WO2019149288A1 - Efficient genetic engineering vector - Google Patents

Abstract

Provided is a nucleotide construct with 5' to 3' as represented by T-F-GOI; T-GOI-F; F-T-GOI; F-GOI-T; GOI-T-F; GOI-F-T; F-GOI; GOI-F; or P-F-GOI; P-GOI-F; F-P-GOI; F-GOI-P; GOI-P-F; GOI-F-P; F-GOI; and GOI-F respectively. T is an exogenous terminator; and F is a gene fragment represented by B-C-Marker-B, wherein B is a site-specific recombination site; C is a recombinant enzyme expression cassette corresponding to the site-specific recombination site; Marker is a marker gene expression cassette; GOI is a foreign gene expression cassette; and P is an exogenous promoter. A large number of foreign genes can be integrated into the genome using the nucleotide construct.

Description

An efficient genetic engineering carrier Technical field

The present invention relates to the field of biotechnology; in particular, the present invention relates to a novel genetic engineering vector which integrates a large number of foreign genes into the genome of a living organism by two recombinations while avoiding disrupting the function of the host cell gene.

Background technique

N-glycosylation is an important modification process after protein translation and plays an important role in the structure and function of proteins. Mammalian cells and yeast cells synthesize nascent peptide chains in the endoplasmic reticulum, and perform the same N-glycosylation initiation step and modification process on the nascent peptide chain. First, the precursor oligosaccharide G1c ₃ Man ₉ GlcNAc ₂ was Attached to the Asn residue in the conserved sequence of the nascent peptide chain Asn-X-Thr/Ser (X is any amino acid other than Pro), followed by glycoside hydrolase such as glucoside hydrolase I, II and mannoside hydrolase I Under the action of the protein, the sugar chain of the protein is processed to form a Man ₈ GlcNAc ₂ sugar chain structure, and then the protein carrying the sugar chain is transported into the Golgi apparatus. However, in mammalian cells and yeast Golgi, the further modification of the protein sugar chain is completely different. In the mammalian cell Golgi, the Man ₈ GlcNAc ₂ sugar chain on the protein first removes three mannose under the action of mannoside hydrolase I (MnsI) to form a Man ₅ GlcNAc ₂ sugar chain structure; Adding N-acetylglucosamine to the GlcNAcMan ₅ GlcNAc ₂ sugar chain structure by the action of glucosyltransferase I (GnTI); then removing two mannose by the action of mannoside hydrolase II (MnsII) to form GlcNAcMan ₃ GlcNAc ₂ sugar chain structure; then add N-acetylglucosamine under the action of N-acetylglucosaminyltransferase II (GnTII) to form GlcNAc ₂ Man ₃ GlcNAc ₂ sugar chain structure; finally in galactose transferase Under the action of (GalT) and sialyltransferase (ST), Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ and Sia ₂ Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ complex glycochain structures were formed. However, in the yeast cell Golgi, the Man ₈ GlcNAc ₂ sugar chain on the protein first receives an α-l,6-mannose under the action of α-l,6-mannosyltransferase (Ochlp) encoded by the OCH1 gene. The Man ₉ GlcNAc ₂ sugar chain structure is formed, and then mannose is continuously added under the action of various other mannose transferases to form a high mannose type sugar chain structure. Therefore, a major defect in the production of proteins from yeast is the formation of a high mannose chain different from the human body by N-glycosylation of the protein, which may alter the structure of the glycoprotein, affect its function, and be immunogenic (Kornfeld, R. & Kornfeld, S. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631-664, 1985).

The genetic engineering technique can be used to engineer the glycosylation pathway of yeast to make the same N-glycosylation modification of the protein. In order to achieve this goal, it is necessary not only to knock out the OCH1 gene of yeast, but also to express a large number of foreign genes, including the appropriate mannoside hydrolase I (MnsI), N-acetylglucosaminyltransferase I (GnTI), and mannoside hydrolysis. Enzyme II (MnsII), N-acetylglucosaminyltransferase II (GnTII), galactose transferase (GalT), sialyltransferase (ST), and genes involved in biosynthesis of galactose and sialic acid (Hamilton SR) , Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, et al. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 2006; 313: 1441-3). In order to integrate these foreign genes into the yeast genome for expression, different vectors, selection markers, and genomic integration sites are required for selection.

Some vectors, including pBLADE-SX, pBLARG-SX, pBLHIS-SX, and pBLURA3-SX, use a biosynthesis gene such as the genes ADE1, ARG4, HIS4, and URA3 to integrate foreign genes in a single exchange. Corresponding to the locus of auxotrophic yeast cells. The integrated vector can be screened in incomplete media by complementation of yeast auxotrophs (prototrophic). For example, the pBLHIS-SX vector carrying the HIS4 gene can integrate the foreign gene into the his4 locus of the histidine-deficient GS115 strain by single-crossover recombination, compensating for histidine deficiency, and using a histidine-free medium. Screened out (Lin Cereghino GP, Lin Cereghino J, Sunga AJ, Johnson MA, Lim M, Gleeson MA, Cregg JM. New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris. Gene. 2001, 263: 159- 69).

Some vectors also use a dominant selection marker gene to integrate a foreign gene into a specific locus of yeast by single-exchange recombination. For example, the GlycoSwitch vector utilizes zeosin, noureothricin, geneticin (G418), hygromycin and other dominant selectable marker genes to exogenous MnsI, GnTI, MnsII, GnTII. And the GalT gene is integrated into the AOX1 or GAPDH gene promoter of Pichia pastoris cells (Jacobs P, Geysens S, Vervecken W, Contreras R, Callewaert N, Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology, Nat Protoc .2009; 4:58-70).

However, when the foreign gene vector is integrated into the target gene locus of the yeast genome by single-crossover recombination, a homologous repeat of the target gene is formed. Homologous recombination can occur again between the target gene sequences repeated in the same direction, and the integrated exogenous vector is excised to restore the original state of the target gene. Moreover, the target gene can be restored to a wild-type or defective state by homologous recombination at different positions between the same repeat sequences, and the selection of screening conditions can not be used to prevent the occurrence of homologous recombination excision of the vector process. Thus, a foreign gene vector integrated into the genome by single-exchange recombination is genetically unstable.

Different from single-exchange recombinant, some vectors utilize the selectable marker gene, and the double-recombinant recombination can stably integrate the foreign gene vector into the yeast genome, which is the preferred choice for genetic engineering. For example, vectors such as pPIC9, pPIC3.5, pHIL-D2, and pHIL-S1 (Invitrogen Corp., Carlsbad, CA) integrate the foreign gene into the AOX1 locus of Pichia pastoris by double-crossover recombination, resulting in a Mut ^S table. Type, growing slowly in medium with methanol. In addition, the URA5 marker gene was used to stably integrate the foreign gene into the OCH1 locus of ura3 auxotrophic Pichia pastoris by double-crossover recombination. (Nett JH, Gerngross TU, Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris, Yeast. 2003, 20: 1279-90).

However, through the double-crossover recombination of these vectors, the foreign genes are mainly integrated into the individual known loci of the genome, and the gene expression and function of destroying these loci are not affected by the survival of the host cells. However, these known loci integration sites in the yeast genome are few and cannot meet the need to integrate large numbers of foreign genes.

Therefore, the current genetic manipulation techniques for large-scale transformation of the genome are subject to many limitations, mainly due to the lack of expression vectors, selection markers, and integration sites for genomic loci that can be used in large quantities.

Therefore, it is necessary to develop new methods and materials to overcome these limitations, integrate a large number of foreign genes into the genome, and avoid destroying the function of genes near the integration site, so that professional technicians can synthesize organisms such as glycosylation engineering. The field completes complex genetic engineering.

Summary of the invention

The object of the present invention is to provide a genetic engineering vector, which can integrate a large number of foreign genes into a genome while avoiding disrupting the function of genes near the integration site and not affecting the survival of the host cells.

