Yeast interspecies shuttle vector and preparation method thereof

Abstract

The present invention relates to the field of biotechnology, and in particular to yeast interspecies shuttle vectors and preparation methods. The present invention specifically includes two yeast interspecies shuttle vector construction strategies and six yeast saccharomyces-other yeast shuttle vectors constructed using the strategies. The construction of yeast saccharomyces-Pichia pastoris and yeast saccharomyces-Schizosaccharomyces pombe shuttle vectors can be completed by using a shuttle vector construction strategy based on the centromere of yeast saccharomyces cerevisiae, and the construction of yeast saccharomyces-Pichia pastoris, yeast saccharomyces-Schizosaccharomyces pombe, yeast saccharomyces-Yarrowia lipolytica and yeast saccharomyces-Pichia pastoris-Yarrowia lipolytica shuttle vectors can be completed by using a shuttle vector construction strategy based on the 2μ sequence of yeast saccharomyces cerevisiae. The present invention can realize the construction of yeast interspecies shuttle vectors and can be used as a bottom-level tool for interspecies transfer of DNA.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a yeast interspecies shuttle vector and a preparation method thereof. Background Art

[0002] The shuttling of DNA between species can expand the application scope of genetic manipulation tools in organisms. For example, yeast-bacteria shuttle vectors can use the high copy number of bacterial plasmids to prepare a large number of vectors and then transform them into yeast cells for gene expression. Yeast-mammalian cell shuttle vectors can realize the construction of artificial chromosomes in yeast and then transfect them into mammalian cells for gene expression. The model organism Saccharomyces cerevisiae has mature genetic manipulation tools and is widely used in DNA assembly and editing. In contrast, some other types of yeast, such as Pichia pastoris, Yarrowia lipolytica and other unconventional yeasts, have relatively scarce genetic manipulation tools, making it difficult to achieve large-scale genetic modification involving multiple genes. Expanding the application scope of genetic manipulation tools for Saccharomyces cerevisiae can improve the scale and efficiency of genetic manipulation of yeast that is difficult to transform.

[0003] DNA shuttling requires the stability of the target DNA in the cells of both the donor and recipient species. Shuttle vectors are one of the basic tools for achieving cross-species DNA transfer, but there are currently no reports on the successful construction of yeast interspecies shuttle vectors. Summary of the invention

[0004] In view of this, the yeast interspecies shuttle vector and preparation method provided by the present invention can realize the construction of the yeast interspecies shuttle vector and can be used as a bottom-level tool for interspecies DNA transfer.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a method for constructing a shuttle vector, comprising integrating a yeast stable element with homology arms, a marker gene with homology arms, a skeleton with homology arms and an acceptable gene element based on yeast homologous recombination to obtain the shuttle vector.

[0007] In some specific embodiments of the present invention, the yeast stabilizing element in the above method comprises one or two or more of the centromere sequence of Saccharomyces cerevisiae chromosome VI, the regional centromere sequence of Pichia pastoris chromosome II, the centromere sequence of Yarrowia lipolytica chromosome I, the Saccharomyces cerevisiae ARS sequence, the Schizosaccharomyces pombe ARS sequence, the Yarrowia lipolytica ARS sequence, the centromere fusion ARS sequence of Saccharomyces cerevisiae chromosome VI, the centromere fusion ARS sequence of Yarrowia lipolytica chromosome I or the Saccharomyces cerevisiae 2μ replication sequence.

[0008] In some specific embodiments of the present invention, the yeast stabilizing element in the above method includes one or two or more of the centromere fusion ARS sequence of chromosome VI of Saccharomyces cerevisiae, the regional centromere sequence of chromosome II of Pichia pastoris, the ARS sequence of Schizosaccharomyces pombe, the centromere fusion ARS sequence of chromosome I of Yarrowia lipolytica, or the 2μ replication sequence of Saccharomyces cerevisiae.

[0009] In some specific embodiments of the present invention, the marker gene in the above method includes Amp R 、His3、Zeo R , Hyg R , Ura4, iRFP or GFP, one or two or more thereof.

[0010] In some specific embodiments of the present invention, the backbone in the above method comprises pRS423 plasmid and / or pRS416 plasmid.

[0011] In some specific embodiments of the present invention, the above method comprises the following steps:

[0012] Step (1): designing primers for amplifying a yeast stable element with homology arms, designing primers for amplifying a marker gene with homology arms, and designing primers for amplifying a backbone with homology arms, amplifying the template, and obtaining a yeast stable element with homology arms, a marker gene with homology arms, and a backbone with homology arms;

[0013] Step (2): introducing the yeast stabilizing element with homology arms, the marker gene with homology arms, the skeleton with homology arms and the acceptable gene elements described in step (1) into yeast, culturing, extracting and obtaining a shuttle vector;

[0014] The template includes one or two or more of a yeast genome, a marker gene or a backbone.

