# US 9,040,264 B2 · Recombinant cyanobacterium expressing a transcription factor domain protein

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- Number: US 9,040,264 B2
- Document ID: US9040264B2
- Status: Granted
- Kind: B2
- Application number: 13/435,315
- Filed: 2012-03-30
- Published: 2012-10-04
- Granted: 2015-05-26
- Earliest claimed: 2011-03-31
- Assignees: ExxonMobil Research and Engineering Company
- Applicants: Jessica Kristof; Rekha Seshadri
- Inventors: Jessica Kristof; Rekha Seshadri
- Web page: https://patentgrep.com/patent/US9040264B2

## CPC

- C12P 7/64: Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C07K 14/195: from bacteria
- 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)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention provides microorganisms such as cyanobacteria genetically engineered to express proteins that include transcription factor domains for upregulation of lipid biosynthetic pathways. In addition to expression a gene encoding a transcription factor domain protein, the recombinant microorganisms can express at least one exogenous gene that encodes a polypeptide for the production of a fatty acid, fatty acid derivative, or triglyceride. Also included are methods of producing a fatty acid, fatty acid derivative, or triglyceride using the engineered microorganisms described herein as well as nucleic acid molecules encoding novel transcription factor domain proteins.

## Claims

### Claim 1

A recombinant cyanobacterium comprising an exogenous nucleic acid molecule encoding a transcription factor domain protein,
wherein the transcription factor domain protein has transcription factor activity and comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, or SEQ ID NO:12, and
wherein the recombinant cyanobacterium produces a greater amount of at least one free fatty acid or at least one fatty acid derivative than does a control cyanobacterium that does not comprise the exogenous nucleic acid molecule encoding the transcription factor domain protein, and
further wherein the exogenous nucleic acid molecule is operably linked to a promoter that is heterologous relative to the cyanobacterium and/or the exogenous nucleic acid molecule.

### Claim 2 (depends on 1)

The cyanobacterium of claim 1, wherein the cyanobacterium further comprises a nucleic acid molecule encoding a thioesterase and/or polypeptide having lipolytic activity.

### Claim 3 (depends on 1)

The cyanobacterium of claim 1, wherein the cyanobacterium further comprises at least one additional exogenous nucleic acid molecule, encoding a protein for the production of a fatty acid or a fatty acid derivative.

### Claim 4 (depends on 3)

The cyanobacterium of claim 3, wherein the cyanobacterium further comprises at least one additional exogenous nucleic acid molecule encoding an enzyme selected from the group consisting of an acetyl CoA carboxylase, a ketoacyl-CoA synthase, an acyl-CoA synthetase, a fatty acyl-CoA/aldehyde reductase, an alcohol-forming fatty acyl-CoA reductase, a fatty aldehyde-forming fatty acyl-CoA reductase, an acyl-ACP reductase, a carboxylic acid reductase, a fatty aldehyde reductase, an alcohol acetyl transferase, an acyl-CoA alcohol transacylase, an acyltransferase, a wax synthase, a fatty aldehyde decarbonylase, and a fatty acid decarboxylase.

### Claim 5 (depends on 1)

The cyanobacterium of claim 1, wherein the cyanobacterium is an Agmenellum, Anabaena, Anabaenopsis, Anacystis, Aphanizomenon, Arthrospira, Asterocapsa, Borzia, Calothrix, Chamaesiphon, Chlorogloeopsis, Chroococcidiopsis, Chroococcus, Crinalium, Cyanobacterium, Cyanobium, Cyanocystis, Cyanospira, Cyanothece, Cylindrospermopsis, Cylindrospermum, Dactylococcopsis, Dermocarpella, Fischerella, Fremyella, Geitleria, Geitlerinema, Gloeobacter, Gloeocapsa, Gloeothece, Halospirulina, Iyengariella, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Micro cystis, Myxosarcina, Nodularia, Nostoc, Nostochopsis, Oscillatoria, Phormidium, Planktothrix, Pleurocapsa, Prochlorococcus, Prochloron, Prochlorothrix, Pseudanabaena, Rivularia, Schizothrix, Scytonema, Spirulina, Stanieria, Starria, Stigonema, Symploca, Synechococcus, Synechocystis, Thermosynechococcus, Tolypothrix, Trichodesmium, Tychonema or Xenococcus species.

### Claim 6 (depends on 1)

A method of producing a free fatty acid or fatty acid derivative, the method comprising culturing the recombinant cyanobacterium of claim 1 in a growth medium under conditions in which the exogenous nucleic acid molecule encoding the transcription factor domain protein is expressed to produce at least one free fatty acid or fatty acid derivative.

