Filamentous fungal mutants with improved homologous recombination efficiency

Patent metadata

Field	Value
Publication number	US-9657301-B2
Application number	US-201514980425-A
Country	US
Kind code	B2
Filing date	Dec 28, 2015
Priority date	Apr 2, 2004
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

The present invention relates to a method for increasing the efficiency of targeted integration of a polynucleotide to a pre-determined site into the genome of a filamentous fungal cell with a preference for NHR, wherein said polynucleotide has a region of homology with said pre-determined site, comprising steering an integration pathway towards HR. The present invention also relates to a mutant filamentous fungus originating from a parent cell, said mutant having an HR pathway with elevated efficiency and/or an NHR pathway with a lowered efficiency and/or a NHR/HR ratio with decreased efficiency as compared to said HR and/or NHR efficiency and/or NHR/HR ratio of said parent cell under the same conditions.

First claim

The invention claimed is:

1. A mutant of a parent filamentous fungal cell, the parent organism having a preference for non-homologous recombination (NHR), wherein the filamentous fungal host cell is a Penicillium species, wherein the ratio of NHR/homologous recombination (HR) is decreased in the mutant as compared to said ratio in said parent organism measured under the same conditions, wherein the mutant is deficient in a gene encoding a component involved in NHR, and/or has a decreased level of a component involved in NHR, wherein the mutant is, optionally inducibly, deficient in at least one filamentous fungal homologue of yeast KU80, yeast KU70, or both, or has, optionally inducibly, a decreased amount of at least one of the proteins encoded by these genes.

2. The mutant according to claim 1 , wherein in the genome of the organism a gene involved in NHR has been replaced by a non-functional variant.

3. The mutant according to claim 1 , wherein the mutant has an increased level of a component involved in HR.

4. The mutant according to claim 1 , wherein the mutant is a recombinant mutant.

5. The mutant according to claim 1 , wherein the mutant is transformed with a DNA construct comprising a DNA sequence comprising a gene of interest encoding a polypeptide of interest.

6. The mutant according to claim 1 , wherein the Penicillium is a Penicillium chrysogenum or Penicillium citrinum species.

7. A method for producing a polypeptide of interest, comprising:
(a) culturing the mutant according to claim 1 , under conditions conducive to expression of the DNA sequence encoding the polypeptide; and
(b) recovering the polypeptide of interest.

8. A method for producing a metabolite, comprising:
(a) culturing the mutant according to claim 1 , under conditions conducive to produce the metabolite; and
(b) recovering the metabolite.

9. The method according to claim 8 , wherein the metabolite is a carotenoid compound or a beta-lactam compound.

10. A method for increasing the efficiency of targeted integration of a polynucleotide to a pre-determined site into the genome of a filamentous fungal cell with a preference for NHR, wherein said polynucleotide has a region of homology with said pre-determined site, wherein said filamentous fungal cell belongs the genus Penicillium , comprising providing a mutant of a parent filamentous fungal cell, wherein the ratio of NHR/HR is decreased in the mutant as compared to said ratio in said parent organism measured under the same conditions, wherein said mutant is deficient in a gene encoding a component involved in NHR, and/or has a decreased level of a component involved in NHR, wherein the mutant is, optionally inducibly, deficient in at least one filamentous fungal homologue of yeast KU80, yeast KU70, or both, or has, optionally inducibly, a decreased amount of at least one of the proteins encoded by these genes.

11. The method of claim 10 , wherein the steering comprises providing a mutant of a parent filamentous fungal cell, wherein the ratio of NHR/HR is decreased in the mutant as compared to said ratio in said parent organism measured under the same conditions.

12. The method of claim 10 , wherein the steering comprises providing a mutant which is deficient in a gene encoding a component involved in NHR, and/or has a decreased level of a component involved in NHR.

13. The method of claim 10 , wherein a gene involved in NHR has been replaced by a non-functional variant.

14. The method according to claim 10 , wherein the mutant has an increased level of a component involved in HR.

Assignees

• Dsm Ip Assets Bv

Inventors

• Dekker Petrus Jacobus Theodorus
• Van Den Berg Marco Alexander

Classifications

• C07K14/38

  from Aspergillus · CPC title

• C07K14/385

  from Penicillium · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/80 Primary

  for fungi · CPC title

• C07K14/37

  from fungi · CPC title