# US 10,190,101 B2 · Production of lactic acid from organic waste or biogas or methane using recombinant methanotrophic bacteria

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- Number: US 10,190,101 B2
- Document ID: US10190101B2
- Status: Granted
- Kind: B2
- Application number: 15/303,188
- Filed: 2015-04-13
- Published: 2017-04-27
- Granted: 2019-01-29
- Earliest claimed: 2014-04-11
- Assignees: String Bio Private Limited
- Applicants: String Bio Private Limited
- Inventors: Ezhilkani Subbian
- Web page: https://patentgrep.com/patent/US10190101B2

## CPC

- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 1/00: Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12P 7/56: Lactic acid
- C12Y 101/01027: L-Lactate dehydrogenase (1.1.1.27)
- 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)}
- C12Y: ENZYMES

## Abstract

The present disclosure provides for production of lactic acid from organic waste or biogas or methane using recombinant methanotrophic bacteria. In one embodiment, the recombinant methanotrophic bacterium includes a heterologous nucleic acid encoding for lactate dehydrogenase (ldh) enzyme. In a further embodiment, the capacity of the recombinant methanotrophic bacterium for producing lactic acid over basal level is increased by overexpression or/and down-regulation or deletion of specified genes. In another embodiment, a process for producing lactic acid from organic waste using the recombinant methanotrophic bacterium is disclosed. The present disclosure provides a cradle to cradle environment-friendly and commercially viable solution for managing organic waste.

## Claims

### Claim 1

A recombinant methanotrophic bacterium capable of producing lactic acid from organic waste or biogas or methane, comprising
a heterologous nucleic acid or gene encoding for lactate dehydrogenase (ldh) enzyme, having the sequence of SEQ ID NO: 1, 3, or 5, or a combination thereof.

### Claim 2 (depends on 1)

The recombinant methanotrophic bacterium as claimed in claim 1, wherein the bacterium is selected from the group consisting of Methylococcus capsulatus, Methylobacterium extorquens, Methylobacterium organophilum, Methylobacterium mesophilicum, Methylobacterium dichloromethanicum, Methylocella silvestris, Methylosinus trichosporium, Methylobacillus flagellatus KT, Methylibium petroleiphilum PM1, Methylobacterium nodulans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylacidiphilum infernorum V4, Methylophilus methylotrophus, Methylomonas methanica, Methylobacterium rhodesianum MB 126, Methylobacter tundripaludum, Methylobacterium sp. 4-46, Methylovorus glucosetrophus SIPS-4, Mycobacterium smegmatis, Methylobacterium rhodesianum, Methylosinus sporium, Methylosinus trichosporium Ob3b, Methylosinus sp strains, Methylocella palustris, Methylobacterium fujisawaense, Methylocystis parvus, Methylovulum miyakonense, Methylobacterium rhodinum, Methylocystis echinoides, Methylomonas rubra, Methylococcus thermophilus, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidithiobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum, Methylobacterium podarium, Methylobacter whittenburyi, Crenothrix polyspora, Clonothrix fusca, Methylobacter bovis, Methylomonas aurantiaca, Methylomonas fodinarum, Methylobacterium variabile, Methylocystis minimus, Methylobacter vinelandii, Methylobacterium hispanicum, Methylomicrobium japanense, Methylococcaceae bacterium, and Methylocystis methanolicus.

### Claim 3 (depends on 1)

The recombinant methanotrophic bacterium as claimed in claim 1, wherein the recombinant methanotrophic bacterium further comprises one or more genes for encoding enzyme(s) which are overexpressed selected from the group consisting of glyceraldehyde 3-phosphate dehydrogenase, glucose-6-phosphate isomerase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, glycerate 2-kinase, hexulose-6-phosphate synthase, Transaldolase, 6-phosphofructokinase, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase, enolase, ribulose phosphate 3-epimerase and methanol dehydrogenase or any combination thereof; genes for encoding enzymes(s) which are down-regulated or deleted selected from the group consisting of acetate kinase, acetate synthase, succinyl CoA synthetase, and malate dehydrogenase or any combination thereof, for increasing production of the lactic acid.

### Claim 4 (depends on 3)

The recombinant methanotrophic bacterium as claimed in claim 3, wherein the gene(s) encoding enzyme(s), responsible for the increased production of lactic acid by overexpression is the sequence of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29, or any combination thereof and wherein their corresponding amino acid sequence is SEQ ID NOS: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, respectively.

### Claim 5 (depends on 1)

The recombinant methanotrophic bacterium as claimed in claim 1, wherein the heterologous nucleic acid or gene encoding for the lactate dehydrogenase enzyme is from a micro-organism selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, and Pectobacterium carotovorum.

### Claim 6 (depends on 1)

The recombinant methanotrophic bacterium as claimed in claim 1, wherein the lactic acid produced from methane or biogas or organic waste is at least 5 fold higher than that by a wildtype strain under similar conditions.

### Claim 7 (depends on 1)

A process for producing lactic acid from organic waste using the recombinant methanotrophic bacterium as claimed in claim 1, said process comprising acts of:
a) receiving organic waste as input;
b) anaerobically digesting the organic waste to biogas;
c) culturing the bacterium in the biogas so generated, for converting the biogas to the lactic acid; and
d) optionally purifying or separating the lactic acid produced from the culture for obtaining the lactic acid.

### Claim 8 (depends on 7)

The process as claimed in claim 7, wherein the biogas produced by anaerobically digesting the organic waste is optionally cleaned to remove carbon dioxide and other impurities present in the biogas to obtain methane for producing lactic acid from methane thereafter.

### Claim 9 (depends on 1)

A process for producing lactic acid from a carbon source using recombinant methanotrophic bacterium as claimed in claim 1, said process comprising acts of:
a) receiving at least one carbon source selected from the group consisting of biogas or methane as input;
b) culturing the bacterium in the input, thereby converting the input into lactic acid; and
c) optionally purifying or separating the lactic acid produced from the culture for obtaining the lactic acid.

### Claim 10 (depends on 9)

The process as claimed in claim 9, wherein the input carbon source has a ratio of methane to carbon dioxide ranging from about 95:5 to about 50:50.

### Claim 11 (depends on 7)

The process as claimed in claim 7, wherein temperature maintained for the act of converting methane or biogas to lactic acid is in the range of about 37° C. to about 50° C., and wherein pH maintained during the act of converting methane or biogas to lactic acid is in the range of about 3 to about 7.

### Claim 12 (depends on 7)

The process as claimed in claim 7, wherein dissolved oxygen concentration for the act of converting methane or biogas to lactic acid is about <20%.