[0015] In some specific embodiments of the present invention, the above method comprises the following steps:

[0016] Step (1): designing primers for amplifying a yeast stable element with homology arms, designing primers for amplifying a marker gene with homology arms, and designing primers for amplifying a backbone with homology arms, amplifying the template, and obtaining a yeast stable element with homology arms, a marker gene with homology arms, and a backbone with homology arms;

[0017] Step (2): introducing the yeast stable element with homology arms, the marker gene with homology arms, the skeleton with homology arms and acceptable gene elements described in step (1) into yeast, culturing, screening and extracting to obtain plasmid 1;

[0018] Step (3): introducing the plasmid 1 in step (2) into Escherichia coli, culturing, extracting, and obtaining plasmid 2;

[0019] Step (4): introducing the plasmid 2 in step (3) into yeast, culturing, extracting, and obtaining a shuttle vector;

[0020] The template includes one or two or more of a yeast genome, a marker gene or a backbone;

[0021] The yeast includes one or two or more of Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe or Yarrowia lipolytica.

[0022] The present invention also provides a shuttle vector, comprising two different yeast stable elements and an acceptable gene element;

[0023] The yeast stabilizing element comprises one or two or more of the centromere sequence of chromosome VI of Saccharomyces cerevisiae, the regional centromere sequence of chromosome II of Pichia pastoris, the centromere sequence of chromosome I of Yarrowia lipolytica, the ARS sequence of Saccharomyces cerevisiae, the ARS sequence of Schizosaccharomyces pombe, the centromere fusion ARS sequence of chromosome VI of Saccharomyces cerevisiae, the centromere fusion ARS sequence of chromosome I of Yarrowia lipolytica or the 2μ replication sequence of Saccharomyces cerevisiae.

[0024] In some specific embodiments of the present invention, the shuttle vector further comprises a marker gene and / or a backbone;

[0025] The marker gene includes Amp R 、His3、Zeo R , Hyg R , one or two or more of Ura4, iRFP or GFP;

[0026] The backbone includes the pRS423 plasmid and / or the pRS416 plasmid.

[0027] In some specific embodiments of the present invention, the above shuttle vector has:

[0028] (1) a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 6; or

[0029] (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (1), and having the same or similar function as (1); or

[0030] (3) a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in (1) or (2);

[0031] The plurality is 2 to 1000.

[0032] The yeast interspecies shuttle vector and preparation method of the present invention have the following effects:

[0033] The construction of Saccharomyces cerevisiae-Pichia pastoris and Saccharomyces cerevisiae-Schizosaccharomyces pombe shuttle vectors can be completed by using the shuttle vector construction strategy based on the centromere of Saccharomyces cerevisiae, and the construction of Saccharomyces cerevisiae-Pichia pastoris, Saccharomyces cerevisiae-Schizosaccharomyces pombe, Saccharomyces cerevisiae-Yarrowia lipolytica and Saccharomyces cerevisiae-Pichia pastoris-Yarrowia lipolytica shuttle vectors can be completed by using the shuttle vector construction strategy based on the 2μ sequence of Saccharomyces cerevisiae. This method can realize the construction of yeast interspecies shuttle vectors and can be used as a bottom-level tool for interspecies DNA transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0035] Figure 1 The shuttle vector construction strategy based on the centromere of Saccharomyces cerevisiae in the present invention is shown; a 504bp sequence of the centromere of chromosome VI of Saccharomyces cerevisiae fused with ARS is used as a stable element of the shuttle vector in Saccharomyces cerevisiae and combined with stable elements of other yeasts. At the same time, a screening tag available for the corresponding yeast needs to be added to the vector to screen the transformants. In order to facilitate the enrichment of the vector in bacteria, the replication start site and screening tag of Escherichia coli also need to be added;

[0036] Figure 2 The PCR verification results of E. coli transformed with the recombinant plasmid are shown;

[0037] Figure 3 The figure shows the Saccharomyces cerevisiae-Pichia pastoris shuttle vector constructed by the Saccharomyces cerevisiae centromere-based shuttle vector construction strategy in the present invention and the results of its shuttle property verification; the upper figure is a schematic diagram of the shuttle vector, wherein the Pichia pastoris stable element is its chromosome II region-type centromere; the lower figure is a diagram showing the results of the vector transformation into BY4741 (the culture dish on the left) and GS115 (the culture dish on the right);

[0038] Figure 4 The results of PCR verification of recombinant plasmid transformation into Pichia pastoris are shown;

[0039] Figure 5 The PCR verification results of E. coli transformed with the recombinant plasmid are shown;