In a first aspect, the invention provides a nucleotide construct having a class I structure as shown below:

5'H-T-F-GOI-3'H;

5'H-T-GOI-F-3'H;

5'H-F-T-GOI-3'H;

5'H-F-GOI-T-3'H;

5'H-GOI-T-F-3'H;

5'H-GOI-F-T-3'H;

5'H-F-GOI-3'H;

5'H-GOI-F-3'H;

Wherein T is an exogenous terminator; F is a gene fragment represented by BC-Marker-B; wherein B is a site-specific recombination site; C is a recombinase expression corresponding to the site-specific recombination site Box; Marker is a marker gene expression cassette; GOI is a foreign gene expression cassette;

or,

The nucleotide construct has a class II structure as shown below:

5'H-F-GOI-P-3'H;

5'H-GOI-F-P-3'H;

5'H-P-F-GOI-3'H;

5'H-P-GOI-F-3'H;

5'H-F-P-GOI-3'H;

5'H-GOI-P-F-3'H;

5'H-F-GOI-3'H;

5'H-GOI-F-3'H;

Wherein P is an exogenous promoter; F, B, C, Marker and GOI are as described above.

It will be understood by those skilled in the art that the nucleotide construct of the present invention, if it contains no terminator or promoter, can be placed anywhere in the genome; that is, it can be placed in the middle of an open reading frame (ORF). Or any location upstream and downstream.

In a specific embodiment, the nucleotide construct has a class I structure as shown below:

5'H-T-F-GOI-3'H;

5'H-T-GOI-F-3'H;

5'H-F-T-GOI-3'H;

5'H-F-GOI-T-3'H;

5'H-GOI-T-F-3'H;

5'H-GOI-F-T-3'H;

Wherein, T, F, and GOI are as described above;

or,

The nucleotide construct has the class II structure shown below

5'H-F-GOI-P-3'H;

5'H-GOI-F-P-3'H;

5'H-P-F-GOI-3'H;

5'H-P-GOI-F-3'H;

5'H-F-P-GOI-3'H;

5'H-GOI-P-F-3'H;

Among them, P, F and GOI are as described above.

In a specific embodiment, the transcription terminator includes, but is not limited to, a transcription terminator such as ADH1, CYC1 and TIF51A of Saccharomyces cerevisiae, a transcription terminator such as ALG6, AOD, AOX1, ARG4, PMA1 and TEF1 of P. pastoris;

Transcription promoters include, but are not limited to, transcription promoters such as ADH1, GAP, PGK1, and TEF1 of Saccharomyces cerevisiae, transcriptional promoters such as GAP, ILV5, PGK1, and TEF1 of Pichia pastoris, and promoters AOX1 and FLD1.

In a specific embodiment, the recombinase includes, but is not limited to, a Flp recombinase or a Cre recombinase; preferably a Flp recombinase.

In a preferred embodiment, the site-specific recombination site and the corresponding recombinase constitute a site-specific recombination system including, but not limited to, Flp-FRT of Saccharomyces cerevisiae, Cre-loxP and Xygosaccharomyces rouxii of bacteriophage P1 R-RS.

In a preferred embodiment, the nucleotide construct may comprise a plurality of exogenous gene expression cassettes or a plurality of marker gene expression cassettes.

In a preferred embodiment, said exogenous gene expression cassette in a nucleotide construct having a class I structure may be free of its own transcription terminator; said in a nucleotide construct having a class II structure The foreign gene expression cassette may contain no transcriptional promoter of its own.

In a preferred embodiment, the marker gene comprises one or more antibiotic resistance genes, preferably a Sh ble gene resistant to zeocin, and resistant to kanamycin (kan) or genetic mold The neo gene of geneticin (G418), which is resistant to the BSD gene of blasticidin (Blasticidin).

In a specific embodiment, the nucleotide construct further comprises a homology arm at the 5' and 3' ends.

In a specific embodiment, the nucleotide construct may further comprise other genes X, preferably bacteria, capable of replicating in a host cell (eg, a bacterium) when the nucleotide construct forms a loop. The origin of replication and the antibiotic resistance gene; and when the nucleotide construct forms a linear construct, the position of the other gene X is anywhere between B of the nucleotide construct.

In a preferred embodiment, the origin of replication includes, but is not limited to, fl-ori, colisin, col El.

In a third aspect, the invention provides an expression vector comprising the nucleotide construct of the first aspect or the second aspect.

In a fourth aspect, the present invention provides a host cell which integrates the nucleotide construct of the first aspect or the second aspect in its genome.

In a preferred embodiment, the host cell includes, but is not limited to, eukaryotic and prokaryotic host cells, such as E. coli, Pseudomonas spp., Bacillus spp. ), Streptomyces spp., fungi and yeast, insect cells, such as Spodoptera frugiperda (SF9), animal cells, such as Chinese hamster ovary cells (CHO), Mouse cells, African green monkey cells, cultured human cells and plant cells.

In a preferred embodiment, the yeasts and fungi include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris , Hansenula polymorpha, Yarrowia lipolytica, Pichia stipitis, and Kluyveromyces lactis, Candida albicas, Aspergillus nidulans, Aspergillus niger, Trichoderma reesei, and the like.

In a preferred embodiment, the host cell is a yeast, more preferably Pichia pastoris.

In a fifth aspect, the invention provides a method of genetically engineering a host cell, the method comprising integrating a foreign gene using the nucleotide construct of the first or second aspect.

In a specific embodiment, the method includes the following steps:

a. constructing the nucleotide construct of the first or second aspect;

b. Functional integration of a nucleotide construct having a class I structure by homologous recombination downstream of a translation termination nonsense codon (eg, TAA, etc.) of the genomic open reading frame (ORF);

Functionally integrating a nucleotide construct having a class II structure upstream of the translation initiation codon (ATG) of the open reading frame (ORF) by homologous recombination; or

A nucleotide construct having a class I or class II structure is functionally integrated at any position in the genome by homologous recombination; that is, in the middle of an open reading frame (ORF), or any position upstream and downstream thereof;

c. Recombinase-mediated recombination removes elements between site-specific recombination sites in a nucleotide construct having a class I or class II structure, thereby only nucleotides having a class I or class II structure A site-specific recombination site and an exogenous gene expression cassette in the construct, and optionally an exogenous transcription terminator or an exogenous transcriptional promoter, are integrated into the genome of the host cell.

In a preferred embodiment, said functional integration of a nucleotide construct having a class I structure downstream of a translation termination nonsense codon (eg, TAA, etc.) of a genomic open reading frame (ORF) means having I The nucleotide construct of the class structure is located in the genomic open reading frame (ORF), the translation of the third nucleotide (+3) of the nonsense codon (eg TAA, etc.) to another downstream open reading frame (ORF) a region other than the transcription promoter or terminator, preferably between 300 nucleotides (+300) downstream thereof; most preferably, the nucleotide construct having the class I structure is in close proximity to the open reading frame of the genome ( Translation of ORF) terminates downstream of the third nucleotide (+3) of a nonsense codon (eg, TAA, etc.);

The functional integration of a nucleotide construct having a class II structure upstream of the translation initiation codon (ATG) of the open reading frame (ORF) of the genome means that the nucleotide construct having the class II structure is located in the open reading of the genome. The region from the first nucleotide (1) of the start codon (ATG) of the frame (ORF) to the transcription promoter or terminator of another open reading frame (ORF) upstream thereof, preferably to its upstream 300 Between nucleotides (-300); most preferably, the nucleotide construct having a class II structure is immediately adjacent to the first nucleotide of the start codon (ATG) of the genomic open reading frame (ORF) (1) Upstream.

In a preferred embodiment, the host cell includes, but is not limited to, eukaryotic and prokaryotic host host cells, such as E. coli, Pseudomonas spp., Bacillus spp .), Streptomyces spp., fungi and yeast, insect cells, such as Spodoptera frugiperda (SF9), animal cells, such as Chinese hamster ovary cell (CHO) , mouse cells, African green monkey cells, cultured human cells and plant cells.

In a preferred embodiment, the yeasts and fungi include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris , Hansenula polymorpha, Yarrowia lipolytica, Pichia stipitis, and Kluyveromyces lactis, Candida albicas, Aspergillus nidulans, Aspergillus niger, Trichoderma reesei, and the like.

In a preferred embodiment, the host cell is a yeast, more preferably Pichia pastoris.

