# US 2023/0265463 A1 · Limiting yeast-produced trehalose in fermentation

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- Number: US 2023/0265463 A1
- Document ID: US20230265463A1
- Status: Published application
- Kind: A1
- Application number: 18/183,669
- Filed: 2023-03-14
- Published: 2023-08-24
- Earliest claimed: 2015-11-06
- Assignees: Lallemand Hungary Liquidity Management LLC
- Applicants: Lallemand Hungary Liquidity Management LLC
- Inventors: Charles F. Rice; Ryan Skinner; Trisha Barrett; Aaron Argyros
- Web page: https://patentgrep.com/patent/US20230265463A1

## CPC

- C12P 7/10: substrate containing cellulosic material
- C12N 1/18: Baker's yeast; Brewer's yeast
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 15/81: for yeasts
- C12P 7/20: Glycerol
- C12Y 302/01028: Alpha,alpha-trehalase (3.2.1.28)
- C12P 19/12: Disaccharides
- 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)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present disclosure relates to recombinant yeast host cells having (i) a first genetic modification for reducing the production of one or more native enzymes that function to produce glycerol or regulating glycerol synthesis and/or allowing the production of an heterologous glucoamylase and (ii) a second genetic modification for reducing the production of one or more native enzymes that function to produce trehalose or regulating trehalose synthesis and/or allowing the expression of an heterologous trehalase. The recombinant yeast host cells can be used to limit the production of (yeast-produced) trehalose (particularly extracellular trehalose) during fermentation and, in some embodiments, can increase the production of a fermentation product (such as, for example, ethanol).

## Claims

### Claim 1

A method of increasing the production of a fermentation product during a fermentation, said method comprising contacting a medium with (i) a recombinant yeast host cell comprising a first genetic modification for expressing a heterologous STL1 protein and (ii) a trehalase, wherein the production of the fermentation product is increased when compared to a control method with a control yeast host cell lacking the first genetic modification.

### Claim 2 (depends on 1)

The method of claim 1, wherein the recombinant yeast host cell comprises a further genetic modification for reducing the production of one or more native enzymes that function to produce glycerol.

### Claim 3 (depends on 2)

The method of claim 2, wherein the recombinant yeast host cell expresses less of a native GPD1 polypeptide than a control yeast host cell lacking the further genetic modification.

### Claim 4 (depends on 2)

The method of claim 2, wherein the recombinant yeast host cell lacks the ability to produce a native GPD1 polypeptide.

### Claim 5 (depends on 2)

The method of claim 2, wherein the recombinant yeast host cell expresses less of a native GPD2 polypeptide than a control yeast host cell lacking the further genetic modification.

### Claim 6 (depends on 2)

The method of claim 2, wherein the recombinant yeast host cell lacks the ability to produce a native GPD2 polypeptide.

### Claim 7 (depends on 1)

The method of claim 1, wherein the recombinant yeast host cell is from the genus Saccharomyces sp.

### Claim 8 (depends on 7)

The method of claim 7, wherein the recombinant yeast host cell is from the species Saccharomyces cerevisiae.

### Claim 9 (depends on 1)

The method of claim 1, wherein the trehalase is extracellular from the recombinant yeast host cell.

### Claim 10 (depends on 1)

The method of claim 1, wherein the trehalase is provided in a purified form.

### Claim 11 (depends on 1)

The method of claim 1, wherein the trehalase belongs to E.C. 3.1.2.1.28.

### Claim 12 (depends on 1)

The method of claim 1, wherein the trehalase is an acid trehalase.

### Claim 13 (depends on 1)

The method of claim 1, wherein the trehalase is a neutral trehalase.

### Claim 14 (depends on 1)

The method of claim 1, wherein the medium comprises trehalose.

### Claim 15 (depends on 1)

The method of claim 1, wherein the medium comprises starch.

### Claim 16 (depends on 15)

The method of claim 15, wherein the starch is provided in a gelatinized or a raw form.

### Claim 17 (depends on 1)

The method of claim 1, wherein the medium is derived from corn.

### Claim 18

A combination comprising (i) a recombinant yeast host cell comprising a first genetic modification for expressing a heterologous STL1 protein and (ii) a trehalase.