[0040] Figure 6The figure shows the Saccharomyces cerevisiae-Schizosaccharomyces pombe shuttle vector constructed by the Saccharomyces cerevisiae centromere-based shuttle vector construction strategy and the results of its shuttle property verification; the upper figure is a schematic diagram of the shuttle vector, in which the Saccharomyces pombe stable element is its ARS sequence; the lower figure is the vector transformed into BY4741 (the culture dish on the left) and 927h - (Petri dish on the right) Results of the strain;

[0041] Figure 7 The PCR verification results of recombinant plasmid transformation into Schizosaccharomyces pombe are shown;

[0042] Figure 8 The shuttle vector construction strategy based on the 2μ sequence of Saccharomyces cerevisiae in the present invention is shown; the 1343bp 2μ sequence of Saccharomyces cerevisiae (including 2μ plasmid ori, STB and FRT sequences) is used as the stable element of the shuttle vector in Saccharomyces cerevisiae and combined with the stable elements of other yeasts. At the same time, the vector needs to be added with a screening tag available for the corresponding yeast to screen the transformants. In order to facilitate the enrichment of the vector in bacteria, the replication start site and screening tag of Escherichia coli need to be added;

[0043] Fig. 9 The PCR verification results of recombinant plasmid transformation into E. coli are shown;

[0044] Fig.10 The figure shows the Saccharomyces cerevisiae-Pichia pastoris shuttle vector constructed by the shuttle vector construction strategy based on the Saccharomyces cerevisiae 2μ sequence in the present invention and the results of its shuttle property verification; the upper figure is a schematic diagram of the shuttle vector, wherein the Pichia pastoris stable element is its chromosome II region type centromere; the lower figure is a diagram showing the results of the vector transformation into BY4741 (the culture dish on the left) and GS115 (the culture dish on the right);

[0045] Fig.11 The results of PCR verification of recombinant plasmid transformation into Pichia pastoris are shown;

[0046] Fig.12 The figure shows the Saccharomyces cerevisiae-Schizosaccharomyces pombe shuttle vector constructed by the shuttle vector construction strategy based on the Saccharomyces cerevisiae 2μ sequence in the present invention and the results of its shuttle property verification; the upper figure is a schematic diagram of the shuttle vector, in which the Saccharomyces pombe stable element is its ARS sequence; the lower figure is the vector transformed into BY4741 (the culture dish on the left) and 927h - (Petri dish on the right) Results of the strain;

[0047] Fig.13The figure shows the Saccharomyces cerevisiae-Yarrowia lipolytica shuttle vector constructed by the shuttle vector construction strategy based on the Saccharomyces cerevisiae 2μ sequence in the present invention and the results of its shuttle property verification; the upper figure is a schematic diagram of the shuttle vector, wherein the stable element of Yarrowia lipolytica is a fusion sequence of the centromere of its chromosome I and the ARS sequence; the lower figure is a diagram showing the results of the vector transformation into BY4741 (the culture dish on the left) and ATCC 201249 (the culture dish on the right) strains;

[0048] Fig.14 The figure shows the Saccharomyces cerevisiae-Pichia pastoris-Yarrowia lipolytica shuttle vector constructed by the shuttle vector construction strategy based on the Saccharomyces cerevisiae 2μ sequence in the present invention and the shuttling verification results thereof; the upper figure is a schematic diagram of the shuttle vector, wherein the Pichia pastoris stable element is the regional centromere of its chromosome II, and the Yarrowia lipolytica stable element is the fusion sequence of the centromere of its chromosome I and the ARS sequence; the lower figure is a diagram showing the results of the vector transformation into BY4741 (the culture dish on the left), GS115 (the culture dish in the middle) and ATCC 201249 (the culture dish on the right) strains. DETAILED DESCRIPTION

[0049] The present invention discloses a yeast interspecies shuttle vector and a preparation method. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] The present invention belongs to the field of shuttle vectors, specifically including two yeast interspecies shuttle vector construction strategies and six saccharomyces cerevisiae-other yeast shuttle vectors constructed by using the strategies. The present invention can be widely used in the fields of DNA assembly, DNA transfer, etc.

[0051] The invention provides two yeast interspecies shuttle vector construction strategies, and uses the two strategies to construct six yeast saccharomyces cerevisiae-other yeast shuttle vectors. The development of yeast interspecies shuttle vectors provides a tool for realizing yeast interspecies DNA shuttle.

[0052] In order to achieve the above-mentioned invention object, the present invention provides the following technical methods:

[0053] Strategy 1: The present invention provides a shuttle vector construction strategy based on the centromere of Saccharomyces cerevisiae. The strategy is applicable to yeast strains whose stabilizing elements are regional centromeres or ARS sequences, and comprises the following steps:

[0054] Step 1: Use PCR amplification to obtain other yeast stable elements with homology arms and screen gene fragments.