In a sixth aspect, the invention provides the use of the nucleotide construct of the first or second aspect or the expression vector of the third aspect for genetic engineering of a host cell.

In a seventh aspect, the present invention provides the use of a host cell modified by the method of the fifth aspect, the strain being applied to the fields of metabolic engineering, systems biology, and synthetic biology; including but not limited to: the strain is used for A biocatalytic reaction, or the strain is used to produce a recombinant protein.

In a preferred embodiment, the glycosylation pattern in the recombinant protein is altered.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1A depicts the integration of the exogenous transcription terminator (TT2) and the foreign gene expression cassette (GOI) behind the translational termination nonsense codon (TAA) of the open reading frame (ORF) of the genome without disrupting the expression of the open reading frame. . In this method, the selectable marker can be used repeatedly to integrate different foreign genes into different open reading frames of the genome. The components of the vector and genomic locus are not drawn to scale;

Figure 1B depicts the integration of the exogenous transcriptional promoter (P2) and the exogenous gene expression cassette (GOI) in front of the start codon (ATG) of the open reading frame (ORF) of the genome without disrupting the expression of the open reading frame. In this method, the selectable marker can be used repeatedly to integrate different foreign genes into different open reading frames of the genome. The components of the vector and genomic locus are not drawn to scale;

Figure 2 depicts a schematic representation of the construction of a pFZ vector, the components of which are not drawn to scale;

Figure 3 depicts a schematic representation of the construction of the pFZ-ARG2-cAtMnsI expression vector, the components of which are not drawn to scale;

Figure 4A depicts a schematic representation of integration of the pFZ-ARG2-cAtMnsI expression vector at the ARG2 locus and excision of Zeocin, the components of the vector and genomic locus are not drawn to scale;

Figure 4B shows the PCR results confirming that the pFZ-ARG2-cAtMnsI expression vector is integrated at the ARG2 locus;

Figure 4C shows that PCR results confirm the excision of the Zeocin resistance gene in the ARG2 locus;

Figure 5 depicts the construction of the pFZ-PNO1-cAtMnsI expression vector, the components of which are not drawn to scale;

Figure 6A depicts a schematic representation of integration of the pFZ-PNO1-cAtMnsI expression vector at the PNO1 locus and excision of Zeocin, the components of the vector and genomic loci are not drawn to scale;

Figure 6B shows the PCR results confirming that the pFZ-PNO1-cAtMnsI expression vector is integrated at the PNO1 locus;

Figure 6C shows that the PCR results confirmed the excision of the Zeocin resistance gene in the PNO1 locus.

Detailed ways

After extensive and intensive research, the inventors unexpectedly discovered a novel genetic engineering vector that integrates a large number of foreign genes into the genome of a living organism through two recombinations. The genetic engineering vector can not only maintain the normal expression of genes near the genome integration site, but also avoid destroying the host cell gene function and not affecting the survival of the host cell, and thus can be widely applied to complete large-scale genome transformation projects. The present invention has been completed on this basis.

The present invention relates to methods and materials for the stable integration of foreign genes into the genome using vectors. The terms used herein are defined according to the following.

"Gene targeting" is a method of integrating a foreign gene (or DNA) on a genome, usually resulting in the transformation, replacement or replication of a target gene. This mechanism applies to all organisms.

Single cross-over recombination and double cross-over recombination are two different ways in which homologous recombination integrates exogenous DNA into the genome. In the single-swap recombination process, when the foreign DNA is paired with the target gene homologous region in the genome, the ends of the linear foreign DNA point to each other, and the DNA is integrated into the genome by single-exchange recombination. This method can be simply referred to as “Ends- In" or "roll in" gene targeting. After the single-transformation recombination, due to the generation of the same repeat sequence, the exogenous DNA can be excised by homologous recombination between the repeat sequences to restore the original state of the target gene. During double-crossover recombination, when the foreign DNA is paired with a homologous region in the genome, the ends of the linear foreign DNA are deviated from each other, and the DNA is inserted by double-crossover recombination between the terminal targeting flanking and the homologous sequence of the host genome. In the genome, this method can be simply referred to as "Ends-out" gene targeting. The "Ends-out" gene target is commonly used in mice and yeast because it can directly replace or delete target genes. However, the probability of "ends-out" occurring is much lower than the "ends-in" event. (Paques and Haber 1999, Microbiology and Molecular Biology Reviews, 63: 349-404). In the present invention, gene targeting refers to "ends-out" gene targeting of double-crossover homologous recombination unless specifically indicated by "ends-in" gene targeting by single-exchange homologous recombination.

"Cell" or "body" is a term used to carry out the gene targeting of the present invention.

Examples of host cells useful in the present invention include typical eukaryotic and prokaryotic hosts, such as E. coli, Pseudomonas spp., Bacillus spp., Streptomyces. (Streptomyces spp.), fungi and yeast, insect cells, such as Spodoptera frugiperda (SF9), animal cells, such as Chinese hamster ovary cells (CHO), mouse cells, Africa Green monkey cells, cultured human cells and plant cells.

Yeasts and fungi include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Hansenula polymorpha , Yarrowia lipolytica, Pichia stipitis, and Kluyveromyces lactis, Candida albicas, Aspergillus nidulans, Aspergillus niger (Aspergillus niger), Trichoderma reesei, etc. Yeast is a preferred host cell of the invention. Pichia pastoris is a more preferred host cell.

"Cell transformation and transfection" refers to the process by which foreign DNA is introduced into cells. Generally refers to the process of integrating foreign DNA into the genome of a cell or introducing a self-replicating plasmid.

The vector DNA is introduced into a host cell for homologous recombination, and transformation and transfection of the host cell can be carried out according to methods well known to those skilled in the art.

Suitable transformation methods include viral infection, transfection, conjugation, protoplast fusion, electroporation, gene gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method will usually depend on the type of cell being transformed and the conditions under which the transformation takes place. A general discussion of these methods can be found in the literature (Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995).

For example, yeast transformation can be carried out using different methods, including spheroidal methods, electroporation, polyethylene glycol methods, alkaline cation methods, etc. [Gregg JM (2010) Pichia Protocols, Second edition. Totowa, New Jersey: Humanna Press ].

The "target gene" or "target gene" refers to a gene or DNA segment in a cell which is altered by the method of the present invention. The target gene may be any DNA fragment in the genome of the cell or an exogenous DNA fragment previously introduced into the body, including but not limited to a polypeptide coding region, an open reading frame (ORF), a control region, an intron, an exon. Or part of them.

The nucleotide designation of the 5' and 3' regions refers to the corresponding start codon of the open reading frame (ORF) as nucleotides 1-3, and the 5' upstream region is numbered with a minus sign; and the corresponding stop codon As nucleotides +1 to +3, the 3' downstream region is numbered by a plus sign.

Targeting vectors can also be referred to as vectors. Vectors for recombinant DNA technology are typically in the form of "plasmids". In the present specification, the terms "vector" and "plasmid" are used interchangeably.

The expression cassette, as part of the vector, produces RNA and protein within the cell. Its composition includes, but is not limited to, a promoter sequence, an open reading frame and a terminator sequence arranged in a known position and orientation.

Herein, a region homologous to a corresponding gene region indicates that the region has at least 90%, preferably at least 92%, more preferably at least 94%, still more preferably at least 96%, still more preferably the base sequence of the gene region. Preferably at least 98%, still more preferably at least 99%, most preferably 100% identical. Such "homologous regions" or "homologous sequences" are preferably derived from the described gene regions.

The length of the homologous recombination region is not particularly limited. The length of this region is preferably adapted to undergo homologous recombination. Therefore, this region is at least 40 base pairs in length.

When transposing a vector of the present invention into bacterial cell passage, it is preferred to include a bacterial origin of replication and an antibiotic resistance gene in the vector to ensure that the vector is retained during bacterial passage. Bacterial origins of replication include fl-ori, colisin, col El, and other starting points known in the art. Antibiotic resistance genes include Ampicillin (Amp), zeocin, kanamycin (kan), Tetracyclines tolerance genes, and other antibiotic resistance known in the art. Sex gene.

