# US 2019/0169586 A1 · Lactic acid-utilizing bacteria genetically modified to secrete polysaccharide-degrading enzymes

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- Number: US 2019/0169586 A1
- Document ID: US20190169586A1
- Status: Published application
- Kind: A1
- Application number: 16/068,410
- Filed: 2017-01-11
- Published: 2019-06-06
- Earliest claimed: 2016-01-11
- Assignees: 3Plw Ltd.
- Applicants: 3Plw Ltd.
- Inventors: Tal SHAPIRA; Amir ORANIM
- Web page: https://patentgrep.com/patent/US20190169586A1

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C02F 3/34: characterised by the microorganisms used
- C12N 1/38: Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound (C12N1/34 takes precedence)
- C12N 9/2411: {Amylases}
- C12N 9/2477: {Hemicellulases not provided in a preceding group}
- C12P 7/56: Lactic acid
- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C02F 2103/32: from the food or foodstuff industry, e.g. brewery waste waters
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C02F: TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE (separation in general B01D; special arrangements on waterborne vessels of installations for treating water, waste water or sewage, e.g. for producing fresh water, B63J; adding materials to water to prevent corrosion C23F; treating radioactively-contaminated liquids G21F9/04; regeneration of reactants for recirculation into processes, see the relevant places for the processes)
- 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)}

## Abstract

Lactic acid (LA)-utilizing bacteria are provided, genetically modified to express and optionally secrete polysaccharide-degrading enzymes, such as cellulases, hemicellulases and amylases, and uses thereof. The polysaccharide-degrading enzymes are advantageous for processing organic waste so that the organic waste can be used as a substrate in industrial fermentation processes, particularly industrial production of discrete lactic acid enantiomer(s). Vectors and constructs useful for genetically modifying the LA-utilizing bacteria are also provided.

## Claims

### Claim at position 1

-45. (canceled)

### Claim 46

A lactic acid (LA)-utilizing bacterium genetically modified to produce one or more exogenous polysaccharide-degrading enzyme, wherein the one or more exogenous polysaccharide-degrading enzyme comprises a cellulase, a hemicellulase, an amylase or combinations thereof.

### Claim 47 (depends on 46)

The LA-utilizing bacterium of claim 46, wherein the polysaccharide-degrading enzyme is from a thermophilic bacterium selected from the group consisting of Clostridium sp., Paenibacillus sp., Thermobifida fusca, Bacillus sp., Geobacillus sp., Chromohalobacter sp. and Rhodothermus marinus.

### Claim 48 (depends on 46)

The LA-utilizing bacterium of claim 46, wherein the polysaccharide-degrading enzyme is from a mesophilic bacterium is selected from the group consisting of Klebsiella sp., Cohnella sp., Streptomyces sp, Acetivibrio cellulolyticus, Ruminococcus albus; Bacillus sp. and Lactobacillus fermentum.

### Claim 49 (depends on 46)

The LA-utilizing bacterium of claim 46, wherein the polysaccharide-degrading enzyme is from a fungus selected from the group consisting of Trichoderma reesei, Humicola insolens, Fusarium oxysporum, Aspergillus oryzae, Penicillium fellutanum and Thermomyces lanuginosu.

### Claim 50 (depends on 46)

The LA-utilizing bacterium of claim 46, wherein the LA-utilizing bacterium is selected from the group consisting of Propionibacterium species, Megasphaera species, Selenomonas species and Veillonella species.

### Claim 51 (depends on 46)

The LA-utilizing bacterium of claim 46, wherein the LA-utilizing bacterium is Propionibacterium freudenreichii.

### Claim 52

A population of lactic-acid (LA)-utilizing bacteria genetically modified to produce a plurality of exogenous polysaccharide-degrading enzymes comprising a cellulase, a hemicellulase, an amylase or combinations thereof, wherein the population comprises a plurality of sub-populations of LA-utilizing bacteria, each sub-population being genetically modified to produce a different polysaccharide-degrading enzyme.

### Claim 53 (depends on 52)

The population of LA-utilizing bacteria of claim 52, wherein the LA-utilizing bacteria comprise Propionibacterium freudenreichii.

### Claim 54 (depends on 46)

A method for processing organic waste, the method comprising contacting the organic waste with the LA-utilizing bacterium of claim 46 under conditions in which lactic acid is consumed by the LA-utilizing bacteria and which are suitable for expression and activity of the polysaccharide-degrading enzymes, wherein said processing eliminates D-lactic acid, L-lactic acid or both from the organic waste and degrades polysaccharides in the waste to release soluble reducing sugars.

### Claim 55 (depends on 54)

The method of claim 54, wherein elimination of lactic acid from the waste is effected concomitant with degradation of polysaccharides into soluble reducing sugars.

### Claim 56 (depends on 54)

The method of claim 54, wherein elimination of lactic acid from the waste is effected prior to degradation of polysaccharides into soluble reducing sugars.

### Claim 57 (depends on 56)

The method of claim 56, wherein the polysaccharide-degrading enzymes have suitable activity at a temperature higher than the suitable growth temperature of the LA-utilizing bacteria, and wherein the method comprises: (i) contacting the organic waste with the LA-utilizing bacteria at a first temperature, the first temperature being suitable for growth of the LA-utilizing bacteria and expression of the polysaccharide-degrading enzymes, for sufficient time to eliminate D-lactic acid, L-lactic acid or both from the waste; and (ii) increasing the temperature to a second temperature, the second temperature being suitable for activity of the polysaccharide-degrading enzymes.

### Claim 58 (depends on 56)

The method of claim 56, wherein the polysaccharide-degrading enzymes have suitable activity at a pH higher or lower than the suitable growth pH of the LA-utilizing bacteria, and wherein the method comprises: (i) contacting the organic waste