High-throughput screening of expressed DNA libraries in filamentous fungi

• US 20060257923A1
• Filed: 07/21/2006
• Published: 11/16/2006
• Est. Priority Date: 04/13/2000
• Status: Active Grant

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Zeijl, Cornelia van, Hondel, Cornelius van den, Emalfarb, Mark A., Punt, Peter J.
• Granted Patent: US 7,794,962 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/1077 Pentosyltransferases (2.4.2)
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• CPC Class Codes: G01N 2333/37 from fungi
• CPC Class Codes: G01N 2500/20 cell-free systems
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Abstract

The invention provides a method for the expression of exogenous DNA libraries in filamentous fungi. The fungi are capable of processing intron-containing eukaryotic genes, and also can carry out post-translational processing steps such as glyclosylation and protein folding. The invention provides for the use of fungi with altered morphology, which permits high-throughput screening and directed molecular evolution of expressed proteins. The same transformed fungi may be used to produce larger quantities of protein for isolation, characterization, and application testing, and may be suitable for commercial production of the protein as well.

Claims

1. A method of screening a plurality of monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies for a metabolite having an activity or property of interest, comprising the steps of:

(a) expressing a plurality of proteins in monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies, by the method of producing a plurality of proteins having an activity or property of interest encoded by a library of DNA vectors, wherein the library of vectors comprises a plurality of different vectors, each different vector comprising a different protein-encoding nucleic acid sequence, said nucleic acid sequence being operably linked to an expression-regulating region and optionally a secretion signal encoding sequence, the method comprising the steps of;

(i) stably transforming a plurality of individual filamentous fungi, wherein the fungi are selected from the group consisting of Aspergillus, Fusarium, Chrysosporium, and Trichoderma, said fungi having a phenotype characterized by growth in suspension and by the production of transferable reproductive elements which are monoclonal and readily dispersed in suspension, with said library of DNA vectors so as to introduce into each of the plurality of the individual fungi at least one protein-encoding nucleic acid sequence;

(ii) culturing the transformed mutant filamentous fungi under conditions conducive to formation of transferable reproductive elements which are monoclonal and readily dispersed in suspension;

(iii) separating from one another a plurality of transferable reproductive elements in suspension;

(iv) transferring the separated transferable reproductive elements to secondary cultures; and

(v) culturing into monoclonal cultures or monoclonal colonies the individual transferable reproductive elements in said secondary cultures, under conditions conducive to expression of the proteins encoded by the protein-encoding nucleic acid sequences; and

(b) screening each individual clonal culture or clonal colony for the activity or property of interest.

4. The method of claim 1, wherein the screening step is carried out by high-thoughput screening.

7. The method of claim 1, wherein the fungus has a phenotype characterized by a culture viscosity, when cultured in suspension, of less than 200 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.

8. The method of claim 1, wherein the fungus has a phenotype characterized by a culture viscosity, when cultured in suspension, of less than 100 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.

9. The method of claim 1, wherein the fungus has a phenotype characterized by culture viscosity, when cultured in suspension, of less than 60 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.

10. The method of claim 1, wherein the fungus has a phenotype characterized by a culture viscosity, when cultured in suspension, of less than 10 cP at the end of fermentation when grown with adequate nutrients under optimal or near-optimal conditions.

11. The method of claim 1, wherein the vectors comprise a fungal signal sequence.

12. The method of claim 11, wherein the fungal signal sequence is the signal sequence of a fungal gene encoding a protein selected from the group consisting of cellulase, P-galactosidase, xylanase, pectinase, esterase, protease, amylase, polygalacturonase and hydrophobin.

13. The method of claim 1, wherein the vectors comprise a nucleotide sequence encoding a selectable marker.

14. The method of claim 1, wherein the vectors comprise an expression-regulating region region operably linked to the protein-encoding nucleic acid sequence.

15. The method of claim 14, wherein the expression regulating region comprises an inducible promoter.

16. The method of claim 1, wherein the fungus is of the class Euascomycetes.

17. The method of claim 16 wherein the fungus is of the order Onygenales.

18. The method of claim 16 wherein the fungus is of the order Eurotiales.

19. The method of claim 1, wherein the fungus is of the division Ascomycota, with the proviso that it is not of the order Saccharomycetales.

26. The method of claim 1, wherein an expressed protein to biomass ratio is at least 1:

1.

27. The method of claim 26, wherein an expressed protein to biomass ratio is at least 2:

1.

28. The method of claim 27, wherein an expressed protein to biomass ratio is at least 6:

1.

29. The method of claim 28, wherein an expressed protein to biomass ratio is at least 8:

1.

