# US 11,999,987 B2 · Bacterial cocultures expressing a bacteriocin system

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- Number: US 11,999,987 B2
- Document ID: US11999987B2
- Status: Granted
- Kind: B2
- Application number: 17/206,943
- Filed: 2021-03-19
- Published: 2021-09-23
- Granted: 2024-06-04
- Earliest claimed: 2020-03-19
- Assignees: Lallemand Hungary Liquidity Management LLC
- Applicants: Lallemand Hungary Liquidity Management LLC
- Inventors: James L. Steele; Brooks Henningsen; Jeffery R. Broadbent; Ekkarat Phrommao; Fernanda Cristina Firmino
- Web page: https://patentgrep.com/patent/US11999987B2

## CPC

- C12P 39/00: Processes involving microorganisms of different genera in the same process, simultaneously
- C07K 14/335: from Lactobacillus (G)
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/88: Lyases (4.)
- C12P 7/06: Ethanol, i.e. non-beverage
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Y 101/01001: Alcohol dehydrogenase (1.1.1.1)
- C12Y 401/01001: Pyruvate decarboxylase (4.1.1.1)
- 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)}
- 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)
- 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 concerns a co-culture of bacterial cells for making a fermented product from a biomass. The co-culture comprising a first recombinant lactic acid bacteria (LAB) cell expressing at least one bacteriocin and a second recombinant lactic acid bacteria (LAB) cell capable of converting, at least in part, the biomass into the fermented product. The second recombinant LAB cell is immune to the bacteriocin produced by the first recombinant LAB cell. The co-culture can be used, optionally in combination with a yeast host cell, to make a fermented product. The present disclosure also provides processes for making the fermented product by using the co-culture as wells kits and media comprising the co-culture.

## Claims

### Claim 1

A co-culture of lactic acid bacterial cells for making ethanol from a biomass, the co-culture comprising:
a first recombinant lactic acid bacteria (LAB) cell:
expressing at least one bacteriocin; and
comprising heterologous nucleic acid molecules encoding polypeptides for converting the biomass into ethanol;
and
a second recombinant LAB cell:
capable of converting the biomass into ethanol; comprising heterologous nucleic acid molecules encoding polypeptides for converting the biomass into ethanol;
and comprising one or more heterologous nucleic acid molecules encoding one or more polypeptides for conferring immunity against the at least one bacteriocin expressed by the first recombinant LAB cell
wherein the polypeptides for converting the biomass into ethanol comprise at least:
a pyruvate decarboxylase; and
an alcohol dehydrogenase,
wherein the at least one bacteriocin comprises a lantibiotic, a cyclic bacteriocin, a Class IIA bacteriocin, a Class IIB bacteriocin, or a combination thereof.

### Claim 2 (depends on 1)

The co-culture of claim 1, wherein the Lantibiotic is nisin and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes NisI; the cyclic bacteriocin is gassericin and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes GaaI; the cyclic bacteriocin is plantaricyclin A and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes PlcD and/or PlcI; the Class IIA bacteriocin is plantaricin 423 and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes PlaB; the Class IIA bacteriocin is pediocin and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes PedB; the Class IIA bacteriocin is lactoccin A and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes LciA; the Class IIA bacteriocin is horediocin A and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes HdrI; or the Class IIB bacteriocin is brochocin and the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin encodes BrcI.

### Claim 3 (depends on 2)

The co-culture of claim 2, wherein nisin has the amino acid sequence of any one of SEQ ID NOS: 7 to 10, is a variant of the amino acid sequence of any one of SEQ ID NOS: 7 to 10 having nisin bacteriocin activity or is a fragment of the amino acid sequence of any one of SEQ ID NOS: 7 to 10 having nisin bacteriocin activity.

### Claim 4 (depends on 2)

The co-culture of claim 2, wherein NisI has the amino acid sequence of SEQ ID NO: 11, is a variant of the amino acid sequence of SEQ ID NO: 11 conferring immunity against nisin or is a fragment of the amino acid sequence of SEQ ID NO: 11 conferring immunity against nisin.

