Methods of introducing multiple expression constructs into a eukaryotic cell

Abstract

The present invention relates to methods of introducing multiple expression constructs into a eukaryotic cell, methods of constructing a eukaryotic cell having multiple target loci for expressing multiple heterologous proteins of interest, eukaryotic cells for expressing multiple heterologous proteins of interest, and methods of production of multiple heterologous proteins of interest.

What is claimed is: 
     
       1. A method of introducing multiple expression constructs into two or more target loci of a eukaryotic cell, said method comprising:
 (a) transforming a population of the eukaryotic cell with one or more first constructs and one or more second constructs, 
 wherein the eukaryotic cell comprises (1) one or more first target loci each comprising a pair of a first recombination recognition site and a second recombination recognition site, and (2) one or more second target loci each comprising a first fragment of a first selectable marker lacking a selectable function; 
 wherein the one or more first constructs each comprises one or more first expression cassettes each comprising a first polynucleotide encoding a first protein of interest, wherein in each of the first constructs the one or more first expression cassettes are flanked on one side by the first recombination recognition site and on the other side by the second recombination recognition site, wherein the first and second recombination recognition sites flanking the first expression cassettes are the same as the first and second recombination recognition sites of the first target loci; 
 wherein the one or more second constructs each comprises one or more second expression cassettes each comprising a second polynucleotide encoding a second protein of interest, wherein in each of the second constructs the one or more second expression cassettes are flanked on one side by a homologous region of the second target locus and on the other side by a second fragment of the first selectable marker that lacks the selectable function, wherein the second fragment comprises a sequence overlapping homologously a sequence of the first fragment of the first selectable marker of the second target loci; and 
 wherein the transforming of the population of the eukaryotic cell with the one or more first constructs and the one or more second constructs is performed sequentially in any order or as a co-transformation; and 
 (b) selecting a transformant using a reconstituted functional first selectable marker wherein the first fragment of the first selectable marker and the second fragment of the first selectable marker undergo recombination to become functional and the homologous region of the one or more second constructs undergo homologous recombination with the same region of the second target loci, wherein each of the second expression cassettes are integrated at the second target loci, and wherein the first and second recombination recognition sites of the first constructs undergo recombination at the same first and second recombination recognition sites of the first target loci driven by at least one recombinase integrating each of the first expression cassettes at the first target loci. 
 
     
     
       2. The method of  claim 1 , wherein the first and second fragments of the first selectable marker each further comprise a repeat sequence 5′ of the first fragment and a repeat sequence 3′ of the second fragment, and wherein the repeat sequences undergo homologous recombination to remove the first selectable marker; or wherein the first and second fragments of the first selectable marker each further comprise repeat sequences allowing removal of a fragment of the first selectable marker by homologous recombination resulting in no selectable function. 
     
     
       3. The method of  claim 1 , wherein the eukaryotic cell is reusable by repeating steps (a) and (b) with one or more different first constructs, second constructs, or first and second constructs each comprising an expression cassette comprising a polynucleotide encoding a different protein of interest. 
     
     
       4. The method of  claim 1 , wherein the recombination recognition sites are selected from the group consisting of B2 system from  Zygosaccharomyces bailii , B3 system from  Zygosaccharomyces bisporus , beta-recombinase-six system from a 25  Bacillus subtilis  plasmid, Bxb1 from phage Bxb1, Cre-lox system of bacteriophase P1, Dre from Bacteriophage D6, FLP-FRT of  Saccharomyces cerevisiae , Delta-gamma-es system from bacterial transposon Tn1000, Gin-gix system from bacteriophase Mu, HK022 from phage HK022, KD system from  Kluyveromyces drosophilarum , Mx9 phage transformation system,  Streptomyces  phage lC31, R-RS system of  Zygosaccharomyces rouxii , Tn3 from  E. coli , Vika recombinase from  Vibrio coralliilyticus , Xis-att system of temperate lactococcal bacteriophage TP901-1; and combinations thereof. 
     
     
       5. The method of  claim 1 , wherein the eukaryotic cell comprises one or more recombinases, which are native, heterologous, or a combination of native and heterologous to the eukaryotic cell. 
     
