Expression of granular starch hydrolyzing enzyme in trichoderma

Patent metadata

Field	Value
Publication number	US-9382563-B2
Application number	US-201314097168-A
Country	US
Kind code	B2
Filing date	Dec 4, 2013
Priority date	Nov 21, 2003
Publication date	Jul 5, 2016
Grant date	Jul 5, 2016

Abstract

The present invention relates to filamentous fungal host cells and particularly Trichoderma host cells useful for the production of heterologous granular starch hydrolyzing enzymes having glucoamylase activity.

First claim

It is claimed:

1. A method for producing a granular starch hydrolyzing enzyme having glucoamylase activity (GSHE) in a filamentous fungal host cell comprising
a) transforming a filamentous fungal host cell with a DNA construct comprising a promoter having transcriptional activity in the filamentous fungal host cell operably linked to a heterologous polynucleotide encoding the GSHE polypeptide of SEQ ID NO: 6,
b) cultivating the transformed filamentous fungal host cell in a suitable culture medium to allow expression of said GSHE, and
c) producing the GSHE.

2. The method according to claim 1 further comprising recovering the produced GSHE.

3. The method according to claim 1 , wherein the filamentous fungal host cell is a Trichoderma cell.

4. The method according to claim 3 , wherein the Trichoderma cell is a T. reesei cell.

5. The method according to claim 1 , wherein the filamentous fungal host cell is an Aspergillus cell.

6. The method according to claim 5 , wherein the Aspergillus cell is an A. awamori, A. niger or A. oryzae cell.

7. The method according to claim 1 , wherein one or more genes encoding cellulytic enzymes has been deleted in the filamentous fungal host cell.

8. The method according to claim 1 , wherein a level of glycosylation of the expressed GSHE from the transformed filamentous fungal host cell is less than the level of glycosylation of the GSHE expressed in a native fungal host.

9. The method according to claim 8 , wherein the native fungal host is a strain of Humicola grisea.

10. The method according to claim 1 , wherein the amount of GSHE expressed is greater than 1.0 g/L of culture media.

11. The method according to claim 1 , wherein the amount of GSHE expressed is greater than 10 g/L of culture media.

12. The method according to claim 1 , wherein at a pH level of about 3.0 to 4.0 enzyme activity is greater for the produced GSHE than enzyme activity of the corresponding GSHE expressed and produced in a native fungal host.

13. The method according to claim 1 , wherein the transformed fungal host cell is cultured under continuous fermentation conditions.

14. The method according to claim 1 , wherein the transformed fungal host is cultured under batch fermentation conditions.

15. A recombinant Trichoderma cell comprising a heterologous polynucleotide encoding a granular starch hydrolyzing enzyme having glucoamylase activity (GSHE), wherein the GSHE comprises the amino acid sequence of SEQ ID NO:6.

16. The recombinant Trichoderma cell of claim 15 , wherein the Trichoderma cell is a T. reesei cell.

17. The recombinant Trichoderma cell of claim 15 , wherein one or more genes encoding cellulytic enzymes has been deleted from the Trichoderma cell.

18. The recombinant cell of claim 15 , wherein the GSHE has the amino acid sequence of SEQ ID NO: 6.

19. The method according to claim 1 further comprising recovering the produced GSHE.

Assignees

• Danisco Us Inc

Inventors

• Baldwin Toby M
• Bower Benjamin S
• Dunn-Coleman Nigel
• Lantz Suzanne E
• Pepsin Michael J

Classifications

• C12P19/24

  produced by the action of an isomerase, e.g. fructose · CPC title

• C12P19/14 Primary

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12P19/04

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title

• C12N9/2428

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12N1/14

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title