A "marker" refers to a gene or sequence whose presence or absence provides a detectable phenotype. One or more markers can be used to select and screen for gene targeting events.

Expression of the marker gene can result in the body having a phenotype that is tolerant or sensitive to a particular set of conditions. These markers include tolerance genes for different antibiotics, such as the Sh ble gene that is resistant to zeocin, the neo gene that is resistant to kanamycin (kan) or geneticin (G418), Blasticidin S deaminase BSD gene resistant to blasticidin, nat gene resistant to nourseosis, hygromycin resistant to hygromycin Phosphotransferase genes, as well as other antibiotic resistance genes known in the art, and the like.

The marker system can also be composed of an auxotrophic mutant organism and a wild-type biological gene, which complements the body's defects in incomplete culture, for example, the ADE1, ARG4, HIS4 and URA3 wild-type genes of Saccharomyces cerevisiae or Pichia pastoris can be used as marker genes. For transformation of ade1, arg4, his4 and ura3 auxotrophic yeast, respectively, as well as other genes known in the art.

Tags can also convey observable and distinguishable features. These markers include fluorescent proteins such as green fluorescent protein (GFP); reporter enzymes such as beta-galactosidase (lacZ), alkaline phosphatase (AP), beta-lactamase, beta-glucuronidase, and valleys. Glutathione S-transferase (GST), luciferase, and other enzymes known in the art.

The present invention develops a vector capable of stably integrating a foreign gene into a genome. The composition of such vectors mainly includes, but is not limited to, a recombinant enzyme expression cassette, a marker gene expression cassette, an origin of replication, a site-specific recombination site, a foreign gene, and a homologous sequence, and may also include transcription initiation. Child or transcription terminator. There may be one or more selection markers in the vector for yeast and E. coli screening. These moieties can be joined to form a circular support which, if desired, can contain other moieties and linkers between the moieties. However, the invention should also include other forms of vectors that are functionally equivalent.

The site-specific recombination site can be excised by site-specific recombinase. As used herein, "site-specific recombinase" refers to any enzyme that is capable of functionally catalyzing recombination between its corresponding site-specific recombination sites. The site-specific recombinase may be a recombinantly expressed polypeptide, fragment, variant or derivative that naturally produces or retains naturally occurring recombinase activity (Craig (1988) Annu. Rev. Genet. 22, 77-105). ). The site-specific recombinase is preferably expressed under the control of an inducible promoter (eg, an inducible promoter such as AOX1, FLD1, NPS, etc.). Any site-specific recombination system can be employed in the present invention. Examples of site-specific recombination systems suitable for the present invention include Flp-FRT of Saccharomyces cerevisiae, Cre-loxP of bacteriophage P1, and R-RS of Xygosaccharomyces rouxii, and the like. Each system consists of a recombinase that catalyzes the recombination between the recognition sites FRT, loxP or RS, respectively. In a specific embodiment, the recombinase is a Flp recombinase or a Cre recombinase. In a preferred embodiment, the recombinase is a Flp recombinase.

The marker gene includes one or several antibiotic resistance genes, preferably a Sh ble gene that is resistant to zeocin, and is resistant to kanamycin (kan) or geneticin (G418). Neo gene.

The origin of replication includes fl-ori, colisin, col El, and other origins of replication known in the art.

After the recombinase expression cassette, the marker gene expression cassette and the replication origin are ligated to each other, two site-specific recombination sites are flanked, and the two site-specific recombination sites are ligated into a circular vector through the multiple cloning site. The foreign gene, the homologous sequence, and the transcriptional promoter or transcriptional terminator are then ligated to the multiple cloning site, respectively.

Here, there is a restriction site between the 5' end of the upstream homologous sequence (5'H) and the 3' end of the downstream homologous sequence (3'H) for linearization. Preferably, the upstream homologous sequence has a half restriction end site (such as GCT) at the 5' end, and the other half restriction endonuclease site (such as AGC) at the 3' end of the downstream homologous sequence, using overlapping PCR The two are spliced, thereby creating a restriction endonuclease (such as AGCGCT for Afe I) for enzymatic linearization of the vector.

According to another aspect, the present invention provides a linear vector which can be linearized by restriction enzyme digestion or can be obtained by genetic chemical synthesis.

Substantial portions of linear vectors include recombinant enzyme expression cassettes, selectable marker expression cassettes, origins of replication, site-specific recombination sites, foreign genes (GOI), homologous sequences, transcriptional promoters, or transcriptional terminators. The linear carrier can contain other moieties, and if desired, a linker can be included between the moieties. Recombinase expression cassettes (eg, FLPs), selectable marker expression cassettes (Marker), and origins of replication (eg, ori) linked in a different order are ligated to site-specific recombination sites (eg, FRT) on their upstream and downstream sides, respectively; The exogenous gene (GOI) and transcription terminator (TT2) or transcriptional promoter (P2) are flanked by a specific recombination site; the linear vector is flanked by homologous sequences (5'H and 3'H). Connections to linear carriers include, but are not limited to, the following:

5'H-TT2-FRT-FLP-Marker-ori-FRT-GOI-3'H;

5'H-TT2-GOI-FRT-FLP-Marker-ori-FRT-3'H;

5'H-FRT-FLP-Marker-ori-FRT-TT2-GOI-3'H;

5'H-GOI-TT2-FRT-FLP-Marker-ori-FRT-3'H;

Wherein, the positions of the recombinase expression cassette (FLP), the Marker, and the origin of replication (ori) between the site-specific recombination sites FRT are interchangeable;

or

5'H-GOI-FRT-FLP-Marker-ori-FRT-P2-3'H;

5'H-FRT-FLP-Marker-ori-FRT-GOI-P2-3'H;

5'H-GOI-P2-FRT-FLP-Marker-ori-FRT-3'H;

5'H-FRT-FLP-Marker-ori-FRT-P2-GOI-3'H;

Among them, the positions of the recombinase expression cassette (FLP), the Marker, and the origin of replication (ori) between the site-specific recombination sites FRT can be interchanged.

In a preferred embodiment, in order to ensure that the function of the insertion site gene is not disrupted when the vector of the present invention is inserted into the genome of the host cell, the following structure is preferably employed:

5'H-TT2-FRT-FLP-Marker-ori-FRT-GOI-3'H;

5'H-TT2-GOI-FRT-FLP-Marker-ori-FRT-3'H;

Wherein, the positions of the recombinase expression cassette (FLP), the Marker, and the origin of replication (ori) between the site-specific recombination sites FRT are interchangeable;

or

5'H-GOI-FRT-FLP-Marker-ori-FRT-P2-3'H;

5'H-FRT-FLP-Marker-ori-FRT-GOI-P2-3'H;

Among them, the positions of the recombinase expression cassette (FLP), the Marker, and the origin of replication (ori) between the site-specific recombination sites FRT can be interchanged.

The linear vector is introduced into a host cell for homologous recombination, and transformation and transfection of the host cell can be carried out according to methods well known to those skilled in the art.

After the linear vector is stably integrated into the host cell genome by double-crossover homologous recombination, the recombinant enzyme that induces expression can accurately and efficiently excise the recombinase, selectable marker and origin of replication at a site-specific recombination site, leaving a site-specific Recombination sites and foreign gene expression cassettes. Thus, the excised selection marker can be used repeatedly to integrate the foreign gene into different sites in the host cell genome without being restricted by the limited selection marker.

Figure 1A depicts one way to integrate a foreign gene downstream of a genomic open reading frame translation termination nonsense codon. The map depicts the 5' regulatory region (5'region) of a locus in the genome and its transcriptional promoter (P1), open reading frame (ORF), 3' region (3'region) and its transcription terminator ( TT1), translation of the open reading frame terminates nonsense codons (eg TAA, TGA, TAG). In the linear vector, a recombinase expression cassette (FLP), a marker gene expression cassette (Marker), and an origin of replication (ori) which can be ligated in different sequences are ligated to a site-specific recombination site (FRT) on the upstream and downstream sides thereof, respectively. The site-specific recombination site is flanked by a transcription terminator (TT2), downstream of the exogenous gene expression cassette (GOI, gene of interest), and the outermost homologous sequence (5'H and 3'H). ). Multiple marker genes and foreign genes can be linked in a linear vector. Transcriptional terminators useful for Pichia pastoris include, but are not limited to, transcriptional terminators such as ADH1, CYC1 and TIF51A of Saccharomyces cerevisiae; transcriptional terminators such as ALG6, AOD, AOX1, ARG4, PMA1 and TEF1 of Pichia pastoris; Other transcription terminators known in the art.

