Methods for transforming and expression screening of filamentous fungal cells with a DNA library
View Patent ↗The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli ; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.
1. A method for expression screening of filamentous fungal transformants, comprising:
(a) isolating single colony transformants of a DNA library of different plasmids introduced into E. coli , wherein each different plasmid comprises a different polynucleotide encoding a different polypeptide operably linked to control sequences for expression and secretion of each different polypeptide in a filamentous fungus;
(b) preparing each different plasmid from each of the single colony E. coli transformants, wherein each single colony comprises a different plasmid comprising a different polynucleotide from the DNA library;
(c) introducing a sample of each of the different plasmid preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual and different plasmid from the DNA library;
(d) growing the individual filamentous fungal transformants obtained from transformation of each of the different plasmid preparations of step (c) on growth medium that selects for filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi, and also enables expression and secretion of each different polypeptide into the growth medium; and
(e) measuring activity or a property of each different polypeptide encoded by each individual and different DNA secreted into the culture medium of step (d).
2. The method of claim 1 , wherein the DNA library is a library of mutants of a gene.
3. The method of claim 2 , wherein the mutated genes encode polypeptide variants.
4. The method of claim 1 , wherein the polypeptide encoded by the polynucleotide is an antibody, antigen, enzyme, hormone, or reporter.
5. The method of claim 4 , wherein the enzyme is selected from the group consisting of an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, protease, ribonuclease, transglutaminase, and xylanase.
6. The method of claim 1 , wherein the polypeptide is a variant.
7. The method of claim 1 , wherein the filamentous fungus is selected from the group consisting of an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium , and Trichoderma strain.
8. The method of claim 1 , further comprising (f) isolating a polynucleotide originating from the DNA library, wherein the polynucleotide encodes a polypeptide of interest.
9. The method of claim 1 , wherein one or more steps are automated.
10. The method of claim 1 , wherein each step is automated.
11. An isolated polynucleotide encoding a polypeptide of interest obtained by the method of claim 8 .
12. A nucleic acid construct comprising the polynucleotide of claim 11 .
13. A recombinant expression vector comprising the polynucleotide of claim 11 .
14. A recombinant host cell comprising the polynucleotide of claim 11 .