# US 2019/0376071 A1 · Glucoamylase-modified yeast strains and methods for bioproduct production

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- Number: US 2019/0376071 A1
- Document ID: US20190376071A1
- Status: Published application
- Kind: A1
- Application number: 16/439,244
- Filed: 2019-06-12
- Published: 2019-12-12
- Earliest claimed: 2015-03-27
- Assignees: Cargill, Incorporated
- Applicants: Cargill, Incorporated
- Inventors: Christopher K. Miller; Ana Negrete-Raymond; Jon Veldhouse; Amit Vas
- Web page: https://patentgrep.com/patent/US20190376071A1

## CPC

- C12N 15/81: for yeasts
- C12P 7/10: substrate containing cellulosic material
- C12Y 302/01003: Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

Genetically engineered yeast with a heterologous glucoamylase and fermentation methods are described. The engineered yeast can have multiple exogenous nucleic acid sequences which each have a different sequence, but that encode the same or a similar glucoamylase protein that is heterologous to the yeast. The engineered yeast exhibit desirable bioproduct production profiles during a fermentation process. A fermentation medium with a starch material can be fermented with the engineered yeast to provide high ethanol titers, low glycerol titers, or both.

## Claims

### Claim at position 1

1-52. (canceled)

### Claim 53

An engineered host cell comprising at least first, second, third, and fourth exogenous nucleic acids each comprising a sequence encoding a common heterologous glucoamylase polypeptide, or encoding two or more different heterologous glucoamylase polypeptides having 90% or greater sequence identity to each other, wherein the first, second, third, and fourth exogenous nucleic acids have nucleic acid sequences that are different from one another.

### Claim 54 (depends on 53)

The host cell of claim 53, wherein the heterologous glucoamylase polypeptide(s) is from a yeast or fungal organism selected from the group consisting of Amorphotheca resinae, Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Aspergillus kawachii, Aspergillus shirousami, Blastobotrys adeninivorans, Candida albicans, Rhizopus oryzae, Schizosaccharomyces pombe, and Saccharomycopsis fibuligera.

### Claim 55 (depends on 53)

The host cell of claim 53, wherein the heterologous glucoamylase polypeptide(s) is an enzymatically active portion of a glucoamylase polypeptide from Saccharomycopsis fibuligera.

### Claim 56 (depends on 53)

The host cell of claim 53, wherein the glucoamylase polypeptide(s) has 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NO: 11.

### Claim 57 (depends on 53)

The host cell of claim 53, wherein the first, second, third, and fourth exogenous nucleic acids encode two or more different glucoamylase polypeptides having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to each other.

### Claim 58 (depends on 53)

The host cell of claim 53, wherein the host cell is a yeast is selected from the group consisting of Saccharomyces cerevisiae, Isaatchenkia orientalis, Candida utilis, Pichia Stipitis, Yarrowia lipolytica, Kluyveromyces marxiannus, and Kluyeromyces lactis.

### Claim 59 (depends on 53)

The host cell of claim 53, wherein the host cell can be used to make a bioproduct selected from the group consisting of alcohols, diols, polyols, fatty acids, monacyl glycerides, diacyl glycerides, triacyl glycerides, sweeteners, sophorolipids, and mixtures thereof.

### Claim 60 (depends on 53)

The host cell of claim 53, wherein the host cell can be used to make ethanol.

### Claim 61 (depends on 53)

The host cell of claim 53, wherein the host cell can be used to make an organic acid or an amino acid.

### Claim 62 (depends on 53)

The host cell of claim 53, wherein the host cell can be used to make lactic acid.

### Claim 63

An engineered host cell comprising two or more exogenous nucleic acids each comprising a sequence encoding a common heterologous glucoamylase polypeptide, or encoding two or more different glucoamylase polypeptides having 90% or greater sequence identity to each other, wherein the two or more exogenous nucleic acids have nucleic acid sequences that are different from one another.

### Claim 64 (depends on 63)

The host cell of claim 63, wherein the heterologous glucoamylase polypeptide(s) is from a yeast or fungal organism selected from the group consisting of Amorphotheca resinae, Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Aspergillus kawachii, Aspergillus shirousami, Blastobotrys adeninivorans, Candida albicans, Rhizopus oryzae, Schizosaccharomyces pombe, and Saccharomycopsis fibuligera.

### Claim 65 (depends on 63)

The host cell of claim 63, wherein the heterologous glucoamylase polypeptide(s) is an enzymatically active portion of a glucoamylase polypeptide from Saccharomycopsis fibuligera.

### Claim 66 (depends on 63)

The host cell of claim 63, wherein the glucoamylase polypeptide(s) has 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to SEQ ID NO: 11.

### Claim 67 (depends on 63)

The host cell of claim 63, wherein the first, second, third, and fourth exogenous nucleic acids encode two or more different glucoamylase polypeptides having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity to each other.

### Claim 68 (depends on 63)

The host cell of claim 63, wherein the host cell is a yeast is selected from the group consisting of Saccharomyces cerevisiae, Isaatchenkia orientalis, Candida utilis, Pichia Stipitis, Yarrowia lipolytica, Kluyveromyces marxiannus, and Kluyeromyces lactis.

### Claim 69 (depends on 63)

The host cell of claim 63, wherein the host cell can be used to make a bioproduct selected from the group consisting of alcohols, diols, polyols, fatty acids, monacyl glycerides, diacyl glycerides, triacyl glycerides, sweeteners, sophorolipids, and mixtures thereof.

### Claim 70 (depends on 63)

The host cell of claim 63, wherein the host cell can be used to make ethanol.

### Claim 71 (depends on 63)

The host cell of claim 63, wherein the host cell can be used to make an organic acid or an amino acid.

### Claim 72 (depends on 63)

The host cell of claim 63, wherein the host cell can be used to make lactic acid.