[0055] Step 2: Use yeast homologous recombination and other methods to assemble other yeast regional stable elements with homology arms and screening genes obtained in step 1 into the Saccharomyces cerevisiae regional centromeric plasmid.

[0056] Step 3: Extract the shuttle vector from Saccharomyces cerevisiae and transform it into other corresponding yeast cells to achieve shuttling.

[0057] In some specific embodiments of the present invention, the yeast stabilizing element in step 1 of the method is a regional centromere or an ARS sequence. The present invention uses the 6655bp regional centromere of chromosome II of Pichia pastoris and the 782bp ARS sequence of Schizosaccharomyces pombe as stabilizing elements, and assembles them with the centromere plasmid of Saccharomyces cerevisiae to construct the Saccharomyces cerevisiae-Pichia pastoris and Saccharomyces cerevisiae-Schizosaccharomyces pombe shuttle vectors.

[0058] Strategy 2: The present invention provides a shuttle vector construction strategy based on a 2micron plasmid (2μ) sequence, which is applicable to yeast strains whose stabilizing elements are regional centromeres, ARS sequences, and dot centromeres, and comprises the following steps:

[0059] Step 1: Use PCR amplification to obtain other yeast stable elements with homology arms and screen gene fragments.

[0060] Step 2: Use yeast homologous recombination and other methods to assemble other yeast region stable elements with homology arms and screening genes obtained in step 1 into the Saccharomyces cerevisiae 2μ plasmid.

[0061] Step 3: Extract the shuttle vector from Saccharomyces cerevisiae and transform it into other corresponding yeast cells to achieve shuttling.

[0062] In some specific embodiments of the present invention, the yeast stable element in step 1 of the method is a regional centromere, an ARS sequence or a point centromere. The present invention uses the 6655bp regional centromere of chromosome II of Pichia pastoris, the 782bp ARS sequence of Schizosaccharomyces pombe and the fusion sequence of the 230bp point centromere of chromosome I of Yarrowia lipolytica and the ARS sequence as stable elements, and assembles them with the 2μ plasmid of Saccharomyces cerevisiae to construct the shuttle vectors of Saccharomyces cerevisiae-Pichia pastoris, Saccharomyces cerevisiae-Schizosaccharomyces pombe and Saccharomyces cerevisiae-Yarrowia lipolytica, and on this basis, further assembles the regional centromere of chromosome II of Pichia pastoris and the point centromere of chromosome I of Yarrowia lipolytica with the 2μ plasmid of Saccharomyces cerevisiae to construct the shuttle vector of Saccharomyces cerevisiae-Pichia pastoris-Yarrowia lipolytica.

[0063] The sequence information involved in the present invention is as follows:

[0064] Shuttle plasmid pScKp1 (SEQ ID NO: 1) based on the centromere of Saccharomyces cerevisiae and Pichia pastoris:

[0065]

[0066]

[0067]

[0068]

[0069] Shuttle plasmid pScSp1 (SEQ ID NO: 2) based on the centromere of Saccharomyces cerevisiae and the ARS of Schizosaccharomyces pombe:

[0070]

[0071]

[0072]

[0073] Shuttle vector pScKp2 (SEQ ID NO: 3) based on the Saccharomyces cerevisiae 2μ sequence and the Pichia pastoris centromere:

[0074]

[0075]

[0076]

[0077]

[0078] Shuttle plasmid pScSp2 (SEQ ID NO: 4) based on the Saccharomyces cerevisiae 2μ sequence and Schizosaccharomyces pombe ARS:

[0079]

[0080]

[0081]

[0082] Shuttle plasmid pScY13 (SEQ ID NO: 5) based on the Saccharomyces cerevisiae 2μ sequence and the Yarrowia lipolytica centromere:

[0083]

[0084]

[0085]

[0086] Shuttle plasmid pSKY3 (SEQ ID NO: 6) based on the Saccharomyces cerevisiae 2μ sequence, Pichia pastoris centromere and Yarrowia lipolytica centromere:

[0087]

[0088]

[0089]

[0090]

[0091] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0092] The present invention will be further described below in conjunction with embodiments:

[0093] Example 1

[0094] The shuttle vector delivery between Saccharomyces cerevisiae and Pichia pastoris is achieved by using a shuttle vector construction strategy based on the centromere of Saccharomyces cerevisiae and the centromere of Pichia pastoris. The following steps are included:

[0095] 1. Obtain the DNA fragment of chromosome II region type centromere of Pichia pastoris with 50 bp homology arms, bleomycin gene expression cassette, iRFP670 fluorescent protein, and pRS413 plasmid backbone (such as Figure 1 The gene elements and expression cassette sequences are shown in SEQ ID NO: 1, wherein positions 1 to 504 indicate the centromere sequence of Saccharomyces cerevisiae, positions 515 to 2123 indicate the iRFP670 fluorescent protein expression sequence, positions 2124 to 3509 indicate the bleomycin gene expression cassette sequence, positions 3542 to 10196 indicate the centromere sequence of chromosome II region of Pichia pastoris, positions 10328 to 11174 indicate the histidine auxotrophy screening tag sequence of Saccharomyces cerevisiae, positions 489 to 539, positions 2097 to 2148, positions 6812 to 6866, and positions 11146 to 11199 indicate primers, and are assembled as homology arm sequences.

