# US 11,306,330 B2 · Glucoamylase engineered yeast and fermentation methods

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- Number: US 11,306,330 B2
- Document ID: US11306330B2
- Status: Granted
- Kind: B2
- Application number: 16/976,550
- Filed: 2019-02-27
- Published: 2021-01-07
- Granted: 2022-04-19
- Earliest claimed: 2018-02-28
- Assignees: Cargill, Incorporated
- Applicants: Cargill, Incorporated
- Inventors: Chris Miller; Brian Rush; Brynne Stanton; Joshua Dunn
- Web page: https://patentgrep.com/patent/US11306330B2

## CPC

- C12P 7/10: substrate containing cellulosic material
- C12N 9/2428: {Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase}
- C12N 1/185: Saccharomyces isolates
- C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
- C12R 2001/865: Saccharomyces cerevisiae
- C12Y 302/01003: Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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

### Claim 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.

### Claim 10 (depends on 9)

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.

### Claim 11 (depends on 9)

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.

### Claim 12 (depends on 9)

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

### Claim 13 (depends on 9)

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.