGGAAGAGGACTATTTGCAAAGGGAAGGGATGCTAAGGTAGAGGGTGAACGTTACAGAAAAGCAGGCTGGGAAGCATATTTGAGAAGATGCGGCCAGCAAAACTAA-3’ (SEQ NO ID:14); Primer tCYC1-F: 5’-ACCAGAAGATTAACTGTTAGTCATGTAATTAGTTATGTCA-3’ (SEQ NO ID:15); Primer tCYC1-R: 5’-ttatccacagaatcaGCAAATTAAAGCCTTCGAGCGTCCC-3’ (SEQ NO ID:16); CYC1 terminator: 5’-ATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAG-3’ (SEQ ID NO:17); C-M1 fragment: 5’-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAGTTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTATCCATGTGTATACTCCGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:18); C-M2 fragment: 5’-CTTTCATCAAATTTAGGGATGCAATCAACTTTCAGTTCATAATTAACATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGTAATGTAACATTTTATATATCAAGCTCTATTTAACCCTCACAGTAGTTGGGCATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTATCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:19); C-M3 fragment: 5'-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGATACAGAATTGTCGTGTCAGTGTATACCATCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3' (SEQ NO ID:20); C-M4 fragment: 5'-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGGTACAGAATTGTCGTGTCAGTGTATACTATCCATGTGTATACTCTGGATATCCCATTTGTATTCCATTATCTACGAAAAGC-3' (SEQ NO ID:21); C-M5 fragment: 5'-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGACACAGAATTGTCGTGTCAGAGTATACTATCCATGTGTATACTCTGGATATCCCATTTGTATTCCATTATCTACGAAAAGC-3' (SEQ NO ID:22); C-M6 fragment: 5’-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTTACGCCTCACAGTAGTTAACATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTATGCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:23); C-M7 fragment: 5’-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAGCATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTATCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:24); C-M8 fragment: 5’-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAATATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAGCATAAAGAGATACAGAATGTCGTGTCAGATAAAATATCCAAGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:25); C-M9 fragment: 5’-GTTTCTTCTGTTGTTGGGATTCCATCGACATTCAGTTCATGATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTATCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:26); C-M10 fragment: 5’-CTTTCATCAAATTTAGGGATGCCATCAACATTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAGCTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTATCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:27); C-M11 fragment: 5’-CTTTCATCAGATTGAGGGATTCCATCTACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATTGCAAAAATTCAGACGGTGGAATGTATCATTGTATTGCAAGCTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGATACAGAATTGTCGTGTCAGTGTATACTATCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3’ (SEQ NO ID:28); C-M12 fragment: 5'-CTTTCATCAAATTTAGGGATGCCATCAACTTTCAGTTCATAATTAATATCTTACCAAATTAGGTAATCTGCAAAAGTTCAGACTGTGAAATGTAACATTTTATATATCAAG CTCTATTTAACGCCTCACAGTAGTTAACATAAAGAGATACAGAATTGTCGTGTCAGTGTATACCATCCATGTGTATACTCTGGATATCCATTTGTATTCCATTATCTACGAAAAGC-3' (SEQ NO ID:29).

[0034] 2. Construction of pARS1 and pCE series vectors 1) Construction of pCE series plasmids: The pCE series plasmids also use the pUC19 plasmid as the basic backbone and are constructed through a modular assembly strategy. This process involves the integration of two functional modules: the yeast replication module (centromere sequence-Link1-ori1001) and the selection marker module (pTEFin-Ura3-tCYC1).

[0035] First, the functional units are connected using overlap PCR technology: The construction of the yeast replication module used Link1-F / R primers to amplify the Link1 ligation sequence using pUC19 as a template. The two fragments were ligated using overlap PCR with primers CEN-F / Link1-R to obtain the centromere sequence-Link1. The centromere sequence-Link1 fragment was ligated with the ori1001 fragment using primers CEN-F / ori1001-R to obtain the centromere sequence-Link1-ori1001. The screening marker module was constructed by overlapping two fragments using primers pTEFin-F / Ura3-R to obtain pTEFin- Ura3 Subsequently, primer pTEFin-F / tCYC1-R was used to ligate it to the terminator CYC1 to obtain pTEFin- Ura3 -tCYC1. Finally, primers pCEV1-F / pCEV1-R and pCEV2-F / pCEV2-R were used to amplify the pUC19 plasmid as a template to obtain the backbones pCE-V1 and pCE-V2. The amplified centromere sequences -Link1-ori1001 and pTEFin- were ligated using Gibson Assembly. Ura3-tCYC1, pCE-V1, and pCE-V2 were mixed in a volume ratio of 2:1:1:1 based on the detected concentrations, and then transformed as described above. The pCE plasmid was successfully constructed, as shown in the schematic diagram below. Figure 1 As shown, the construction methods for the 12 plasmids pCE-M1 to pCE-M12 carrying different mutant centromere sequences are the same as above.