[0096] The PCR reaction system is:

[0097] Table 1: KOD One TM PCR Master Mix PCR Reaction System

[0098]

[0099] X, fill the total reaction volume to 50.0μL. Y, the amount of template added is adjusted according to the type of template to achieve the best effect. Generally speaking, the amount of genomic DNA added is about 200.0ng / 50.0μL, the amount of plasmid DNA added is about 50.0ng / 50.0μL, the amount of bacterial liquid added is about 4.0μL / 50.0μL, and the volume of solid colony template is negligible.

[0100] The PCR reaction program is:

[0101] Table 2: KOD One TM PCR Master Mix PCR Reaction Procedure

[0102]

[0103] The extension reaction time is determined according to the length of the amplified fragment: 1s / kb for fragments below 1kb, 5s / kb for fragments between 1 and 10kb, and 10s / kb for fragments above 10kb.

[0104] 2. The above DNA fragments were co-transformed into Saccharomyces cerevisiae BY4741 for homologous recombination assembly. The transformation method of Saccharomyces cerevisiae is as follows:

[0105] (1) Pick a single colony into 5.0 mL of liquid culture medium and culture overnight at 30°C in a shaking incubator.

[0106] (2) 100.0 μL of bacterial solution was transferred to 5.0 mL of liquid culture medium and cultured in a shaking incubator at 30°C until OD 660 =1.0.

[0107] (3) Take 1.0 mL of bacterial solution and place it in a 1.5 mL centrifuge tube. Centrifuge at 3000 g for 1 min to collect the cells.

[0108] (4) Wash twice with 1.0 mL of sterile water.

[0109] (5) Resuspend the cells in 1.0 mL of 0.1 M LiOAc and place on ice for 5 min.

[0110] (6) Prepare yeast transformation system: 620.0 mL 50% PEG3350, 40.0 μL ssDNA, and 90.0 μL 1.0 M LiOAc were added to the system in sequence and vortexed to mix. (ssDNA was pre-boiled at 100°C for 10 min and cooled on ice before use.)

[0111] (7) The bacterial solution was centrifuged at 3000 g for 1 min to collect the cells, and the cells were resuspended in 100.0 μL 0.1 M LiOAc. The bacterial solution was added to the transformation system, and the centrifuge tube was inverted several times to mix well.

[0112] (8) Incubate in a 30°C incubator for 30 min.

[0113] (9) Add 90.0 μL DMSO, invert the tube several times to mix, and heat shock in a 42°C water bath for 18 min.

[0114] (10) Collect cells by centrifugation at 3000 g for 1 min, and add 400.0 μL CaCl 2 Resuspend and let stand for 5 to 10 minutes.

[0115] (11) Collect the cells by centrifugation at 3000 g for 1 min, resuspend in 100.0 μL sterile water, spread the bacterial solution on the corresponding screening plate, and incubate in a 30°C incubator. (If antibiotic screening is used, use a non-resistant medium to incubate for 4 h before spreading the plate.)

[0116] 3. Perform PCR verification and Sanger sequencing on the assembled interface of the grown colonies, select the correct colonies to extract the plasmid, and then transfer them into E. coli for enrichment and extract the E. coli plasmid. The PCR verification results are shown in Figure 2 The E. coli transformation method is as follows:

[0117] (1) Pick a single colony and place it in 5.0 mL of screening liquid culture medium and culture it in a shaking incubator at 37°C overnight.

[0118] (2) Transfer 100.0 μL of bacterial solution into 50.0 mL of screening medium and culture at 37°C in a shaking incubator for 2.5-3 h.

[0119] (3) Place the bacterial solution on ice for 15 minutes. Keep the temperature at 4°C for subsequent operations.

[0120] (4) Collect the cells by centrifugation at 3000 g for 10 min.

[0121] (5) Wash the cells three times with 50 mL of 10% glycerol at 4°C.

[0122] (6) Resuspend the cells in 500.0 μL of 4°C glycerol and dispense 100.0 μL of cells into one tube.

[0123] (7) Add 3.0 ng of the DNA to be transformed into a tube of competent cells, mix well, and add it to a 1 mm gap electroporation cuvette precooled at -20°C.

[0124] (8) Adjust the electroporator and use the bacterial program for electroporation.

