Heterologous alpha amylase expression in Aspergillus

• US 20060134747A1
• Filed: 11/08/2005
• Published: 06/22/2006
• Est. Priority Date: 05/27/2004
• Status: Active Grant

• Current Assignee: Kathleen A. Clarkson, Nigel Dunn-Coleman, Suzanne E. Lantz, Toby M. Baldwin
• Original Assignee: Kathleen A. Clarkson, Nigel Dunn-Coleman, Suzanne E. Lantz, Toby M. Baldwin
• Inventors: Lantz, Suzanne E., Dunn-Coleman, Nigel, Clarkson, Kathleen A., Baldwin, Toby M.
• Granted Patent: US 7,332,319 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 15/8245 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• CPC Class Codes: C12N 9/2417 from microbiological source
• CPC Class Codes: C12N 9/242 Fungal source
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 19/22 produced by the action of a beta-amylase, e.g. maltose
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to the co-expression and production of a heterologous alpha amylase and an endogenous glucoamylase in an Aspergillus strain and enzyme compositions including the same.

Claims

1. An Aspergillus fungal strain comprising DNA encoding a glucoamylase and DNA encoding a heterologous alpha amylase having at least 90% sequence identity to SEQ ID NO:

3, wherein both the glucoamylase and the heterologous alpha amylase are expressed and secreted by the Aspergillus fungal strain.

2. The Aspergillus strain of claim 1, wherein the heterologous alpha amylase has at least 95% sequence identity to SEQ ID NO:

3.

3. The Aspergillus strain of claim 1, wherein the strain is an A. niger, A. nidulans, A. oryzae or A. awamori strain.

4. The Aspergillus strain of claim 3, wherein the Aspergillus strain is an A. niger strain.

5. The Aspergillus strain of claim 3, wherein the strain is an overproducing glucoamylase strain.

6. A culture medium comprising the Aspergillus strain of claim 1.

7. An enzyme composition comprising the Aspergillus strain of claim 1.

8. An enzyme composition comprising the culture medium of claim 6.

21. A method of hydrolyzing starch comprising contacting a substrate containing starch with the enzyme composition of claim 7 under suitable conditions for starch hydrolysis and obtaining hydrolyzed starch.

22. The method according to claim 21, wherein the starch is granular starch.

23. The method according to claim 22, wherein the contacting is at a temperature below the gelatinization temperature of the granular starch in the substrate.

24. The method according to claim 21, wherein the substrate is a grain or fractionated part thereof.

9. An Aspergillus strain comprising a) DNA encoding an endogenous glucoamylase and b) DNA encoding a heterologous alpha amylase having at least 95% sequence identity to SEQ ID NO:

3, wherein the Aspergillus strain expresses both the glucoamylase and the heterologous alpha amylase and overproduces the glucoamylase.

10. A culture medium comprising the Aspergillus strain of claim 9.

11. A method of producing an alpha amylase in an Aspergillus cell which comprises, culturing an Aspergillus cell which is capable of expressing a heterologous alpha amylase polypeptide having at least 90% sequence identity to SEQ ID NO:

3 or SEQ ID NO;

4 under conditions suitable for the expression and production of the heterologous alpha amylase and producing the heterologous alpha amylase.

12. The method according to claim 11, wherein the Aspergillus cell further comprises a polynucleotide which codes for an endogenous glucoamylase and wherein said glucoamylase is expressed and produced.

13. The method according to claim 12, wherein the Aspergillus is an A. niger strain.

14. The method according to claim 12 further comprising recovering the produced alpha amylase and glucoamylase.

15. The method according to claim 12, wherein the Aspergillus strain is an overproducing glucoamylase strain.

16. A method for producing an acid stable alpha amylase and a glucoamylase in a filamentous fungal host cell comprising

a) co-expressing a glucoamylase and an alpha amylase in a filamentous host by transforming the filamentous fungal cell with a DNA construct comprising a promoter having transcriptional activity in the host cell operably linked to a polynucleotide encoding an alpha amylase having at least 90% sequence identity to SEQ ID NO:

3, wherein said host cell further expresses and produces a glucoamylase,

b) cultivating the transformed host cell in a suitable culture medium to allow expression of the alpha amylase and the glucoamylase, and

c) producing the alpha amylase and the glucoamylase.

17. The method according to claim 16 further comprising recovering the produced glucoamylase and alpha amylase.

18. The method according to claim 16, wherein the filamentous host cell is an Aspergillus strain.

19. The method according to claim 18, wherein the Aspergillus strain is an A. niger strain.

20. The method according to claim 18, wherein the Aspergillus strain is an overproducing glucoamylase strain.