     
       6. The method of  claim 5 , wherein the recombinase is a Bxb1 recombinase, a Cre recombinase, a CinH recombinase, a Flp flippase, a HK022 integrase, a ParA recombinase, a Tn1721 recombinase, a Tn5053 recombinase, a TP901-1 integrase, an U153 recombinase, a λ integrase, or a ϕC31 recombinase. 
     
     
       7. The method of  claim 1 , wherein the selectable markers are selected from the group consisting of ADE2, ARO4-OFP, FLD1, HIS3, LEU2, LYS2, MET3, TRP1, URA3, adeA, adeB, amdS, argB, bar, bleR, bsd, fcy1, hpt, hpt-tk, nat1, niaD, ptr1, pyrG, sC, tk, Tn903kan r , trpC, and beta-tubulin. 
     
     
       8. A method of constructing a eukaryotic cell having multiple target loci for expressing multiple heterologous proteins of interest, comprising:
 (a) transforming a population of the eukaryotic cell with a first construct comprising (1) a 5′ homologous region of a first target locus, (2) a first recombination recognition site, (3) a first selectable marker conferring a first selectable function, (4) a second recombination recognition site, and (5) a 3′ homologous region of the first target locus; 
 (b) selecting a first transformant having the first construct integrated at the first target locus using the first selectable marker for selection of the first transformant, wherein the 5′ homologous region and the 3′ homologous region of the first construct undergo homologous recombination with the 5′ homologous region and the 3′ homologous region of the first target locus integrating the first construct at the first target locus, and wherein the first recombination recognition site and the second recombination recognition site are integrated at the first target locus; 
 (c) transforming a population of the first transformant with a second construct comprising (1) a 5′ homologous region of a second target locus, (2) a first fragment of a second selectable marker that lacks a second selectable function, (3) a third selectable marker conferring a third selectable function, and (4) a 3′ homologous region of the second target locus; 
 (d) selecting a second transformant having the second construct integrated at the second target locus using the third selectable marker for selection of the second transformant, wherein the 5′ homologous region and the 3′ homologous region of the second construct undergo homologous recombination with the 5′ homologous region and the 3′ homologous region of the second target locus integrating the second construct at the second target locus, and wherein the first fragment of the second selectable marker that lacks a second selectable function is integrated at the second target locus; 
 (e) co-transforming a population of the second transformant with (i) a third construct comprising (1) the first recombination recognition site, (2) one or more first expression cassettes each comprising a first polynucleotide encoding a first protein of interest, and (3) the second recombination recognition site, and (ii) a fourth construct comprising (1) the 5′ homologous region of the second target locus, (2) one or more second expression cassettes each comprising a second polynucleotide encoding a second protein of interest, and (3) a second fragment of the second selectable marker that lacks the second selectable function wherein the second fragment comprises a sequence overlapping homologously a sequence of the first fragment of the first selectable marker; and 
 (f) selecting a third transformant using the second selectable marker wherein the first integrated fragment and the second fragment of the second selectable marker become functional upon recombination, wherein the 5′ homologous region of the fourth construct undergoes homologous recombination with the 5′ homologous region of the second target locus, wherein the one or more second expression cassettes are integrated at the second target locus, and wherein the first recombination recognition site and the second recombination recognition site undergo recombination at the first target locus driven by at least one recombinase integrating the one or more first expression cassettes at the first target locus. 
 
     
     
       9. The method of  claim 8 , wherein steps (a) and (b) are performed before or after steps (c) and (d). 
     
     
       10. The method of  claim 8 , wherein the first selectable marker further comprises a first repeat 5′ of the first selectable marker and a second repeat 3′ of the first selectable marker, wherein the first and second repeat sequences of the integrated first construct undergo homologous recombination to remove the first selectable marker. 
     
     
       11. The method of  claim 8 , wherein the third selectable marker further comprises a third repeat 5′ of the third selectable marker and a fourth repeat 3′ of the third selectable marker, wherein the third and fourth repeat sequences of the second construct undergo homologous recombination to remove the third selectable marker. 
     
     
       12. The method of  claim 8 , wherein steps (a)-(f) are repeated at at least two additional target loci with different recombination recognition sites, different polynucleotides encoding proteins of interest, and the same or a different second selectable marker; or wherein steps (a)-(b) or (c)-(d) are repeated at one additional target locus with different recombination recognition sites, different polynucleotides encoding proteins of interest, and the same or a different selectable marker. 
     
