High-throughput screening of expressed DNA libraries in filamentous fungi

• US 7,122,330 B2
• Filed: 04/13/2001
• Issued: 10/17/2006
• Est. Priority Date: 04/13/2000
• Status: Expired due to Term

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Mark Aaron Emalfarb
• Inventors: van den Hondel, Cornelius, van Zeijl, Cornelia, Emalfarb, Mark A., Punt, Peter J.
• Primary Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US09/834,434
• Publication Number:
  US 20030162218A1
• Time in Patent Office: 2,013 Days
• Field of Search: 435/6, 435/7, 435/69.1, 435/471, 435/254.11, 435/7.31, 435/7.2, 435/7.1, 435/DIG.47, 536/23.1, 530/350
• US Class Current: 435/7.31
• 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 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:

(a) stably transforming a plurality of individual filamnentous 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;

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

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

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

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

2. A 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:

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

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

3. A method of producing a DNA molecule encoding a protein 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 claim 1;

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

(c) isolating DNA from a clonal culture or clonal colony exhibiting the activity or property of interest.

4. A method of producing the nucleotide sequence of a DNA molecule encoding a protein having an activity or property of interest, comprising the steps of:

(a) isolating DNA from a clonal culture or clonal colony exhibiting the activity or property of interest, by the method of claim 3; and

(b) sequencing said DNA.

5. A method of producing the amino acid sequence of a protein having an activity or property of interest, comprrising the steps of:

(a) producing the DNA sequence of the protein having an activity or property of interest, by the method of claim 4; and

(b) converting said DNA sequence into an amino acid sequence.

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

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

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

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

10. The method of any one of claims 1–

5, wherein the fungus is Chrysosporium strain UV18-25 having accession number VKM F-3631 D.

11. The method of any one of claims 1–

5, wherein the fungus is Chrysosporium lucknowense strain C1 having accession number VKM F-3500 D.

12. The method of claim 1, wherein steps (b) through (e) are repeated more than once.

13. The method of any one of claims 1–

5, wherein the fungi are Aspergillus.

14. The method of any one of claims 1–

5, wherein the fungi are Fusarium.

15. The method of any one of claims 1–

5, wherein the fungi are Chrysosporium.

16. The method of any one of claims 1–

5, wherein the fungi are Trichoderma.