[0036] The primer sequences used in the above steps are as follows: Primer Link1-F: 5'-CATTATCTACGAAAAGCGGTACCGAGCTCGAATTCA-3' (SEQ ID NO:30); Primer Link1-R: 5'-AGTAGTATGCGGCCGCAGATCTGGCCCTTTCGTCTCGCGCGTTTCGGT-3' (SEQ ID NO: 31); Primer pCEV1-F: 5'-ATCCTTTTATCAGACATAGACGAAAGGGCCTCGTGATACG-3' (SEQ ID NO:32); Primer pCEV1-R: 5'-GCCGCCAACCCGGTCTCTGGGGATAACGCAGGAAAGAACA-3' (SEQ ID NO: 33); Primer pCEV2-F: 5'-TCGAAGGCTTTAATTTGCTGATTCTGTGGATAACCGTATT-3' (SEQ IDNO: 34); Primer pCEV2-R: 5'-CCCTAAATTTGATGAAAGGGGGATCCTCTAGAGTCGACCT-3' (SEQ ID NO:35).

[0037] 2) Construction of pARS1 plasmid carrying the traditional lipolytic yeast replication system ARS1: First, using pUC19 (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) as a template, the backbones pARS-V1 and pARS-V2 were amplified using primers pARSV1-F / pARSV1-R and pARSV2-F / pARSV2-R, respectively. Then, using the Gibson Assembly ligation method, the pARS-V1 and pARS-V2 backbone fragments, ARS1, and pTEFin-Ura3-tCYC1 obtained from overlap amplification were mixed at a volume ratio of 1.5:1.5:1:1 according to the detected concentrations. Next, the enzyme reagent for seamless ligation assembly technology (NEB, Gibson Assembly Cloning Kit, catalog number E5510) was added. After incubation at 50°C for 35 min, the ligation product was transformed into [the desired product] using a heat shock method. E. coli DH5α was successfully used to construct the pARS1 plasmid, as shown in the schematic diagram below. Figure 2 As shown.

[0038] The primer sequences used in the above steps are as follows: Primer pARSV1-F: 5'-CTTGCTTGAATATACAGTAGTATGGACGAAAGGGCCTCGT-3' (SEQ IDNO: 36); Primer pARSV1-R: 5'-GCCAACCCGGTCTCTGGGGATAACGCAGGAAAGAACATGT-3' (SEQ IDNO: 37); Primer pARSV2-F: 5'-CGCTCGAAGGCTTTAATTTGCTGATTCTGTGGATAACCGT-3' (SEQ IDNO: 38); Primer pARSV2-R: 5'-ATGAAATTGTAAATGAGGGGGATCCTCTAGAGTCGACCTG-3' (SEQ ID NO: 39).

[0039] 3. Strain construction Yeast lipolyticis Po1f was cultured in 5 mL YPD at 30℃ and 220 rpm until mid-stage. The culture was centrifuged at 650 g for 8 min, the supernatant was discarded, and 5 mL of Frozen-EZ yeast solution 1 (ZYMO RESEARCH Frozen-EZ Yeast Transformation II™) was added for resuspending. After centrifugation again, the supernatant was discarded, and 500 μL of Frozen-EZ yeast solution 2 (ZYMO RESEARCH Frozen-EZ Yeast Transformation II™) was added for resuspending. The resulting cells were aliquoted to obtain competent yeast cells. Subsequently, 14 vectors (pCE, pCE-M1 to pCE-M12, and pARS1) were transformed into Po1f competent cells. 20 µL of competent cells were thoroughly mixed with 1 µg of plasmid DNA, and 200 µL of Frozen-EZ yeast solution 3 (ZYMO RESEARCH Frozen-EZ Yeast Transformation II™) was added to assist transformation. The transformation products were plated onto SD-Ura-deficient medium and cultured at 30°C for 2–4 days. Clean single colonies from the transformation plates were then picked for colony PCR verification, yielding 14 yeast strains (Po-CE, Po-CEM1 to Po-CEM12, and Po-ARS1) containing free expression vectors with different replication elements.

[0040] 4. Plasmid stability determination To determine the stability of different free expression vectors in yeast, 14 *Yarrowia lipolyticis* strains containing different replication element vectors were plated onto SD-Ura plates and incubated at 30°C for 2 days. For each strain, three single colonies from the selection plate were selected as replicates and inoculated into SD-Ura liquid medium for overnight incubation. When OD... 600 When the concentration approaches 1, take 10 μL of bacterial suspension, dilute it 500 times, and then take 50 μL of the dilution and spread it onto SD-Ura defective plates and YPD plates respectively for cultivation. Incubate the plates at 30℃ for 48 h, count the colonies, and calculate plasmid stability as: (CFU / YPD plate × 100%). The results are shown below. Figure 3 As shown.

[0041] Depend on Figure 3It can be seen that, after screening, three strains with relatively high stability were initially obtained: Po-CE, Po-CEM6, and Po-CEM3, with plasmid stability of 72.4%, 48.9%, and 25.84%, respectively. In contrast, the plasmid stability of the Po-ARS1 strain carrying the traditional replication element ARS1 free vector was only 12.4%. Specifically, the C-M3 strain carried in pCE-M3 mutated from thymine to cytosine at position 174 compared to the original centromere sequence, and the C-M6 strain carried in pCE-M6 mutated from adenine to thymine at position 119 compared to the original centromere sequence, and an additional guanine base was added at position 177.