### Claim 7 (depends on 6)

The method of claim 6, wherein the cyanobacterium is cultured phototrophically.

### Claim 8 (depends on 6)

The method of claim 6, wherein the amount of the fatty acid or fatty acid derivative produced is at least 10% more than the amount of the fatty acid or fatty acid derivative produced by the control cyanobacterium cultured under substantially identical conditions.

### Claim 9 (depends on 6)

The method of claim 6, wherein the cyanobacterium is selected from the group consisting of Agmenellum, Anabaena, Anabaenopsis, Anacystis, Aphanizomenon, Arthrospira, Asterocapsa, Borzia, Calothrix, Chamaesiphon, Chlorogloeopsis, Chroococcidiopsis, Chroococcus, Crinalium, Cyanobacterium, Cyanobium, Cyanocystis, Cyanospira, Cyanothece, Cylindrospermopsis, Cylindrospermum, Dactylococcopsis, Dermocarpella, Fischerella, Fremyella, Geitleria, Geitlerinema, Gloeobacter, Gloeocapsa, Gloeothece, Halospirulina, Iyengariella, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Microcystis, Myxosarcina, Nodularia, Nostoc, Nostochopsis, Oscillatoria, Phormidium, Planktothrix, Pleurocapsa, Prochlorococcus, Prochloron, Prochlorothrix, Pseudanabaena, Rivularia, Schizothrix, Scytonema, Spirulina, Stanieria, Starria, Stigonema, Symploca, Synechococcus, Synechocystis, Thermosynechococcus, Tolypothrix, Trichodesmium, Tychonema and Xenococcus.

### Claim 10 (depends on 6)

The method of claim 6, further comprising isolating at least one free fatty acid or at least one fatty acid derivative from the cyanobacteria or from the growth medium.

### Claim 11

An expression cassette comprising a nucleic acid molecule encoding: (a) a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO:2, wherein the nucleic acid molecule encoding the polypeptide is operably linked to a heterologous promoter, and wherein the polypeptide has histidine kinase activity; or (b) a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, or SEQ ID NO:12, wherein the nucleic acid molecule encoding a lithe polypeptide is operably linked to a heterologous promoter, and wherein the polypeptide has transcription factor activity.

### Claim 12 (depends on 11)

The expression cassette according to claim 11, wherein the nucleic acid molecule encodes a polypeptide comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, or SEQ ID NO:12.

### Claim 13 (depends on 11)

The expression cassette of claim 11, wherein the promoter is inducible.

### Claim 14 (depends on 11)

The expression cassette of claim 11, wherein the expression cassette is in an expression vector.

### Claim 15 (depends on 11)

The expression cassette of claim 11, wherein the expression cassette is in an integration vector.

### Claim 16 (depends on 1)

The cyanobacterium of claim 1, wherein the cyanobacterium comprises an exogenous nucleic acid molecule encoding a polypeptide having an amino acid sequence with at least about 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2 and an exogenous nucleic acid molecule encoding a polypeptide having an amino acid sequence with at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:4.

### Claim 17

A recombinant cyanobacterium comprising an exogenous nucleic acid molecule encoding a transcription factor domain protein,
wherein the transcription factor domain protein has transcription factor activity and comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16, and
wherein the recombinant cyanobacterium further comprises at least one additional exogenous nucleic acid molecule encoding an enzyme selected from the group consisting of an acetyl CoA carboxylase, a ketoacyl-CoA synthase, an acyl-CoA synthetase, a fatty acyl-CoA/aldehyde reductase, an alcohol-forming fatty acyl-CoA reductase, a fatty aldehyde-forming fatty acyl-CoA reductase, an acyl-ACP reductase, a carboxylic acid reductase, a fatty aldehyde reductase, an alcohol acetyl transferase, an acyl-CoA alcohol transacylase, an acyltransferase, a wax synthase, a fatty aldehyde decarbonylase, and a fatty acid decarboxylase, and
further wherein the recombinant cyanobacterium produces a greater amount of at least one free fatty acid or at least one fatty acid derivative than does a control cyanobacterium that does not comprise the exogenous nucleic acid molecule encoding the transcription factor domain protein, and
still further wherein the exogenous nucleic acid molecule is operably linked to a promoter that is heterologous relative to the cyanobacterium and/or the exogenous nucleic acid molecule.

### Claim 18 (depends on 17)

The recombinant cyanobacterium of claim 17, wherein the transcription factor domain protein has 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:16.