High-throughput screening of expressed DNA libraries in filamentous fungi

• US 7,794,962 B2
• Filed: 07/21/2006
• Issued: 09/14/2010
• Est. Priority Date: 04/13/2000
• Status: Expired due to Fees

• Current Assignee: Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Punt, Peter J., van den Hondel, Cornelius, van Zeijl, Cornelia, Emalfarb, Mark A.
• Primary Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US11/490,761
• Publication Number:
  US 20060257923A1
• Time in Patent Office: 1,516 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, 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, comprising the steps of:

(a) stably transforming a plurality of low viscosity filamentous fungi of less than about 200 cP, wherein the fungi are of a genus selected from the group consisting of;

Aspergillus, Trichoderma, Chrysosporium, Neurospora, Rhizomucor, Hansenula, Humicola, Mucor, Tolypocladium, Fusarium, Penicillium, Talaromyces, Emericella, and Hypocrea, said fungi having a phenotype characterized by growth in suspension and by the production of transferable reproductive elements in suspension with a library of vectors which comprises a plurality of different vectors, each different vector comprising a different mutant protein-encoding nucleic acid sequence so as to introduce into each of a plurality of individual fungi at least one heterologous protein-encoding nucleic acid sequence;

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

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

(d) culturing into monoclonal 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;

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

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

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

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

(i) repeating steps (a) through (e), until the property or activity of interest is optimized.

2. The method of claim 1, further comprising between steps (f) and (g) the steps of:

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

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

evaluating the isolated protein for the property of interest.

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

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

5. The method of claim 1 wherein the fungus is of a genus selected from the group consisting of Aspergillus, Fusarium, Chrysosporium, and Trichoderma.

6. The method of claim 5, wherein the fungus is Chrysosporium strain UV18-25 having accession number VKM F-3631 D.

7. 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 1, culturing an individual clonal culture or clonal colony isolated in the final step (f), and isolating the expressed protein from the culture.