[0125] (9) Quickly add 1.0 mL of LB liquid medium to resuspend the cells in a 1.5 mL centrifuge tube.

[0126] (10) Incubate at 37°C on a shaker for 1 h. Collect the cells by centrifugation at 3000 g for 1 min, resuspend in 100 μL of sterile water, spread the bacterial solution on an LB plate with the corresponding resistance, and culture in a 37°C incubator.

[0127] 4. The extracted plasmid was transformed into Pichia pastoris, and the correct strain was obtained by PCR verification to obtain the shuttle plasmid pScKp1 (such as Figure 3 The plasmid sequence is shown in SEQ ID NO: 1). The Pichia pastoris transformation method is as follows:

[0128] (1) Pick a single colony into 5.0 mL of liquid culture medium and culture overnight at 30°C in a shaking incubator.

[0129] (2) 100.0 μL of bacterial solution was transferred to 50.0 mL of liquid culture medium and cultured in a shaking incubator at 30°C until OD 660 =1.0.

[0130] (3) Transfer 15.0 mL of bacterial culture to a 50 mL centrifuge tube and centrifuge at 3000 g for 5 min to collect the cells.

[0131] (4) Resuspend the cells in 4.8 mL 1M sorbitol, and add 2.3 mL sterile water, 800.0 μL 1M LiOAc, 80.0 μL 1M Tris-HCl (pH=7.5), and 80.0 μL 1M DTT in sequence. Mix well and incubate in a 30°C incubator for 30 min.

[0132] (5) Collect the cells by centrifugation at 3000 g for 5 min and wash them three times with 1.0 mL of pre-cooled 1.0 M sorbitol.

[0133] (6) Resuspend the cells in 200.0 μL of pre-chilled 1.0 M sorbitol.

[0134] (7) Add 3.0 ng of DNA to be transformed into the cells and incubate on ice for 5 min.

[0135] (8) Transfer the mixture to a pre-cooled 2 mm gap electroporation cuvette and perform electroporation using the fungus program.

[0136] (9) Immediately add 1.0 mL of pre-chilled 1.0 M sorbitol to resuspend the cells.

[0137] (10) Take 100.0-200.0 μL of bacterial solution and apply it to the corresponding plate. (If antibiotic screening is used, it is necessary to use a non-resistance medium to incubate for 1 hour before applying the plate.)

[0138] PCR validation results are shown in Figure 4 .

[0139] Example 2

[0140] The shuttle vector delivery between Saccharomyces cerevisiae and Saccharomyces pombe is achieved by using a shuttle vector construction strategy based on the centromere of Saccharomyces cerevisiae and the ARS of Saccharomyces pombe. The following steps are included:

[0141] 1. Obtain the fragments of the pombe ARS sequence, hygromycin gene expression cassette and GFP fluorescent protein DNA fragments, pRS416 plasmid backbone and the like with 50 bp homology arms by PCR. The sequences of each gene element and expression cassette are shown in SEQ ID NO: 2, wherein positions 8150 to 8653 represent the centromere sequence of Saccharomyces cerevisiae, positions 48 to 1072 represent the uracil auxotrophy screening tag sequence of Saccharomyces cerevisiae, positions 1921 to 3403 represent the GFP fluorescent protein expression sequence, positions 3405 to 5017 represent the hygromycin gene expression cassette sequence, positions 5044 to 5825 represent the pombe ARS sequence, positions 1871 to 1920, positions 3380 to 3429, and positions 5890 to 5939 represent primers, and assemble as homology arm sequences. The reaction system and reaction procedure of PCR are shown in Tables 1 and 2 of Example 1.

[0142] 2. The above DNA fragments were co-transformed into Saccharomyces cerevisiae BY4741 for homologous recombination assembly. The transformation method of Saccharomyces cerevisiae was the same as that in Example 1.

[0143] 3. Perform PCR verification and Sanger sequencing on the assembled interface of the grown colonies, select the correct colonies to extract the plasmid, and then transfer them into E. coli for enrichment and extract the E. coli plasmid. The PCR verification results are shown in Figure 5 The E. coli transformation method was the same as in Example 1.

[0144] 4. The extracted plasmid was transformed into Schizosaccharomyces pombe, and the correct strain was obtained by PCR verification to obtain the shuttle plasmid pScSp1 (such as Figure 6 The plasmid sequence is shown in SEQ ID NO: 2). The transformation method of Schizosaccharomyces pombe is as follows:

[0145] (1) Pick a single colony into 5.0 mL of liquid culture medium and culture overnight at 30°C in a shaking incubator.

[0146] (2) 100.0 μL of bacterial solution was transferred to 10.0 mL of liquid culture medium and cultured in a shaking incubator at 30°C until OD 660 =1.0.

