# US 11,111,474 B2 · Bacterial strain producing 2,3-butanediol and other metabolites

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- Number: US 11,111,474 B2
- Document ID: US11111474B2
- Status: Granted
- Kind: B2
- Application number: 16/316,961
- Filed: 2017-07-18
- Published: 2019-08-01
- Granted: 2021-09-07
- Earliest claimed: 2016-07-19
- Assignees: Fundacion Tecnalia Research & Innovation
- Applicants: Fundacion Tecnalia Research & Innovation
- Inventors: Tomás Roncal Martínez; Susana Caballero Román; María Del Mar Díaz De Guereñu Zabarte; Inés Rincón Arroyo; Soraya Prieto Fernández; José Ramón Ochoa Gómez
- Web page: https://patentgrep.com/patent/US11111474B2

## CPC

- C12N 1/205: Bacterial isolates
- C12N 1/20: Bacteria; Culture media therefor
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/03: Bacteria
- C12P 7/16: Butanols
- C12P 7/18: polyhydric
- C12P 7/26: Ketones
- C12P 7/56: Lactic acid
- C12R 2001/46: Streptococcus {; Enterococcus; Lactococcus}
- 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)}
- 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 present invention provides a modified strain of the bacterium Lactococcus lactis obtainable by a method that comprises a step of fusion of two protoplasts from two Lactococcus lactis parental strains which, compared to the wild type strain of Lactococcus lactis from which they derive, show: (a) an increased ability to produce acetoin and/or 2,3-butanediol (2,3-BDO), and (b) a decreased ability to produce lactic acid, when cultured under aerobic conditions, and wherein the modified strain of Lactococcus lactis has an increased ability to produce 2,3-BDO of at least 20 times the amount produced by the wild type strain, an increased ability to produce acetoin of at least 20 times the amount produced by the wild type strain and a decreased ability to produce lactic acid of at least 10 times the amount produced by the wild type strain, when cultured under aerobic conditions. Also provided are methods to produce acetoin and 2,3-BDO.

## Claims

### Claim 1

A Lactococcus lactis strain 43103 deposited at the Spanish Type Culture Collection under accession number CECT 9139, wherein said strain has an increased ability to produce acetoin and 2,3-butanediol and a decreased ability to produce lactic add when cultured under aerobic conditions.

### Claim 2 (depends on 1)

A method of production of 2,3-butanediol that comprises the aerobic fermentation of a carbohydrate-containing culture medium by the bacterial strain as defined in claim 1.

### Claim 3 (depends on 2)

The method of 2,3-butanediol production according to claim 2, comprising the following steps: (a) Pre-culture of the Lactococcus lactis strain 43103 deposited at the Spanish Type Culture Collection under accession number CECT 9139; (b) Inoculation of a carbohydrate-containing culture medium with the pre-culture obtained in step (a); (c) Fermentation of carbohydrates present in the culture medium inoculated in step (b) at 20-40° C.; pH 5.5-6.0 and 5-100% dissolved oxygen concentration; and (d) Separation of cells from the fermented broth resulting from step (c).

### Claim 4 (depends on 3)

The method of 2,3-butanediol production according to claim 3, further comprising a step of purification of the 2,3-butanediol present in the cell-free fermentation broth resulting from step (d).

### Claim 5 (depends on 1)

A method of production of acetoin, that comprises the aerobic fermentation of a carbohydrate-containing culture medium by the bacterial strain as defined in claim 1.

### Claim 6 (depends on 5)

The method of acetoin production according to claim 5, comprising the following steps: (a) Pre-culture of the Lactococcus lactis strain 43103 strain deposited at the Spanish Type Culture Collection under accession number CECT 9139; (b) Inoculation of a carbohydrate-containing culture medium with the pre-culture obtained in step (a); (c) Fermentation of carbohydrates present in the culture medium inoculated in step (b) at 20-40° C.; pH 6.5-7.5 and 30-100% dissolved oxygen concentration; and (d) Separation of cells from the fermented broth resulting from step (c).

### Claim 7 (depends on 6)

The method of acetoin production according to claim 6, further comprising a step of purification of the acetoin present in the cell-free fermentation broth resulting from step (d).

### Claim 8 (depends on 1)

A method of production of lactic add that comprises the anaerobic fermentation of a carbohydrate-containing culture medium by the bacterial strain as defined in claim 1.