Chinese Patent Application No. 201510218188.1 indicates that the linear vector can be efficiently integrated downstream of the translation termination nonsense codon by double exchange recombination. After integration, the open reading frame's own transcription terminator (TT1) was replaced with an exogenous transcription terminator (TT2). It transcribes mRNA similar to wild-type, translates wild-type proteins, and functions normally. Therefore, the protein encoded by the open reading frame near the integration site will not be destroyed by the integration of the foreign gene.

The recombinant enzyme that induces expression can efficiently and efficiently excise the recombinant enzyme expression cassette (FLP), the selectable marker gene expression cassette (Marker), and the origin of replication (ori) at the site-specific recombination site, leaving the transcription terminator (TT2), A site-specific recombination site (FRT) and a foreign gene (GOI). Thus, the excised marker gene can be used repeatedly, and the foreign gene is integrated at different sites in the host cell genome without being restricted by the limited selection marker.

During the integration and self-resection of linear vectors, the transcription and translation of the open reading frame near the genomic integration site remains intact and functions properly. Thus, any open reading frame in the host cell genome, including genes necessary for survival, can be used as a site for integration of foreign genes. At the same time, the excised marker gene can be used repeatedly by self-resection of the linear vector.

In addition, the foreign gene can also be omitted from its transcription terminator, and the transcript terminator (TT1) of the open reading frame is utilized after genome integration.

Figure 1B depicts one way to integrate a foreign gene upstream of the translation initiation codon of the open reading frame of the genome. The map depicts the 5' regulatory region (5'region) of a locus in the genome and its transcriptional promoter (P1), open reading frame (ORF), 3' region (3'region) and its transcription terminator ( TT1), the translation start codon of the open reading frame (such as ATG). In the linear vector, a recombinase expression cassette (FLP), a marker gene expression cassette (Marker), and an origin of replication (ori) which can be ligated in different sequences are ligated to a site-specific recombination site (FRT) on the upstream and downstream sides thereof, respectively. The site-specific recombination site is flanked by a foreign gene expression cassette (GOI), a downstream flanking transcriptional promoter (P2), and an outermost homologous sequence (5'H and 3'H). Multiple marker genes and foreign genes can be linked in a linear vector. The transcriptional promoters that can be used for Pichia pastoris include, but are not limited to, promoters such as ADH1, GAP, PGK1, and TEF1 of Saccharomyces cerevisiae, promoters such as GAP, ILV5, PGK1, and TEF1 of Pichia pastoris, and induction of AOX1 and FLD1. Sons, as well as other transcriptional promoters known in the art.

Chinese Patent Application No. 201510218188.1 indicates that the linear vector can be efficiently integrated upstream of the translation initiation codon by double-crossover recombination. After integration, the open reading frame's own transcriptional promoter (P1) was replaced with an exogenous transcriptional promoter (P2). It transcribes mRNA similar to wild-type, translates wild-type proteins, and functions normally. Therefore, the protein encoded by the open reading frame near the integration site will not be destroyed by the integration of the foreign gene.

Recombinant enzymes that induce expression can efficiently and efficiently excise recombinase (FLP), selectable marker (Marker) and origin of replication (ori) at site-specific recombination sites, leaving a foreign gene (GOI), a site-specific Reproductive recombination site (FRT) and exogenous transcriptional promoter (P2). Thus, the excised marker gene can be used repeatedly, and the foreign gene is integrated at different sites in the host cell genome without being restricted by the limited selection marker.

During the integration and self-resection of linear vectors, the transcription and translation of the open reading frame near the genomic integration site remains intact and functions properly. Thus, any open reading frame in the host cell genome, including genes necessary for survival, can be used as a site for integration of foreign genes. At the same time, the excised marker gene can be used repeatedly by self-resection of the linear vector.

In addition, the foreign gene may also be omitted from its transcriptional promoter, and the transcript promoter (P1) using the open reading frame after genome integration.

In addition to integrating foreign genes upstream and downstream of the open reading frame, it can also be integrated in the middle of an open reading frame, disrupting the expression and biological function of open reading frames. Therefore, the integrated open reading frame should be a non-essential gene for the host cell.

The method and vector developed by the present invention are capable of stably integrating a foreign gene into the genome of a host cell, while minimizing or even affecting the expression and function of an open reading frame near the genomic integration site. Thus, any open reading frame in the host cell genome, including genes necessary for survival, can be used as a site for integration of foreign genes. In this method, the excised marker gene can be used repeatedly. Therefore, this method can stably integrate a large number of foreign genes into different sites of the genome, and can be used for large-scale transformation of genomes, overcoming several major limitations of existing methods: including limited marker genes and limited Genomic integration sites, etc.

The technology of the present invention has a wide range of applications. Those skilled in the art can make certain modifications or improvements without departing from the basic principles of the invention, and the invention can be applied to other host cells by these modifications. In addition, the skilled artisan can apply the invention to other aspects including, but not limited to, genomic engineering in related fields such as metabolic engineering, systems biology, and synthetic biology. These modifications, improvements, and applications are also within the intended scope of the invention.

Advantages of the invention:

1. The novel genetic engineering vector of the present invention is capable of integrating a large number of foreign genes into the genome of a living organism without affecting the survival of the host cells;

2. Any open reading frame in the genome can be used as an integration site for the foreign gene;

3. The method and material provided by the present invention is capable of effectively recycling selection markers; and

4. The genetic engineering vector and method of the present invention can be widely applied to complete large-scale genomic transformation projects.

The technical solutions of the present invention are further described below in conjunction with the specific embodiments, but the following embodiments are not intended to limit the present invention, and all the application methods according to the principles and technical means of the present invention are within the scope of the present invention. For example, although embodiments of the invention are carried out in the preferred Pichia pastoris, the invention is also carried out in a different host via a genomic database. Genomic data can be obtained from published scientific literature and professional websites. For example, the public database NCBI ( www.ncbi.nlm.nih.gov ) provides detailed genomic details for prokaryotes, eukaryotes, and viruses.

The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer.

Example

material

Chemical reagents, enzymes, media and solutions for the generation, validation and application of genetic recombination are commonly used and are well known to those skilled in the art of molecular and cell biology; they are available from a number of companies, including Thermo Fisher Scientific, Invitrogen , Sigma, New England BioLabs, Takara Biotechnology, Toyobo, TransGen Biotech, and Generay Biotechnology, among others. Many of them are available in the form of kits.

The pPIC9K, pPICZα and POG44 vectors were obtained from Invitrogen.

E. coli strain Trans1-T1 was obtained from TransGen Biotech.

method

Unless otherwise indicated, the methods of the invention are carried out according to standard methods well known to those skilled in the art of molecular and cell biology, including polymerase chain reaction (PCR), restriction enzyme cloning, DNA purification, bacteria, yeast, cell culture, Transformation, transfection, and Western blotting, etc., as described in the following manual: Sambrook J et al. (Molecular Cloning A Laboratory Manual (Third Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001), Ausubel F M Et al. (Current Protocols in Molecular Biology, Wiley InterScience, 2010), and Gregg JM (Pichia Protocols, (Second edition), Totowa, New Jersey: Humanna Press, 2010).

The E. coli strain Trans1-T1 was used to construct and amplify the vector. The strain was extracted with Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract and 5 g/L sodium chloride) or LB plate (10 g/L tryptone, 5 g/L yeast extract) with appropriate antibiotics. Incubate with 5 g/L sodium chloride, 20 g/L agar). The concentration of antibiotic added was as follows: 100 mg/L ampicillin, 50 mg/L kanamycin, 25 mg/L Zeocin.

