# US 10,480,016 B2 · Genetically engineered microorganisms for biological oxidation of hydrocarbons

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- Number: US 10,480,016 B2
- Document ID: US10480016B2
- Status: Granted
- Kind: B2
- Application number: 14/435,714
- Filed: 2013-10-15
- Published: 2015-08-20
- Granted: 2019-11-19
- Earliest claimed: 2012-10-15
- Assignees: Calysta, Inc.
- Applicants: Calysta, Inc.
- Inventors: Joshua A. Silverman; Drew D. Regitsky
- Web page: https://patentgrep.com/patent/US10480016B2

## CPC

- C12P 7/04: acyclic
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/0073: {with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13}
- C12P 7/06: Ethanol, i.e. non-beverage
- C12P 7/16: Butanols
- C12P 17/02: Oxygen as only ring hetero atoms
- Y02E 50/10: Biofuels, e.g. bio-diesel
- Y02P 20/582: Recycling of unreacted starting or intermediate materials
- 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)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present disclosure relates to genetically engineered microorganisms for biological oxidation of hydrocarbons, including production of alcohols from alkanes or epoxides from alkenes, and related methods and systems.

## Claims

### Claim 1

A non-naturally occurring bacterium, comprising:
a host methanotrophic bacterium having an exogenous nucleic acid encoding an enzyme with methane monooxygenase (MMO) activity,
wherein the MMO comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOS: 99-102, 174, 197, and 649-653;
wherein at least one alcohol dehydrogenase enzyme is inactivated in the non-naturally occurring bacterium as compared to the host methanotrophic bacterium; and
wherein the non-naturally occurring bacterium is capable of converting methane into methanol.

### Claim 2 (depends on 1)

The non-naturally occurring bacterium of claim 1, wherein the methanotrophic bacterium is selected from Methylococcus capsulatus Bath, Methylomonas sp. 16a, Methylosinus trichosporium OB3b, Methylosinus sporium, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus,Methylobacteriumorganophilum,Methylomonas sp. AJ-3670, or Methylomicrobium alcaliphilum.

### Claim 3 (depends on 1)

The non-naturally occurring bacterium of claim 1, wherein the bacterium is capable of utilizing H2 as a reducing agent for converting methane into methanol.

### Claim 4 (depends on 3)

The non-naturally occurring bacterium of claim 3, wherein the bacterium comprises a nucleic acid encoding a hydrogenase enzyme for utilizing H2 as a reducing agent for converting methane into methanol.

### Claim 5 (depends on 1)

The non-naturally occurring bacterium of claim 1, wherein the enzyme with methane monooxygenase activity comprises the amino acid sequence of SEQ ID NO: 174 or 197.

### Claim 6 (depends on 1)

The non-naturally occurring bacterium of claim 1, wherein the at least one alcohol dehydrogenase is inactivated by genetic modification.

### Claim 7 (depends on 1)

The non-naturally occurring bacterium of claim 1, wherein the at least one inactivated alcohol dehydrogenase includes methanol dehydrogenase.

### Claim 8 (depends on 1)

The non-naturally occurring bacterium of claim 1, wherein the at least one alcohol dehydrogenase comprises an amino acid sequence having at least 70% identity to any one of SEQ ID NOS: 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, and 665.

### Claim 9 (depends on 1)

The non-naturally occurring bacterium of claim 1, wherein the non-naturally occurring bacterium is capable of converting ethane into ethanol, propane into propanol, butane into butanol, ethylene into ethylene oxide, propylene into propylene oxide, butene into butene oxide, a mixed alkene substrate into a mixed epoxide product, or a mixed alkane gas substrate into a mixed alcohol product.

### Claim 10 (depends on 9)

The non-naturally occurring bacterium of claim 9, wherein the non-naturally bacterium is capable of converting a mixed alkane gas substrate into a mixed alcohol product and the mixed alkane gas substrate is wet natural gas or a partially separated derivative thereof.

### Claim 11 (depends on 1)

A method for converting methane into methanol, ethane into ethanol, propane into propanol, or butane into butanol, the method comprising:
exposing the non-naturally occurring bacterium of claim 1 or a cell-free fraction comprising the MMO and derived from the non-naturally occurring bacterium of claim 1 to: (a) O2; (b) a gas substrate comprising methane, ethane, propane, or butane, respectively; and (c) a reducing agent;
wherein the methane gas is converted to methanol, the ethane gas is converted to ethanol, the propane gas is converted to propanol, and the butane gas is converted to butanol, respectively.

### Claim 12 (depends on 11)

The method of claim 11, wherein the non-naturally occurring bacterium or cell-free fraction comprising the MMO and derived from the non-naturally occurring bacterium is immobilized on a solid matrix in a substantially non-aqueous state.

### Claim 13 (depends on 11)

The method of claim 11, wherein the reducing agent is H2 gas or formate.

### Claim 14 (depends on 13)

The method of claim 13, wherein H2 gas is mixed with the gas substrate or provided to the non-naturally occurring bacterium following the gas substrate.

### Claim 15 (depends on 11)

The method of claim 11, wherein O2 is mixed with the gas substrate or provided to the non-naturally occurring bacterium prior to the gas substrate.

### Claim 16 (depends on 1)

A method for converting ethylene into ethylene oxide, propylene into propylene oxide, or butene into butene oxide, the method comprising:
exposing the non-naturally occurring bacterium of claim 1 or a cell-free fraction comprising the MMO and derived from the non-naturally occurring bacterium of claim 1 to: (a) O2; (b) a gas substrate comprising ethylene, propylene, or butene, respectively; and (c) a reducing agent;
wherein the ethylene gas is converted to ethylene oxide, the propylene gas is converted to propylene oxide, and the butene gas is converted to butene oxide, respectively.