[0147] (3) Collect the cells by centrifugation at 1700 g for 10 min.

[0148] (4) Wash once with 10.0 mL of sterile water.

[0149] (5) Resuspend the cells in 4.5 mL of 0.1 M LiOAc, add 50.0 μL of 1×TE and mix well. Centrifuge at 1700 g for 5 min.

[0150] (6) Resuspend the cells in 100.0 μL 0.1 M LiOAc, add 1.0 μL 1× TE and mix well, and incubate at 30°C on a shaker for 1 h.

[0151] (7) Add 5.0 μL ssDNA solution and mix well.

[0152] (8) Add 1.0 μg of the DNA to be transformed, mix well, and incubate at 30°C on a shaker for 30 min.

[0153] (9) Prepare yeast transformation system: 560.0 μL 50% PEG3350, 70.0 μL 1.0 M LiOAc, 7.0 μL 1×TE, 63.0 μL sterile water. Add bacterial solution to the transformation system and mix well.

[0154] (10) Place in a 30°C incubator for 1 hour.

[0155] (11) Heat shock at 42°C for 15 min.

[0156] (12) Collect the cells by centrifugation at 1000 g for 2 min. Resuspend in 100.0 μL sterile water and apply to the plate.

[0157] PCR validation results are shown in Figure 7 .

[0158] Example 3

[0159] The shuttle vector delivery between Saccharomyces cerevisiae and Pichia pastoris is achieved by using a shuttle vector construction strategy based on the Saccharomyces cerevisiae 2μ sequence and the Pichia pastoris centromere. The following steps are included:

[0160] 1. Obtain the Pichia pastoris chromosome II regional centromere with 50 bp homology arms, the bleomycin gene expression cassette, and the pRS423 plasmid backbone (such as Figure 8 The gene elements and expression cassette sequences are shown in SEQ ID NO: 3, wherein positions 12055 to 13397 show the 2μ sequence of Saccharomyces cerevisiae, positions 21 to 1386 show the bleomycin gene expression cassette sequence, positions 1419 to 8073 show the centromere sequence of chromosome II region of Pichia pastoris, positions 8205 to 9051 show the histidine auxotrophy screening tag sequence of Saccharomyces cerevisiae, positions 1 to 50, 4676 to 4725, and 9023 to 9076 show primers, and are assembled as homology arm sequences. The reaction system and reaction procedure of PCR are shown in Tables 1 and 2 of Example 1.

[0161] 2. The above DNA fragments were co-transformed into Saccharomyces cerevisiae BY4741 for homologous recombination assembly. The transformation method of Saccharomyces cerevisiae was the same as that in Example 1.

[0162] 3. Perform PCR verification and Sanger sequencing on the assembled interface of the grown colonies, select the correct colonies to extract the plasmid, and then transfer them into E. coli for enrichment and extract the E. coli plasmid. The PCR verification results are shown in Fig. 9 The E. coli transformation method was the same as in Example 1.

[0163] 4. The extracted plasmid was transformed into Pichia pastoris, and the correct strain was obtained by PCR verification to obtain the shuttle plasmid pScKp2 (such as Fig.10 The plasmid sequence is shown in SEQ ID NO: 3). The Pichia pastoris transformation method is the same as in Example 1. The PCR verification results are shown in Fig.11 .

[0164] Example 4

[0165] The shuttle vector delivery between Saccharomyces cerevisiae and Saccharomyces pombe was achieved by using the shuttle vector construction strategy based on the 2μ sequence of Saccharomyces cerevisiae and the ARS of Saccharomyces pombe. The specific operation method is the same as that in Example 2, and the correct strain is obtained to obtain the shuttle plasmid pScSp2 (such as Fig.12 The plasmid sequence is shown in SEQ ID NO: 4).

[0166] Example 5

[0167] The shuttle vector delivery between Saccharomyces cerevisiae and Yarrowia lipolytica was achieved by using a shuttle vector construction strategy based on the 2μ sequence of Saccharomyces cerevisiae and the centromere of Yarrowia lipolytica. The specific operation method is the same as that in Example 1, wherein the transformation method of Yarrowia lipolytica is as follows:

[0168] The transformation of Yarrowia lipolytica was completed using the Frozen-EZ Yeast Transformation II Kit. The operation process is detailed as follows:

[0169] (1) Streak the Yarrowia lipolytica glycerol strain on a YPD plate and culture at 30°C to obtain a single colony.

[0170] (2) Pick a single colony from the plate, transfer it to 10.0 mL of liquid YPD medium, and culture it in a 30°C incubator at 220 rpm for 24 h.

[0171] (3)OD 600 The bacterial culture was measured after 24 h of culture, and the OD 600 The initial value was 0.1-0.3, and then transferred to 10.0 mL of fresh liquid YPD medium and cultured until the OD 600 Reach 0.8-1.0.