Pichia strain utilizes YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) and YPD plate (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, 20 g/L) Agar) culture. Use amino acid-free YNB medium (67 g/L yeast nitrogen base, 5 g/L glucose) and amino acid-free YNB plate (67 g/L yeast nitrogen source, 5 g/L glucose, 20 g/L agar) ) to select Pichia pastoris strains. Some Pichia pastoris auxotrophic strains utilize SC medium (8g/L SC without histidine and uracil, 20g/L glucose) and SC plates (8g/L SC without histidine and uracil, 20g/ L-glucose, 20 g/L agar) was selected and antibiotics were added as needed. The concentration of antibiotic added was as follows: 250 mg/L G-418 sulfate, 100 mg/L Zeocin.

Genomic DNA in Pichia pastoris was extracted by cleavage with lithium acetate-SDS followed by ethanol precipitation, which is described in the following publication: Looke et al. 2011, Biotechniques. 50: 325-328.

MicroPulser ^TM using electroporation apparatus, according to manufacturer's instruction manual (BioRad) operation, transformed Pichia pastoris by electroporation.

Example 1

Construction of pFZ vector

Figure 2 depicts a schematic of the construction of a pFZ vector.

PCR1, using the POG44 vector as a template, using the FLP F (SEQ ID NO: 1) and FLP R (SEQ ID NO: 2) primer pairs (the primer pair has 5'AOX1 and AOX1TT homologous sequences for homologous recombination) The FLP gene coding region was amplified by PCR.

PCR2, using the pPICZα vector as a template, PCR amplification of the AOX1TT-Zeocin-ori-5'AOX1 fragment using the PICZ F (SEQ ID NO: 3) and PICZ R (SEQ ID NO: 4) primer pairs.

The above two PCR products (1, 2) were ligated into a circular FLP expression vector (pPICZ-FLP) using EZfusion homologous recombinase (Generay).

PCR3, using the FLP expression vector as a template, using SSKFRT F (SEQ ID NO: 5, the primer has Sph I, Sal I and Kpn I restriction sites and FRT sequences) and FLP R (SEQ ID NO: 2) Primer pairs were used to PCR amplify the FRT-5'AOX1-FLP fragment.

PCR4, using FLP expression vector as template, using FLP F (SEQ ID NO: 1) and FRTAAS R (SEQ ID NO: 6, this primer has Apa I, Afl II, and Sph I restriction sites, and FRT The sequence pair primer pair was used for PCR amplification of the FLP-AOX1TT-Zeocin-ori-FRT fragment.

After digestion of the PCR (3, 4) product with restriction enzymes, the EcoR I/Sph I fragments of FRT-5'AOX1-FLP and FLP-AOX1TT-Zeocin-ori-FRT were circularized with T4 ligase to generate a pFZ vector.

Example 2

Construction of pFZ-ARG2-cAtMnsI expression vector

Figure 3 depicts an example of constructing a pFZ-ARG2-cAtMnsI expression vector.

PCR1, using Pichia pastoris genomic DNA as a template, ARG2 3'H F (SEQ ID NO: 7, this primer has a Sph I restriction enzyme cleavage site) and ARG2 3'H R (SEQ ID NO: 8, which Primers have overlapping sequences of ARG2 5' homologous sequences for fusion PCR) primer pairs for PCR amplification of ARG2 3' homologous sequence (3'H) (719 bp).

PCR2, using Pichia pastoris genomic DNA as a template, using ARG2 5'H F (SEQ ID NO: 9, this primer has an overlapping sequence of ARG2 3' homologous sequences for fusion PCR) and ARG2 5'H R (SEQ ID NO: 10, the primer has an ADH1TT overlapping sequence for fusion PCR) primer pair for PCR amplification of the ARG2 5' homologous sequence (5'H) (978 bp).

PCR3, using S. cerevisiae genomic DNA as a template, using ADH1TT F (SEQ ID NO: 11, this primer has an overlapping sequence of ARG2 5' homologous sequences for fusion PCR) and ADH1TT R (SEQ ID NO: 12, the primer has The Kpn I restriction enzyme site primer pair was used for PCR amplification of the ADH1 transcription terminator (ADH1TT).

PCR4, using the ARG2 3'H F (SEQ ID NO: 7) and ADH1TT R (SEQ ID NO: 12) primer pairs, the above three PCR products (1, 2, 3) were ligated by overlap-extension PCR. This resulted in a fusion fragment of ARG2-3'H-5'H-ADH1TT with an Afe I restriction enzyme site between 3'H and 5'H.

After digesting the product with restriction enzymes, the SphI/KpnI fragment of ARG2-3'H-5'H-ADH1TT was ligated to the same site of the pFZ vector with T4 ligase to generate a pFZ-ARG2 vector.

PCR4, using the synthetic hybrid cAtMns expression cassette (SEQ ID NO: 13) as a template, ApaGAP F (SEQ ID NO: 14, this primer has an Apa I restriction site) and TIF51ASph R (SEQ ID NO: 15, the primer has a Sph I restriction site) primer pair for PCR amplification of the hybrid cAtMnsI gene expression cassette.

After digestion of the product with restriction enzymes, the ApaI/SphI fragment of the hybrid cAtMnsI was ligated to the same site of the pFZ-ARG2 vector with T4 ligase to generate the hybrid cAtMnsI expression vector pFZ-ARG2-cAtMnsI.

Example 3

Integration of pFZ-ARG2-cAtMnsI vector at the ARG2 locus and excision of the marker gene

The och1 gene was knocked out by the method described in Chinese Patent Application No. 201510220631.9 to obtain Pichia pastoris och1 knockout ((JC301-och1: loxP) (ade1 his4 ura3 och1::loxP).

PCR1, using Pichia pastoris genomic DNA as a template, primer sets of ADE1F (SEQ ID NO: 16) and ADE1R (SEQ ID NO: 17) were used for PCR amplification of the open reading frame of the ADE1 gene.

The PCR product of the ADE1 open reading frame was transformed into cells of Pichia pastoris och1 knockout (ade1 his4 ura3 och1::loxP) by electroporation using a MicroPulserTM electroporation apparatus according to the manufacturer's instructions for use (BioRad, USA). The transformed cells were grown on SC plates supplemented with 20 mg/L histidine and 50 mg/L uracil to select adenine protozoa och1 knockout (his4 ura3 och1::loxP).

Templated by synthetic codon-optimized mouse interleukin-22 (mIL-22, the DNA sequence of the his-tagged mouse IL-22 mature peptide optimized for yeast codons as shown in SEQ ID NO: 18) With MIL22F (SEQ ID NO:

19)/MIL22R (SEQ ID NO: 20) primer pair for PCR amplification. The PCR product was digested with Xho I and Not I restriction enzymes and cloned into the Xho I/Not I site of pPIC9K to generate a mIL-22 expression vector capable of expressing and secreting His-tagged mIL-22. The expression vector was linearized with restriction enzyme Sac I and electroporated into och1 knockout (his4ura3och1::loxP). The transformed cells were cultured at 25 °C on YPD plates supplemented with 250 mg/L of G418 sulfate. The linearized vector was integrated at the AOX1 locus by single-crossover recombination to obtain a mIL-22 expressing bacterium (his4 ura3 och1::loxP, mIL22).

Figure 4A depicts a schematic representation of integration of the pFZ-ARG2-cAtMnsI expression vector at the ARG2 locus and excision of the Zeocin marker gene.

The pFZ-ARG2-cAtMnsI expression vector was digested with restriction enzyme Afe I to generate a linear vector. In the linear vector, the ligated recombinase FLP expression cassette, the Zeocin marker gene, and the origin of replication (ori) are ligated to the FRT site-specific recombination sites on their upstream and downstream sides, respectively. The upstream side of the site-specific recombination site is further flanked by the ADH1 transcription terminator (ADH1TT), the downstream side is further flanked by a hybrid cAtMnsI gene expression cassette, and the outermost ARG2-specific homologous sequence (5'H and 3'H) ), ensuring integration of the ARG2 locus in the genome by double-crossover homologous recombination.