     
       13. The method of  claim 8 , wherein the recombination recognition sites are selected from the group consisting of B2 system from  Zygosaccharomyces bailii , B3 system from  Zygosaccharomyces bisporus , beta-recombinase-six system from a 25  Bacillus subtilis  plasmid, Bxb1 from phage Bxb1, Cre-lox system of bacteriophase P1, Dre from Bacteriophage D6, FLP-FRT of  Saccharomyces cerevisiae , Delta-gamma-es system from bacterial transposon Tn1000, Gin-gix system from bacteriophase Mu, HK022 from phage HK022, KD system from  Kluyveromyces drosophilarum , Mx9 phage transformation system,  Streptomyces  phage lC31, R-RS system of  Zygosaccharomyces rouxii , Tn3 from  E. coli , Vika recombinase from  Vibrio coralliilyticus , Xis-att system of temperate lactococcal bacteriophage TP901-1; and combinations thereof. 
     
     
       14. The method of  claim 8 , wherein the eukaryotic cell comprises one or more recombinases, which are native, heterologous, or a combination of native and heterologous to the eukaryotic cell. 
     
     
       15. The method of  claim 14 , wherein the recombinase is a Bxb1 recombinase, a Cre recombinase, a CinH recombinase, a Flp flippase, a HK022 integrase, a ParA recombinase, a Tn1721 recombinase, a Tn5053 recombinase, a TP901-1 integrase, an U153 recombinase, a λ integrase, or a ϕC31 recombinase. 
     
     
       16. The method of  claim 8 , wherein the recombination is homologous recombination or recombinase-mediated recombination. 
     
     
       17. A method of introducing multiple expression constructs into a eukaryotic cell, said method comprising:
 (a) transforming a population of the eukaryotic cell with one or more first constructs and one or more second constructs, 
 wherein the eukaryotic cell comprises (1) one or more first target loci each comprising a pair of a first recombination recognition site and a second recombination recognition site, wherein the first recombination recognition site and the second recombination recognition site are TP901-1 sites of the Xis-att system, and (2) one or more second target loci each comprising a pair of a third recombination recognition site and a fourth recombination recognition site, wherein the third recombination recognition site and the fourth recombination recognition site are flippase recognition sites of the FLP-FRT system; 
 wherein the one or more first constructs each comprises one or more first expression cassettes each comprising a first polynucleotide encoding a first protein of interest, wherein in each of the first constructs the one or more first expression cassettes are flanked on one side by the first recombination recognition site and on the other side by the second recombination recognition site, wherein the first and second recombination recognition sites flanking the first expression cassettes are the same as the first and second recombination recognition sites of the first target loci; 
 wherein the one or more second constructs each comprises one or more second expression cassettes each comprising a second polynucleotide encoding a second protein of interest, and wherein in each of the second constructs the one or more second expression cassettes are flanked on one side by the third recombination recognition site and on the other side by the fourth recombination recognition site, wherein the third and fourth recombination recognition sites flanking the second expression cassettes are the same as the third and fourth recombination recognition sites of the second target loci; and 
 wherein one or more of the first constructs and second constructs comprise one or more first selectable markers; 
 (b) selecting a transformant using the one or more first selectable markers, wherein each of the second expression cassettes are integrated at the second target loci by recombination of the third and fourth recombination recognition sites, and wherein each of the first expression cassettes are integrated at the first target loci by recombination of the first and second recombination recognition sites driven by at least one recombinase. 
 
     
     
       18. The method of  claim 17 , wherein the one or more first target loci each further comprises a second selectable marker between the first and second recombination recognition sites, and/or wherein the one or more second target loci each further comprises a third selectable marker between the third and fourth recombination recognition sites. 
     
     
       19. The method of  claim 17 , wherein the eukaryotic cell comprises one or more recombinases, which are native, heterologous, or a combination of native and heterologous to the eukaryotic cell. 
     
     
       20. The method of  claim 19 , wherein the recombinase is a Bxb1 recombinase, a Cre recombinase, a CinH recombinase, a Flp flippase, a HK022 integrase, a ParA recombinase, a Tn1721 recombinase, a Tn5053 recombinase, a TP901-1 integrase, an U153 recombinase, a λ integrase, or a ϕC31 recombinase.