30. The method of claim 1, wherein the transferable reproductive elements are individual fungal cells.

31. The method of claim 1, wherein the transferable reproductive elements are spores.

32. The method of claim 1, wherein the transferable reproductive elements are hyphal fragments.

33. The method of claim 1, wherein the transferable reproductive elements are micropellets.

34. The method of claim 1, wherein the transferable reproductive elements are protoplasts.

35. A method for obtaining a protein having an activity or property of interest, comprising the steps of:

(a) screening a plurality of proteins encoded by a library of DNA vectors for an activity or property of interest, by the method of screening a plurality of proteins encoded by a library of DNA vectors for an activity or property of interest, comprising the steps of;

(i) producing the plurality of proteins in monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies, by the method of claim 1;

and

(ii) screening individual clonal cultures or clonal colonies for the activity or property of interest.

(b) culturing on appropriate scale the monoclonal culture or monoclonal colony expressing the activity or property of interest, under conditions conducive to expression of the heterologous proteins encoded by the heterologous protein-encoding nucleic acid sequences; and

(c) isolating the expressed protein.

38. A library of transformed filamentous fungi, prepared by the method of claim 34.

39. A method for obtaining a transformed filamentous fungal host expressing a protein having an activity or property of interest, comprising the steps of:

(a) screening a plurality of proteins encoded by a library of DNA vectors for an activity or property of interest, by the method of screening a plurality of proteins encoded by a library of DNA vectors for an activity or property of interest, comprising the steps of;

(i) producing the plurality of proteins in monoclonal filamentous fungal cultures or monoclonal filamentous fungal colonies, by the method of claim 1;

and

(ii) screening individual clonal cultures or clonal colonies for the activity or property of interest; and

(b) isolating the monoclonal culture or monoclonal colony expressing the activity or property of interest.

40. The method of claim 1, wherein the fungus is Aspergillus niger strain pcIA, and decendants.

2. A method of optimizing a protein'"'"'s activity or property of interest, comprising the steps of:

(a) providing a library of vectors which comprise DNA sequences encoding mutant forms of the protein;

(b) providing a filamentous fungus having a phenotype characterized by growth in suspension and by the production of transferable reproductive elements in suspension;

(c) stably transforming said filamentous fungus with said library of DNA vectors so as to introduce into each of a plurality of individual fungi at least one heterologous protein-encoding nucleic acid sequence;

(d) culturing the transformed filamentous fungi under conditions conducive to the formation of transferable reproductive elements;

(e) separating from one another a plurality of transferable reproductive elements;

(f) culturing into clonal cultures or clonal colonies the individual transferable reproductive elements, under conditions conducive to expression of the heterologous proteins encoded by the heterologous protein-encoding nucleic acid sequences;

(g) screening each individual organism, clonal culture, or clonal colony for an expressed protein having the activity or property of interest;

(h) isolating one or more individual organisms, clonal cultures, or clonal colonies that express a protein exhibiting the activity or property of interest;

(i) mutating the DNA from the isolated individual organisms, clonal cultures, or clonal colonies that encodes the protein exhibiting the activity or property of interest;

(j) providing a library of vectors which comprise the mutated DNA sequences obtained in step (i); and

(k) repeating steps (b) through (g), until the property or activity of interest either reaches a desirable level or no longer improves.

3. The method of claim 2, further comprising between steps (h) and (i) the steps of:

culturing one or more of the individual organisms, clonal cultures, or clonal colonies isolated in step (h);

isolating the expressed protein exhibiting the activity or property of interest; and

evaluating the isolated protein for the property of interest.

5. The method of claim 2, wherein the screening step is carried out by high-thoughput screening.

6. The method of claim 3, wherein the screening step is carried out by high-thoughput screening.

20. The method of claim 2, wherein the fungus is of a genus selected from the group consisting of:

Aspergillus, Trichoderma, Chrysosporium, Neurospora, Rhizomucor, Hansenula, Humicola, Mucor, Tolypocladium, Fusarium, Penicillium, Talaromyces, Emericella and Hypocrea.

21. The method of claim 20 wherein the fungus is of a genus selected from the group consisting of Aspergillus, Fusarium, Chrysosporium, and Trichoderma.

22. The method of claim 21, wherein the fungus is Chrysosporium strain UV18-25 having accession number VKM F-3631 D.

23. The method of claim 21, wherein the fungus is Trichoderma longibrachiatum strain X-252.

24. The method of claim 21, wherein the fungus is Aspergillus sojae strain pclA.

25. The method of claim 21, wherein the fungus is Aspergillus niger strain pclA.

36. A method for obtaining a protein having an activity or property of interest, comprising optimizing the activity or property of interest by the method of claim 2, culturing on an appropriate scale an individual organism, clonal culture, or clonal colony isolated in the final step (h), and isolating the expressed protein from the culture.

37. A method of making a library of transformed filamentous fungi, comprising the steps of:

(a) providing a filamentous fungus having a phenotype characterized by growth in suspension and characterized by the production of transferable reproductive elements in suspension; and

(b) stably transforming said filamentous fungus with a library of DNA vectors so as to introduce into each of a plurality of the individual fungi at least one heterologous protein-encoding nucleic acid sequence;

wherein the library of DNA vectors comprises a plurality of different vectors, each different vector comprising a different protein-encoding nucleic acid sequence, said nucleic acid sequence being operably linked to an expression-regulating region and optionally a secretion signal encoding sequence.