The linear vector was transformed into mIL-22 expressing bacteria (his4 ura3 och1::loxP, mIL22) by electroporation. The transformed cells were plated on YPD plates supplemented with 100 mg/L Zeocin, and grown at 25 ° C to select expression bacteria resistant to Zeocin. On the transformation plate, colonies were randomly picked and cultured to extract genomic DNA for PCR to verify integration of the genome. Two primer pairs, C1 (SEQ ID NO: 21, located in the ORF of genomic ARG2, downstream of the 5'H homology region) / C2 (SEQ ID NO: 22, located within ADH1TT) and C3 (SEQ ID NO: 23, located TIF51A TT)/C4 (SEQ ID NO: 24, located downstream of the 3'H homology region of genomic ARG2) was used for PCR to verify that the linear vector was integrated at the ARG2 locus of the genome. Successful PCR amplification of the 1270 and 1076 bp bands indicated that the linear vector was successfully integrated into the genomic ARG2 open reading frame by the homologous recombination termination of the nonsense codon (Fig. 4B). Thus, a hybrid cAtMnsI integrated strain (his4ura3och1::loxP, ARG2::Zeocin-cAtMnsI, mIL22) was constructed.

The hybrid cAtMnsI integrator (his4ura3och1::loxP, ARG2::Zeocin-AtMnsI, mIL22) was cultured in 5 ml of YPD medium at 28 ° C, shaking at 225 rpm for 24 hours. The cells were pelleted by centrifugation at 3000 g for 5 minutes, resuspended in 5 ml of BMGY medium, and cultured at 28 ° C, shaking at 225 rpm for 24 hours. Then, the cells were pelleted by centrifugation at 3000 g for 5 minutes, resuspended in 5 ml of BMMY medium, and shake cultured at 225 rpm at 28 ° C to induce FLP expression. The culture was mixed twice daily with 50 ul of 100% methanol (1% final concentration) and induction was maintained for an additional 72 hours. The bacterial solution was spread on a YPD plate, grown at 25 ° C, and then the grown colonies were spotted on a 100 mg/L Zeocin YPD plate to confirm Zeocin sensitivity. Methanol-induced expression of FLP recombinase cleaves FLP recombinase expression cassette, Zeocin selectable marker and replication origin by mediating FRT site-specific recombination, and is stable downstream of ARG2 open reading frame translation termination nonsense codon (TAA) An ADH1 transcriptional terminator (ADH1TT), a site-specific recombination site (FRT) and a cAtMnsI gene expression cassette are left. The predicted 1712 bp band was amplified by PCR from two colony genomes sensitive to Zeocin on YPD plates using C1/C5 (SEQ ID NO: 25, located in the P GAP promoter) primers to verify accurate excision between FRTs in the genome ( Figure 4C). Thus, a hybrid cAtMnsI expressing strain (his4ura3och1::loxP, ARG2::cAtMnsI, mIL22) was constructed.

In the process of linear vector integration and self-resection, the transcriptional terminator of the ARG2 open reading frame was replaced with the exogenous ADH1 transcription terminator (ADH1TT), and transcription and translation were never disrupted, functioning normally, without Growth in the YNB medium of the lysine did not reveal Arg2 auxotrophy.

The excised Zeocin selectable marker can be used repeatedly to integrate the foreign gene into different sites in the host cell genome without being restricted by the limited selection marker.

The hybrid cAtMnsI expressing bacteria (his4ura3och1::loxP, ARG2::cAtMnsI, mIL22) were cultured in 5 ml of YPD medium at 28 ° C, shaking at 225 rpm for 24 hours. The cells were pelleted by centrifugation at 3000 g for 5 minutes, resuspended in 5 ml of BMGY medium, and cultured at 28 ° C, shaking at 225 rpm for 24 hours. Then, the cells were pelleted by centrifugation at 3000 g for 5 minutes, resuspended in 5 ml of BMMY medium, and cultured at 28 ° C, shaking at 225 rpm to induce mIL-22 expression. The culture was mixed twice daily with 50 ul of 100% methanol (1% final concentration) and induction was maintained for an additional 72 hours. Subsequently, the culture supernatant was harvested by centrifuging the cells at 3000 g for 10 minutes. The His-tagged mIL-22 protein was purified from the supernatant by Ni-affinity chromatography. The sugar chain was released and isolated from the His-tagged mIL-22 protein by treatment with N-glycosidase F (PNGaseF) (New England Biolabs, Beverly, MA) using previously reported methods (Gregg JM (2010) Pichia Protocols, Second edition.Totowa,

New Jersey: Humanna Press). The molecular weight of the sugar chain was determined using an Ultraflex MALDI-TOF (bruker daltonics, Bremen, Germany) mass spectrometer. The results showed that the hybrid cAtMnsI expressing bacteria (his4ura3och1::loxP, ARG2::

mAt-22 produced by cAtMnsI, mIL22), its N-glycan chain appeared Man ₅ GlcNAc ₂ (m/z: 1257) and Man ₈ GlcNAc ₂ (m/z: 1743), indicating that hybrid cAtMnsI can convert Man ₈ GlcNAc ₂ Converted to Man ₅ GlcNAc ₂ .

Example 4

Construction of pFZ-PNO1-cAtMnsI expression vector

Figure 5 depicts an example of constructing a pFZ-PNO1-cAtMnsI expression vector.

PCR1, using Pichia pastoris genomic DNA as a template, with PNO1 3'H F (SEQ ID NO: 26, the primer has a Sph I restriction enzyme cleavage site) and PNO1 3'H R (SEQ ID NO: 27, The primers have overlapping sequences of PNO1 5' homologous sequences for fusion PCR) primer pairs for PCR amplification of the PNO1 3' homologous sequence (3'H) (985 bp).

PCR2, using Pichia pastoris genomic DNA as a template, with PNO1 5'H F (SEQ ID NO: 28, this primer has a PNO1 3' homologous sequence overlapping sequence for fusion PCR) and PNO1 5'H R (SEQ ID NO) :29, the primer has a Kpn I restriction site) primer pair for PCR amplification of the PNO1 5' homologous sequence (5'H) (933 bp).

PCR3, using the PNO1 3'H F (SEQ ID NO: 26) and PNO1 5'H R (SEQ ID NO: 29) primer pairs, the above two PCR products (1, 2) were ligated by overlap-extension PCR. This resulted in a fusion fragment of PNO1 3'H-5'H with an Afe I restriction enzyme site between 3'H and 5'H.

After digestion of the product with restriction enzymes, the SphI/KpnI fragment of PNO1 3'H-5'H was ligated to the same site of the pFZ vector with T4 ligase to generate a pFZ-PNO1 vector.

PCR4, using the synthetic hybrid cAtMns expression cassette as a template, ApaGAP F (SEQ ID NO: 14, the primer has an Apa I restriction site) and TIF51ASph R (SEQ ID NO: 15, the primer has Sph I The restriction enzyme cleavage site primer pair was used to PCR amplify the hybrid cAtMnsI gene.

After digestion of the product with restriction enzymes, the ApaI/SphI fragment of cAtMnsI was ligated to the same site of the pFZ-PNO1 vector with T4 ligase to generate a hybrid cAtMnsI expression vector pFZ-PNO1-cAtMnsI.

Example 5

Integration of pFZ-PNO1-cAtMnsI vector at the PNO1 locus and excision of the marker gene

Figure 6A depicts a schematic representation of integration of the pFZ-PNO1-cAtMnsI expression vector at the PNO1 locus and excision of the Zeocin marker gene.

The pFZ-PNO1-cAtMnsI expression vector was digested with restriction enzyme Afe I to generate a linear vector. In the linear vector, the ligated recombinase FLP expression cassette, the Zeocin marker gene, and the origin of replication (ori) are ligated to the FRT site-specific recombination sites on their upstream and downstream sides, respectively. The downstream side of the site-specific recombination site is further flanked by a hybrid cAtMnsI gene expression cassette, flanked by PNO1-specific homologous sequences (5'H and 3'H), ensuring integration into the genome by double-crossover homologous recombination PNO1 locus. These linear vectors were transformed into mIL-22 expressing bacteria (his4ura3och1::loxP, mIL22) by electroporation using a MicroPulserTM electroporation apparatus. The transformed cells were plated on YPD plates supplemented with 100 mg/L Zeocin, and grown at 25 ° C to select expression bacteria resistant to Zeocin. On the transformation plate, colonies were randomly picked and cultured to extract genomic DNA for PCR to verify integration of the genome. Two primer pairs, C6 (SEQ ID NO: 30, located upstream of the 5' homology region in the genome) / C7 (SEQ ID NO: 31, located within 5' AOX1) and C3/C8 (SEQ ID NO: 32, located Downstream of the 3' homology region in the genome) was used for PCR to verify that the linear vector was integrated at the PNO1 locus of the genome. Successful PCR amplification of the 1411 and 1369 bp bands indicated that the linear vector was successfully integrated into the genome by homologous recombination replacing the ORF of PNO1 (Fig. 6B). Thus, pno1::cAtMnsI integrated bacteria (his4ura3och1::loxP, pno1::Zeocin-cAtMnsI, mIL22) was constructed.