[0042] Subsequently, the three groups of strains with high plasmid stability were passaged for assays. Each group of strains was inoculated into 5 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 96 hours, with samples taken every 12 hours. After sampling, the cultures were diluted and spread onto selection medium and YPD medium as described above, and the percentage of colonies carrying the plasmid was calculated. The results are shown below. Figure 4 As shown.

[0043] Figure 4 Passage experiments showed that, in the 12-hour culture, the stability of the three plasmids pCE, pCE-M6, and pCE-M3 was 68.8%, 67%, and 42.69%, respectively. During the 12-24 hour culture period, the plasmid loss rate was relatively fast, and the stability decreased by 31.9%, 38.63%, and 33.7%, respectively. After 24 hours of passaging, the mitotic capacity of the plasmids tended to stabilize, with pCE and pCE-M6 showing higher stability than pCE-M3. Therefore, the genetic stability of the two novel *Yarrowia lipolytica* free expression vectors, pCE and pCE-M6, is significantly better than that of the traditional high-loss-rate plasmid pARS1.

[0044] 5. Production Test To determine the effectiveness of free expression vectors pCE and pCE-M6 in the actual production of natural active products, *Cyclocarya paliurus* (…) was used. Hypoxylon 1,8-Cineole synthase gene from sp.) Hyp3 ,tomato( Solanum lycopersicum ) nerolithyl pyrophosphate synthase gene SltNPPS Mutant farnesyl pyrophosphate synthase gene from endogenous Yersinia lipophilia ERG20 F88W-N119W The complete 1,8-cineole biosynthesis module was assembled onto a vector to obtain pCE-C and pCEM6-C. These two vectors were then transformed into *Yarrowia lipolytica* po1f to obtain YL-CE and YL-CEM6, respectively. The yeast transformation included the following steps: 1) Take 20 µL of aliquoted Yersinia lipophila competent cells from the -80℃ freezer and mix them with 0.2~1 µg of plasmid DNA (volume less than 5 µL) in a clean bench; 2) In a sterile 1.5 mL centrifuge tube, mix the above mixture with 200 μL of Frozen-EZ yeast solution by pipetting and aspiration. 3) Incubate the mixture at 30°C for 1-2 hours. During incubation, gently tap the mixture with your finger or vortex it at low speed 2-3 times to ensure that the plasmid DNA and the yeast competent cells are thoroughly mixed. 4) After incubation, spread the competent mixture onto a solid culture medium plate in a clean bench. After spreading evenly, seal the plate and invert it in a 30°C constant temperature incubator for 2-3 days. 5) Inoculate the colonies transformed with the corresponding plasmid into liquid culture medium and incubate overnight in a shaker at 30°C and 200 rpm; wait for OD... 600 At approximately 2 hours, inoculate into 20 mL of culture medium at a specific inoculation rate to allow the initial OD to reach a certain level. 600 The concentration was 0.1, and a 20% (v / v) dodecane capping layer was added to capture volatile monoterpenes produced during growth. After incubation at 30°C and 200 rpm for 96 h, the bacterial culture was centrifuged at 10000 × g for 8 min. The upper dodecane phase was carefully aspirated to determine the product yield. The results are as follows: Figure 5 As shown.

[0045] Depend on Figure 5 The results showed that YL-CEM6 produced 5.63 mg / L of 1,8-cineole, representing a 63.67% increase in yield compared to YL-CE.

[0046] In summary, this invention has successfully developed a free expression vector pCE-M6 carrying the mutant element C-M6, which can serve as a convenient molecular tool for the synthetic biology production and research of Yersinia lipophila.

[0047] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A nucleic acid molecule, characterized in that, The DNA sequence of the nucleic acid molecule is SEQ ID NO:

23.

2. A carrier, characterized in that, The carrier comprises the nucleic acid molecule of claim 1.

3. The carrier according to claim 2, characterized in that, The vector comprises the following elements: centromere, autonomously replicating sequence, resistance gene, promoter, selection marker gene, and terminator, wherein the centromere is the nucleic acid molecule as described in claim 1.

4. The carrier according to claim 3, characterized in that, The autonomous replication sequence includes ori1001 or ARS.

5. The carrier according to claim 3, characterized in that, The resistance gene includes the AmpR gene or the Kmr gene.

6. The carrier according to claim 3, characterized in that, The selection marker gene includes the URA3 gene.

7. A recombinant yeast, characterized in that, The recombinant yeast comprises the vector according to any one of claims 2 to 6.

8. A reagent kit, characterized in that, The kit includes at least one of (1) to (2): (1) The nucleic acid molecule according to claim 1; (2) The carrier according to any one of claims 2 to 6.

9. A method for producing proteins in *Yarrowia lipolyticis*, characterized in that, The method includes expressing the vector of any one of claims 2 to 6 in the Yersinia lipophila.

10. The use of the nucleic acid molecule of claim 1 or the kit of claim 8 in constructing a stable Yersinia lipophila vector.

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