# US 10,308,963 B2 · Processes of producing ethanol using a fermenting organism

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- Number: US 10,308,963 B2
- Document ID: US10308963B2
- Status: Granted
- Kind: B2
- Application number: 15/553,206
- Filed: 2016-02-26
- Published: 2018-03-15
- Granted: 2019-06-04
- Earliest claimed: 2015-02-27
- Assignees: Novozymes A/S; Microbiogen Pty. Ltd.
- Applicants: Novozymes A/S; Microbiogen Pty. Ltd.
- Inventors: Jennifer Headman; Jeremy Saunders; Ryan Schron; Paul Victor Attfield; Philip John Livingstone Bell
- Web page: https://patentgrep.com/patent/US10308963B2

## CPC

- C12P 7/06: Ethanol, i.e. non-beverage
- C12N 1/18: Baker's yeast; Brewer's yeast
- C12N 1/185: Saccharomyces isolates
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12R 2001/865: Saccharomyces cerevisiae
- 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
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention relates to improved processes of producing ethanol from starch-containing material wherein saccharification and/or fermentation is done at a temperature below the initial gelatinization temperature in the presence of glucoamylase and alpha-amylase, and optionally a protease and/or a cellulolytic enzyme composition; wherein the fermenting organism is a Saccharomyces yeast strain providing a higher ethanol yield boost and lower glycerol production compared to ETHANOL RED™ under the same fermentation conditions. The invention also relates to Saccharomyces yeast strains and derivatives thereof, as well as compositions comprising such yeast strains, suitable for use in a process of the invention.

## Claims

### Claim 1

A process of producing ethanol from starch-containing material, the process comprising:
(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature; and
(b) fermenting using a fermentation organism;
wherein saccharification and/or fermentation is done in the presence of a glucoamylase and an alpha-amylase;
wherein the fermenting organism is:
Saccharomyces cerevisiae MBG4911 (deposited as V15/001459 at National Measurement Institute, Victoria, Australia);
Saccharomyces cerevisiae MBG4913 (deposited as V15/001460 at National Measurement Institute, Victoria, Australia); or
Saccharomyces cerevisiae MBG4914 (deposited as V15/001461 at National Measurement Institute, Victoria, Australia).

### Claim 2 (depends on 1)

The process of claim 1, wherein the fermenting organism is capable of growing on xylose as a sole carbon source.

### Claim 3 (depends on 1)

The process of claim 1, wherein the fermenting organism provides an ethanol yield boost of at least 0.5% after 88 hours compared to the ETHANOL RED™ Saccharomyces cerevisiae strain deposited as V14/007039 at National Measurement Institute, Victoria, Australia under the same conditions.

### Claim 4 (depends on 1)

The process of claim 1, wherein the fermenting organism provides a lower glycerol production of at least 5% after 88 hours compared to the ETHANOL RED™ Saccharomyces cerevisiae strain deposited as V14/007039 at National Measurement Institute, Victoria, Australia under the same conditions.

### Claim 5

A Saccharomyces yeast strain selected from:
Saccharomyces cerevisiae MBG4911 (deposited as V15/001459 at National Measurement Institute, Victoria, Australia);
Saccharomyces cerevisiae MBG4913 (deposited as V15/001460 at National Measurement Institute, Victoria, Australia); and
Saccharomyces cerevisiae MBG4914 (deposited as V15/001461 at National Measurement Institute, Victoria, Australia).

### Claim 6 (depends on 5)

The yeast strain of claim 5, wherein the strain is capable of growing on xylose as a sole carbon source.

### Claim 7 (depends on 5)

The yeast strain of claim 5, wherein the strain is capable of providing an ethanol yield boost of at least 0.5% after 88 hours compared to the ETHANOL RED™ Saccharomyces cerevisiae strain deposited as V14/007039 at National Measurement Institute, Victoria, Australia under the same conditions.

### Claim 8 (depends on 5)

The yeast strain of claim 5, wherein the strain is capable of providing a lower glycerol production of at least 5% after 88 hours compared to the ETHANOL RED™ Saccharomyces cerevisiae strain deposited as V14/007039 at National Measurement Institute, Victoria, Australia under the same conditions.

### Claim 9

A method of producing a derivative of strain Saccharomyces cerevisiae MBG4911, MBG4913 or MBG4914, the method comprising:
(a) providing:
(i) a first yeast strain; and
(ii) a second yeast strain, wherein the second yeast strain is strain Saccharomyces cerevisiae MBG4911, MBG4913 or MBG4914;
(b) culturing the first yeast strain and the second yeast strain under conditions which permit combining of DNA between the first and second yeast strains;
(c) screening or selecting for a derivative strain.

### Claim 10 (depends on 9)

The method of claim 9, wherein step (c) comprises screening or selecting for a hybrid strain which exhibits one or more defining characteristic of strain Saccharomyces cerevisiae MBG4911, MBG4913 or MBG4914.

### Claim 11 (depends on 9)

The method of claim 9, comprising the further step of:
(d) repeating steps (b) and (c) with the screened or selected strain from step (c) as the first and/or second strain, until a derivative is obtained which exhibits the defining characteristics of strain Saccharomyces cerevisiae MBG4911, MBG4913 or MBG4914.

### Claim 12 (depends on 9)

The method of claim 9, wherein the culturing step (b) comprises:
(i) sporulating the first yeast strain and the second yeast strain;
(ii) hybridizing germinated spores produced by the first yeast strain with germinated spores produced by the second yeast strain.

### Claim 13 (depends on 5)

A method of producing ethanol, comprising incubating the Saccharomyces yeast strain of claim 5 with a substrate comprising a fermentable sugar under conditions which permit fermentation of the fermentable sugar to produce ethanol.

### Claim 14 (depends on 5)

A composition comprising a Saccharomyces yeast strain of claim 5 and one or more components selected from surfactants, emulsifiers, gums, swelling agents, and antioxidants.

### Claim 15 (depends on 5)

A method of producing a recombinant derivative of the Saccharomyces yeast of claim 5, the method comprising introducing a nucleic acid into the Saccharomyces yeast of claim 5 using recombinant DNA technology.

### Claim 16 (depends on 15)

The method of claim 15, wherein the nucleic acid is introduced into Saccharomyces cerevisiae MBG4911 (deposited as V15/001459 at National Measurement Institute, Victoria, Australia).