The hybrid cAtMnsI integrator (his4ura3och1::loxP, pno1::Zeocin-AtMnsI, mIL22) was cultured in 5 ml of YPD medium at 28 ° C, shaking at 225 rpm for 24 hours. The cells were pelleted by centrifugation at 3000 g for 5 minutes, resuspended in 5 ml of BMGY medium, and cultured at 28 ° C, shaking at 225 rpm for 24 hours. Then, the cells were pelleted by centrifugation at 3000 g for 5 minutes, resuspended in 5 ml of BMGY medium, and shake cultured at 225 rpm at 28 ° C to induce FLP expression. The culture was mixed twice daily with 50 μl of 100% methanol (1% final concentration) and induction was maintained for an additional 72 hours. The bacterial solution was spread on a YPD plate, grown at 25 ° C, and then the grown colonies were spotted on a 100 mg/L Zeocin YPD plate to confirm Zeocin sensitivity. Methanol-induced expression of FLP recombinase cleaves FLP recombinase expression cassette, Zeocin selectable marker and replication origin by mediating FRT site-specific recombination, and is replaced by a site-specific open reading frame at the PNO1 locus of the genome. The sexual recombination site FRT and the hybrid cAtMnsI gene. The predicted 1367 bp band was PCR amplified from the two colony genomes sensitive to Zeocin on YPD plates using C6/C5 primers to verify accurate excision between FRTs in the genome (Fig. 6C). Thus, a hybrid cAtMnsI expressing strain (his4ura3och1::loxP, pno1::cAtMnsI, mIL22) was constructed. The N-glycan chain of mIL-22 expressed by this bacterium was analyzed by MALDI-TOF and showed Man ₅ GlcNAc ₂ (m/z: 1257) and Man ₈ GlcNAc ₂ (m/z: 1743), indicating that the hybrid cAtMnsI can Man ₈ GlcNAc _{2 was} converted to Man ₅ GlcNAc ₂ .

The sequences used in the present invention are summarized in the following table:

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (12)

1. A nucleotide construct having the class I structure shown below:

   5'H-T-F-GOI-3'H;

   5'H-T-GOI-F-3'H;

   5'H-F-T-GOI-3'H;

   5'H-F-GOI-T-3'H;

   5'H-GOI-T-F-3'H;

   5'H-GOI-F-T-3'H;

   5'H-F-GOI-3'H;

   5'H-GOI-F-3'H;

   among them,

   T is an exogenous terminator;

   F is a gene fragment represented by B-C-Marker-B; wherein B is a site-specific recombination site; C is a recombinase expression cassette corresponding to the site-specific recombination site; and Marker is a marker gene expression cassette;

   GOI is a foreign gene expression cassette;

   or,

   The nucleotide construct has a class II structure as shown below:

   5'H-F-GOI-P-3'H;

   5'H-GOI-F-P-3'H;

   5'H-P-F-GOI-3'H;

   5'H-P-GOI-F-3'H;

   5'H-F-P-GOI-3'H;

   5'H-GOI-P-F-3'H;

   5'H-F-GOI-3'H;

   5'H-GOI-F-3'H;

   among them,

   P is an exogenous promoter;

   F, B, C, Marker and GOI are as described above.
2. The nucleotide construct of claim 1 having the class I structure shown below:

   5'H-T-F-GOI-3'H;

   5'H-T-GOI-F-3'H;

   5'H-F-T-GOI-3'H;

   5'H-F-GOI-T-3'H;

   5'H-GOI-T-F-3'H;

   5'H-GOI-F-T-3'H;

   Wherein T, F, and GOI are as claimed in claim 1;

   or,

   The nucleotide construct has the class II structure shown below

   5'H-F-GOI-P-3'H;

   5'H-GOI-F-P-3'H;

   5'H-P-F-GOI-3'H;

   5'H-P-GOI-F-3'H;

   5'H-F-P-GOI-3'H;

   5'H-GOI-P-F-3'H;

   Wherein P, F and GOI are as claimed in claim 1.
3. The nucleotide construct according to claim 1 or 2, wherein the transcription terminator comprises, but is not limited to, a transcription terminator such as ADH1, CYC1 and TIF51A of Saccharomyces cerevisiae, ALG6, AOD of Pichia pastoris, Transcriptional terminators such as AOX1, ARG4, PMA1 and TEF1;

   Transcriptional promoters include, but are not limited to, transcriptional promoters such as ADH1, GAP, PGK1 and TEF1 of Saccharomyces cerevisiae, transcriptional promoters such as GAP, ILV5, PGK1, and TEF1 of Pichia pastoris, and promoters AOX1 and FLD1;

   The site-specific recombination site and the corresponding recombinase constitute a site-specific recombination system including, but not limited to, Flp-FRT of Saccharomyces cerevisiae, Cre-loxP of bacteriophage P1 and R-RS of Xygosaccharomyces rouxii;

   The marker gene includes one or more antibiotic resistance genes, preferably a Sh ble gene that is resistant to zeocin, and is resistant to kanamycin (kan) or geneticin (G418). The neo gene is resistant to the BSD gene of blasticidin (Blasticidin) and the like.
4. The nucleotide construct of claim 1 or 2, wherein the recombinase comprises, but is not limited to, a Flp recombinase or a Cre recombinase; preferably a Flp recombinase.
5. The nucleotide construct according to claim 1 or 2, wherein the nucleotide construct further comprises a homology arm at the 5' end and the 3' end.
6. A nucleotide construct according to any one of claims 1 to 5, wherein the nucleotide construct may further comprise a host cell when the nucleotide construct forms a loop Other genes X required for replication in (eg, bacteria), preferably bacterial origins of replication and antibiotic resistance genes; and when the nucleotide construct forms a linear construct, the positions of the other genes X are Any position between B of the nucleotide construct.
7. An expression vector comprising the nucleotide construct of any of claims 1-6.
8. A host cell which integrates the nucleotide construct of any of claims 1-6 in its genome.
9. A method of genetically engineering a host cell, the method comprising integrating a foreign gene with the nucleotide construct of any one of claims 1-6.
10. The method of claim 9 comprising the steps of:

    a construction of the nucleotide construct of any of claims 1-6;

    b. Functional integration of a nucleotide construct having a class I structure by homologous recombination downstream of a translation termination nonsense codon (eg, TAA, etc.) of the genomic open reading frame (ORF);

    Functionally integrating a nucleotide construct having a class II structure upstream of the translation initiation codon (ATG) of the open reading frame (ORF) by homologous recombination; or

    A nucleotide construct having a class I or class II structure is functionally integrated at any position in the genome by homologous recombination, ie, in the middle of an open reading frame (ORF), or any position upstream and downstream thereof;

    c. Recombinase-mediated recombination removes elements between site-specific recombination sites in a nucleotide construct having a class I or class II structure, thereby only nucleotides having a class I or class II structure A site-specific recombination site and an exogenous gene expression cassette in the construct, and optionally an exogenous transcription terminator or an exogenous transcriptional promoter, are integrated into the genome of the host cell.
11. Use of the nucleotide construct of any of claims 1-6 or the expression vector of claim 7 for genetic engineering of a host cell.
12. The use of a host cell engineered by the method of claim 9 or 10, which is used in the fields of metabolic engineering, systems biology and synthetic biology; including but not limited to: the strain is used in a biocatalytic reaction, or The strain is used to produce a recombinant protein.

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