Modified glucoamylase enzymes and yeast strains having enhanced bioproduct production

Patent metadata

Field	Value
Publication number	US-10364421-B2
Application number	US-201615548812-A
Country	US
Kind code	B2
Filing date	Feb 5, 2016
Priority date	Feb 6, 2015
Publication date	Jul 30, 2019
Grant date	Jul 30, 2019

Abstract

The invention is directed to non-natural yeast able to secrete significant amounts of glucoamylase into a fermentation media. The glucoamylase can promote degradation of starch material generating glucose for fermentation to a desired bioproduct, such as ethanol. The glucoamylase can be provided in the form of a glucoamylase fusion protein having a S. cerevisiae mating factor alpha 2 (Sc MFα2) or repressible acid phosphatase (Sc PHO5) secretion signal.

First claim

What is claimed is:

1. A polypeptide comprising (a) a secretion signal amino acid sequence having 90% or greater identity to SEQ ID NO:10 or SEQ ID NO:11 and (b) a glucoamylase amino acid sequence from a yeast, fungal, or bacterial glucoamylase polypeptide, wherein the polypeptide has glucoamylase activity, and
wherein the polypeptide comprises SEQ ID NO: 13.

2. A polypeptide comprising:
(a) a secretion signal amino acid sequence comprising SEQ ID NO:10 or SEQ ID NO:11 and
(b) a glucoamylase amino acid sequence from a yeast, fungal, or bacterial glucoamylase polypeptide, wherein the polypeptide has glucoamylase activity, and wherein the glucoamylase amino acid sequence in (b) comprises amino acids 19-515 of SEQ ID NO:12.

3. A polypeptide comprising (a) a secretion signal amino acid sequence having 90% or greater identity to SEQ ID NO:10 or SEQ ID NO:11 and (b) a glucoamylase amino acid sequence from a yeast, fungal, or bacterial glucoamylase polypeptide, wherein the polypeptide has glucoamylase activity, and
wherein the polypeptide comprises SEQ ID NO:14.

4. The polypeptide of claim 1 further comprising a third sequence that is different than SEQ ID NO:10, amino acids 1-18 of SEQ ID NO: 12, or the glucoamylase amino acid sequence, wherein the third sequence is positioned between SEQ ID NO:10 and the glucoamylase amino acid sequence.

5. A host cell that expresses the polypeptide of claim 1 .

6. The host cell of claim 5 wherein the host cell is a strain of Saccharomyces cerevisiae.

7. The host cell of claim 6 which is (a) tolerant to growth in fermentation medium having a concentration of ethanol of greater than 90 g/L, (b) tolerant to growth in at temperatures of greater than 33° C., such as in the range of 34° C.-40° C., or both (a) and (b).

8. The polypeptide of claim 4 , wherein the third sequence is a linker sequence that prevents interactions between the secretion signal amino acid sequence and the glucoamylase amino acid sequence.

9. The polypeptide of claim 3 further comprising a third sequence that is different than SEQ ID NO:11, amino acids 1-18 of SEQ ID NO: 12, or the glucoamylase amino acid sequence, wherein the third sequence is positioned between SEQ ID NO:11 and the glucoamylase amino acid sequence.

10. The polypeptide of claim 9 , wherein the third sequence is a linker sequence that prevents interactions between the secretion signal amino acid sequence and the glucoamylase amino acid sequence.

Assignees

• Cargill Inc

Inventors

• Miller Christopher K
• Negrete-Raymond Ana
• Veldhouse Jon
• Vas Amit

Classifications

• C12Y301/03002

  Acid phosphatase (3.1.3.2) · CPC title

• C12P19/14

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

• C12P7/06

  Ethanol, i.e. non-beverage · CPC title

• C12N9/2428 Primary

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

• C12N9/16

  acting on ester bonds (3.1) · CPC title