[0172] (4) Collect fresh bacteria in a clean bench. Pipette the cultured fresh bacterial liquid into a sterile centrifuge tube, centrifuge at 6000 rpm for 3 minutes, discard the supernatant, and repeat the collection of bacteria 3-4 times.

[0173] (5) Wash the collected cells twice with 1.0 mL of sterile water and discard the supernatant.

[0174] (6) Add 600.0 μL of EZ1 solution, let stand at room temperature for 30 min, centrifuge at 6000 rpm for 3 min, and discard the supernatant.

[0175] (7) Add 60.0 μL of EZ2 solution to resuspend the cells, then add the DNA to be transformed. Let stand at room temperature for 5 min, add 600.0 μL of EZ3 solution, and mix thoroughly.

[0176] (8) Place the plate in a 30°C incubator and incubate at 220 rpm for 3 h, mixing by inverting the plate every 30 min.

[0177] (9) Centrifuge at 6000 rpm for 3 min, save 100.0 μL of resuspended cells, spread on the corresponding SC plate, and culture in a 30°C incubator for 48 h. Obtain the correct strain and obtain pScYl3 (such as Fig.13 The plasmid sequence is shown in SEQ ID NO: 5).

[0178] Example 6

[0179] The shuttle vector delivery between Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica was achieved by using the shuttle vector construction strategy based on the 2μ sequence of Saccharomyces cerevisiae, Pichia pastoris centromere and Yarrowia lipolytica centromere. The specific operation method is the same as in Example 1, wherein the Pichia pastoris transformation method is the same as in Example 1, and the Yarrowia lipolytica transformation method is the same as in Example 5. Obtain the correct strain and obtain pSKY3 (such as Fig.14 The plasmid sequence is shown in SEQ ID NO: 6).

[0180] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Methods for constructing shuttle vectors, It is characterized in that The method comprises integrating a yeast stable element with homology arms, a marker gene with homology arms, a backbone with homology arms and an acceptable gene element based on yeast homologous recombination to obtain a shuttle vector; The yeast stabilizing element comprises two of the centromere fusion ARS sequence of chromosome VI of Saccharomyces cerevisiae, the regional centromere sequence of chromosome II of Pichia pastoris, the ARS sequence of Schizosaccharomyces pombe, the centromere fusion ARS sequence of chromosome I of Yarrowia lipolytica or the 2μ replication sequence of Saccharomyces cerevisiae; The marker gene includes Amp R 、His3、Zeo R , Hyg R , one or two or more of Ura4, iRFP or GFP; The backbone includes a pRS423 plasmid and / or a pRS416 plasmid; The shuttle vector is pScKp1, pScSp1, pScKp2, pScSp2, pScY13 or pSKY; The sequence of pScKp1 is SEQ ID NO: 1; The sequence of pScSp1 is SEQ ID NO: 2; The sequence of pScKp2 is SEQ ID NO: 3; The sequence of pScSp2 is SEQ ID NO: 4; The sequence of pScY13 is SEQ ID NO: 5; The sequence of the pSKY3 is SEQ ID NO:

6.

2. The method according to claim 1, It is characterized in that The steps include: Step (1): designing primers for amplifying a yeast stable element with homology arms, designing primers for amplifying a marker gene with homology arms, and designing primers for amplifying a backbone with homology arms, amplifying the template, and obtaining a yeast stable element with homology arms, a marker gene with homology arms, and a backbone with homology arms; Step (2): introducing the yeast stabilizing element with homology arms, the marker gene with homology arms, the skeleton with homology arms and the acceptable gene elements in step (1) into yeast, culturing, extracting and obtaining the shuttle vector; The template includes one or two or more of a yeast genome, a marker gene or a backbone.

3. The method according to claim 2, It is characterized in that The steps include: Step (1): designing primers for amplifying a yeast stable element with homology arms, designing primers for amplifying a marker gene with homology arms, and designing primers for amplifying a backbone with homology arms, amplifying the template, and obtaining a yeast stable element with homology arms, a marker gene with homology arms, and a backbone with homology arms; Step (2): introducing the yeast stable element with homology arms, the marker gene with homology arms, the skeleton with homology arms and acceptable gene elements described in step (1) into yeast, culturing, screening and extracting to obtain plasmid 1; Step (3): introducing the plasmid 1 in step (2) into Escherichia coli, culturing, extracting, and obtaining plasmid 2; Step (4): introducing the plasmid 2 in step (3) into yeast, culturing, extracting, and obtaining the shuttle vector; The template includes one or two or more of a yeast genome, a marker gene or a backbone; The yeast includes one or two or more of Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe or Yarrowia lipolytica.

4. Shuttle vector, It is characterized in that The sequence is a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 6.

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