### Claim 5 (depends on 2)

The co-culture of claim 2, wherein the heterologous nucleic acid molecule encoding one or more polypeptides for conferring immunity against the at least one bacteriocin further encodes NisE, NisF and/or NisG.

### Claim 6 (depends on 5)

The co-culture of claim 5, wherein NisE has the amino acid sequence of SEQ ID NO: 13, is a variant of the amino acid sequence of SEQ ID NO: 13 having nisin transporter activity or is a fragment of the amino acid sequence of SEQ ID NO: 13 having nisin transporter activity.

### Claim 7 (depends on 5)

The co-culture of claim 5, wherein NisF has the amino acid sequence of SEQ ID NO: 12, is a variant of the amino acid sequence of SEQ ID NO: 12 having nisin transporter activity or is a fragment of the amino acid sequence of SEQ ID NO: 12 having nisin transporter activity.

### Claim 8 (depends on 5)

The co-culture of claim 5, wherein NisG has the amino acid sequence of SEQ ID NO: 14, is a variant of the amino acid sequence of SEQ ID NO: 14 having nisin permease activity or is a fragment of the amino acid sequence of SEQ ID NO: 14 having nisin permease activity.

### Claim 9 (depends on 1)

The co-culture of claim 1, wherein the first recombinant LAB cell expresses a plurality of bacteriocins.

### Claim 10 (depends on 1)

The co-culture of claim 1, wherein the pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 1, is a variant of the amino acid sequence of SEQ ID NO: 1 having pyruvate decarboxylase activity or is a fragment of the amino acid sequence of SEQ ID NO: 1 having pyruvate decarboxylase activity.

### Claim 11 (depends on 1)

The co-culture of claim 1, wherein the alcohol dehydrogenase has the amino acid sequence of SEQ ID NO: 3, is a variant of the amino acid sequence of SEQ ID NO: 3 having alcohol dehydrogenase activity or is a fragment of the amino acid sequence of SEQ ID NO: 3 having alcohol dehydrogenase activity.

### Claim 12 (depends on 1)

The co-culture of claim 1, wherein the first recombinant LAB cell and/or the second recombinant LAB cell comprises:
at least one inactivated or deleted native gene coding for a lactate dehydrogenase;
inactivated or deleted native ldh1, ldh2, ldh3, ldh4 and dhic genes;
at least one inactivated or deleted native gene coding for a mannitol-1-phosphate 5-dehydrogenase; and/or
inactivated or deleted native mltD1 and mltD2 genes.

### Claim 13 (depends on 1)

The co-culture of claim 1, wherein the first recombinant LAB cell is from the genus Lactococcus or from the species Lactococcus lactis.

### Claim 14 (depends on 1)

The co-culture of claim 1, wherein the second recombinant LAB cell is from the genus Lactobacillus or from the species Lactobacillus paracasei.

### Claim 15 (depends on 1)

A combination comprising the co-culture of claim 1 and a yeast cell.

### Claim 16 (depends on 1)

A process for converting a biomass into a fermentation product, wherein the process comprises contacting the biomass with the co-culture of claim 1 under conditions to allow the conversion of the biomass into said fermentation product of claim 1.

### Claim 17 (depends on 16)

The process of claim 16, wherein the biomass comprises corn.

### Claim 18 (depends on 16)

The process of claim 16, wherein said fermentation product is ethanol.

### Claim 19 (depends on 1)

The co-culture of claim 1, wherein the at least one bacteriocin is native to the first recombinant LAB cell.

### Claim 20 (depends on 1)

The co-culture of claim 1, wherein the first recombinant LAB cell further comprises a heterologous nucleic acid molecule encoding the at least one bacteriocin and one or more polypeptides for conferring immunity against the at least one bacteriocin.

### Claim 21 (depends on 15)

The combination of claim 15 wherein the yeast cell is from the genus Saccharomyces sp.

### Claim 22 (depends on 15)

The combination of claim 15 wherein the yeast cell is from the species Saccharomyces cerevisiae.