# US 11,028,402 B2 · Polypeptide and yeast cell compositions and methods of using the same

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- Number: US 11,028,402 B2
- Document ID: US11028402B2
- Status: Granted
- Kind: B2
- Application number: 16/378,106
- Filed: 2019-04-08
- Published: 2019-10-17
- Granted: 2021-06-08
- Earliest claimed: 2018-04-06
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Chris Todd Hittinger; EmilyClare Patricia Baker
- Web page: https://patentgrep.com/patent/US11028402B2

## CPC

- C12C 11/00: Fermentation processes for beer (preparation of wine C12G1/00)
- C12N 15/81: for yeasts
- C12G 1/0203: {by microbiological or enzymatic treatment}
- C12G 3/02: by fermentation
- C12N 1/16: Yeasts; Culture media therefor
- C12P 7/00: Preparation of oxygen-containing organic compounds
- C07K 14/395: from Saccharomyces
- C12C: BEER; PREPARATION OF BEER BY FERMENTATION (ageing or ripening by storing C12H1/22; methods for reducing the alcohol content after fermentation C12H3/00; methods for increasing the alcohol content after fermentation C12H6/00; venting devices for casks, barrels or the like C12L9/00); PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12G: WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES (beer C12C); PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- 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)}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Polypeptides comprising maltose/maltotriose transporters are provided. Additionally, polynucleotides, DNA constructs, and vectors encoding a maltose/maltotriose transporter, or yeast cells harboring such polynucleotides are provided. The yeast cell may be a Saccharomyces eubayanus cell modified to increase the expression or transport activity of a maltose/maltotriose transporter at the plasma membrane of the cell. Further, methods are provided for making a fermentation product by culturing any one of the yeast cells described herein with a fermentable substrate. Finally, methods are provided to select for and isolate maltotriose-utilizing strains of Saccharomyces eubayanus.

## Claims

### Claim 1

A polynucleotide encoding a polypeptide comprising at least 95% sequence identity to SEQ ID NO: 4 and comprising at least six amino acid substitutions to SEQ ID NO: 4 at positions selected from the group consisting of S468, I503, G504, N505, V508, I512, N522, F534, L536, V538, and I540; or a functional fragment of the polypeptide thereof, wherein the polypeptide or functional fragment thereof has maltotriose transporter activity.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the polynucleotide comprises SEQ ID NO: 9.

### Claim 3 (depends on 1)

A DNA construct comprising a promoter operably linked to the polynucleotide of claim 1.

### Claim 4 (depends on 3)

A vector comprising the DNA construct of claim 3.

### Claim 5 (depends on 1)

A Saccharomyces yeast cell comprising the polynucleotide of claim 1.

### Claim 6

A Saccharomyces yeast cell modified to increase as compared to a control yeast cell the expression or maltose/maltotriose transport activity of a maltose/maltotriose transporter polypeptide, wherein the yeast cell comprises a polynucleotide encoding a polypeptide having at least 95% sequence identity to SEQ ID NO: 4 and comprising at least six amino acid substitutions to SEQ ID NO: 4, wherein the amino acid substitutions are at positions selected from the group consisting of S468, I503, G504, N505, V508, I512, N522, F534, L536, V538, and I540, or a functional fragment thereof having maltotriose transporter activity.

### Claim 7 (depends on 6)

The yeast cell of claim 6, wherein the increased expression or increased maltose/maltotriose transport activity of the maltose/maltotriose transporter polypeptide is at the plasma membrane of the yeast cell.

### Claim 8 (depends on 6)

The yeast cell of claim 6, wherein the expression or maltose/maltotriose transport activity of the maltose/maltotriose transporter polypeptide is increased by at least 30% as compared to the control yeast cell.

### Claim 9 (depends on 6)

The yeast cell of claim 6, wherein the yeast cell is selected from the species consisting of Saccharomyces eubayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces arboricola, Saccharomyces kudriavzevii, Saccharomyces jurei, and Saccharomyces uvarum.

### Claim 10 (depends on 5)

A method for making a fermentation product comprising culturing the yeast cells of claim 5 with a fermentable substrate to produce the fermentation product.

### Claim 11 (depends on 10)

The method of claim 10, wherein the fermentable substrate comprises wort or malt extract.

### Claim 12 (depends on 10)

The method of claim 10, wherein the fermentation product is selected from the group consisting of a beer product, a wine product, an alcoholic beverage, a biochemical, and a biofuel.