Glucoamylase engineered yeast and fermentation methods

Patent metadata

Field	Value
Publication number	US-11306330-B2
Application number	US-201916976550-A
Country	US
Kind code	B2
Filing date	Feb 27, 2019
Priority date	Feb 28, 2018
Publication date	Apr 19, 2022
Grant date	Apr 19, 2022

Abstract

The invention is directed to an engineered yeast including an exogenous nucleic acid encoding a glucoamylase comprising SEQ ID NO:1 and SEQ ID NO:4, or a variant thereof. The engineered yeast are able to provide glucoamylase into a fermentation media and cause degradation of starch material generating glucose for fermentation to a desired bioproduct, such as ethanol. High titers of bioproduct (e.g., 70 g/kg of ethanol) can be achieved, along with low residual glucose levels. Further the yeast exhibit good growth and bioproduct product at temperatures of 32° C. or greater.

First claim

What is claimed is:

1. An engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 90% or greater sequence identity to SEQ ID NO:1, wherein the yeast is capable of producing ethanol at a rate of 1 g/L*h or greater during a fermentation process.

2. The engineered yeast of claim 1 , wherein the yeast is capable of producing at least 70 g/kg of ethanol in a fermentation medium made from a glucose polymer-containing feedstock having a dextrose equivalent (DE) of not greater than 50.

3. The engineered yeast of claim 1 , wherein the yeast is capable of producing at least 70 g/kg of ethanol in a fermentation medium made from corn mash having a DE of 30+/−2, wherein the fermentation medium comprises 32% dry wt corn, and a pH 5.8, 35 ppm CaCl, 1900 ppm urea, 5 ppm ampicillin, wherein the staring yeast concentration is 0.1 (OD600), and fermentation is carried out at 48 hrs at 30° C. with agitation.

4. The engineered yeast of claim 1 , wherein the glucoamylase comprises a sequence having 95% or greater sequence identity to SEQ ID NO:1.

5. The engineered yeast of claim 1 wherein there are 2-8 copies of the exogenous nucleic acid in the cell.

6. The engineered yeast of claim 1 which is a Saccharomyces cerevisiae yeast.

7. The engineered yeast of claim 1 which is tolerant to growth in a fermentation medium having a concentration of ethanol of greater than 90 g/L.

8. The engineered yeast of claim 1 which is tolerant to growth at temperatures in the range of greater than 31° C.-35° C.

9. A fermentation method for producing a bioproduct, comprising:
forming a fermentation medium from a glucose polymer-containing feedstock; and
fermenting the fermentation medium using an engineered yeast comprising an exogenous nucleic acid encoding a glucoamylase comprising a sequence having 90% or greater sequence identity to SEQ ID NO:1, wherein fermenting produces a bioproduct and ethanol at a rate of 1 g/L*h or greater.

10. The fermentation method of claim 9 , wherein the glucose polymer-containing feedstock or the fermentation medium, at the beginning of fermentation, has a DE of about 50 or less.

11. The fermentation method of claim 9 , wherein the glucose polymer-containing feedstock comprises glucose polymer having a degree of polymerization of 4 or greater and present in an amount of 75% weight or greater total fermentable carbohydrates in the feedstock.

12. The fermentation of any of claim 9 , wherein ethanol is produced to a concentration of 70 g/L or greater in the medium.

13. The fermentation method of any of claim 9 , comprising adding supplemental glucoamylase to the feedstock, or supplemental glucoamylase to the medium during the fermentation period.

Assignees

• Cargill Inc

Inventors

• Miller Chris
• Rush Brian
• Stanton Brynne
• Dunn Joshua

Classifications

• C12P2203/00

  Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source · CPC title

• C12Y302/01003

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

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12N9/2428 Primary

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

• C12P7/10 Primary

  substrate containing cellulosic material · CPC title