# US 2014/0154806 A1 · Tetraselmis promoters and terminators for use in eukaryotic cells

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- Number: US 2014/0154806 A1
- Document ID: US20140154806A1
- Status: Published application
- Kind: A1
- Application number: 13/693,585
- Filed: 2012-12-04
- Published: 2014-06-05
- Earliest claimed: 2012-12-04
- Assignees: ExxonMobil Research and Engineering Company
- Applicants: ExxonMobil Research and Engineering Company
- Inventors: Jane C. Schneider; Soyan Lieberman; Bo Liu; Eric R. Moellering; John H. Verruto; Amanda Skaggs
- Web page: https://patentgrep.com/patent/US20140154806A1

## CPC

- C12N 15/8221: {Transit peptides}
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

The present invention provides novel promoter and terminator sequences for use in gene expression in eukaryotic cells, such as algal cells. The invention further provides expression cassettes comprising a promoter, as described herein, operably linked to a gene. The invention further provides expression vectors and host eukaryotic cells, such as algal cells, for expressing a protein encoded by the gene; and methods for stably transforming eukaryotic algae such as Tetraselmis with transgenes.

## Claims

### Claim 1

An expression cassette comprising a heterologous nucleic acid sequence, operably linked to a promoter comprising a nucleotide sequence selected from the group consisting of a nucleotide sequence having at least 195% identity to at least 100 contiguous nucleotides of SEQ ID NO:1; a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:2; a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:3; a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:4; a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:5; a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:6; a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:7; and a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:8.

### Claim 2 (depends on 1)

The expression cassette according to claim 1, wherein the expression cassette comprises a sequence selected from the group consisting of a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:1; a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:2; a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:3; a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:4; a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:5; a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:6; a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:7; and a nucleotide sequence comprising at least 100 contiguous nucleotides of SEQ ID NO:8.

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The expression cassette according to claim 1, wherein the promoter is a constitutive promoter.

### Claim 5 (depends on 1)

The expression cassette according to claim 1, wherein the heterologous nucleic acid sequence is further operably linked to a terminator having a nucleotide sequence selected from the group consisting of a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:9; and a nucleotide sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:10.

### Claim 6

(canceled)

### Claim 7 (depends on 1)

The expression cassette according to claim 1, wherein the heterologous nucleic acid sequence encodes a polypeptide or a functional RNA sequence.

### Claim 8

(canceled)

### Claim 9 (depends on 7)

The expression cassette according to claim 7, wherein the heterologous nucleic acid sequence encodes (a) a protein associated with lipid biosynthesis, (b) a protein having lipolytic activity, (c) a protein that participates in photosynthesis, (d) a protein associated with carbon fixation, (e) a transporter protein, (f) a dehydrogenase, (g) a transcription factor, (h) a cell signaling protein, or (i) a functional RNA.

### Claim 10 (depends on 7)

The expression cassette according to claim 7, wherein the heterologous nucleic acid sequence encodes a functional RNA selected from the group consisting of an antisense sequence, a micro RNA, a shRNA, an siRNA, and a ribozyme.

### Claim 11 (depends on 1)

A vector comprising an expression cassette according to claim 1.

### Claim 12 (depends on 11)

The vector according to claim 11, further comprising a selectable marker or reporter gene.

### Claim 13 (depends on 11)

The vector according to claim 11, further comprising a nucleic acid sequence that comprises a terminator having a nucleotide sequence selected from the group consisting of a nucleic acid sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:9 and a nucleic acid sequence having at least 95% identity to at least 100 contiguous nucleotides of SEQ ID NO:10; wherein the terminator is operably linked to the heterologous gene coding sequence and/or the terminator is downstream of the heterologous gene coding sequence.

### Claim 14 (depends on 11)

The vector according to claim 11, wherein the vector includes at least one origin of replication.

### Claim 15 (depends on 12)

The vector according to claim 12, further comprising an additional promoter operably linked to the selectable marker or reporter gene.

### Claim 16 (depends on 12)

The vector according to claim 12, wherein the selectable marker or reported gene is selected from the group consisting of a gene conferring resistance to an antibiotic, a gene conferring resistance to an herbicide, a gene encoding acetyl CoA carboxylase (ACCase), a gene encoding acetohydroxy acid synthase (ahas), a gene encoding acetolactate synthase, a gene encoding aminoglycoside phosphotransferase, a gene encoding anthranilate synthase, a gene encoding bromoxynil nitrilase, a gene encoding cytochrome P450-NADH-cytochrome P450 oxidoreductase, a gene encoding dalapon dehalogenase, a gene encoding dihydropteroate synthase, a gene encoding a class I 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a gene encoding a class II EPSPS (aroA), a gene encoding a non-class I/II EPSPS, a gene encoding glutathione reductase, a gene encoding glyphosate acetyltransferase, a gene encoding glyphosate oxidoreductase, a gene encoding hydroxyphenylpyruvate dehydrogenase, a gene encoding hydroxy-phenylpyruvate dioxygenase, a gene encoding isoprenyl pyrophosphate isomerase, a gene encoding lycopene cyclase, a gene encoding phosphinothricin acteyl transferase, a gene encoding phytoene desaturase, a gene encoding prenyl transferase, a gene encoding protoporphyrin oxidase, a gene encoding superoxide dismutase, arg7, his3, hisD, hisG, manA, nit1, trpB, uidA, xy1A, a dihydrofolate reductase gene, a mannose-6-phosphate isomerase gene, a nitrate reductase gene, an ornithine decarboxylase gene, a thymidine kinase gene, a 2-deoxyglucose resistance gene; an R-locus gene, a tyrosinase gene, lacZ, an alkaline phosphatase gene, an α-amylase gene, a horseradish peroxidase gene, an α-galactosidase gene, a luciferin/luciferase gene, a beta-glucuronidase gene (GUS), and a gene encoding a fluorescent protein.

### Claim 17 (depends on 11)

A method for transforming a eukaryotic cell comprising:
introducing a vector according to claim 11 into the eukaryotic cell; and
selecting for a transformed eukaryotic cell.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the vector is introduced by a biolistic procedure.

### Claim 19 (depends on 18)

The method according to claim 18, wherein about 300 psi or more of pressure is used to impel biolistic microcarriers coated with vector DNA into the eukaryotic cell.

### Claim 20 (depends on 17)

The method according to claim 17, wherein the transformed eukaryotic cell is stably transformed.

### Claim 21 (depends on 17)

The method according to claim 17, wherein the eukaryotic cell is selected from the group consisting of a fungal cell, a heterokont cell, an algal cell, and a plant cell.

### Claim 22 (depends on 21)

The method according to claim 21, wherein the eukaryotic cell is an algal cell.

### Claim 23 (depends on 22)

The method according to claim 22, wherein the algal cell is from a species selected from the group consisting of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, and Volvox.

### Claim 24 (depends on 23)

The method according to claim 23, wherein the algal cell is a Tetraselmis algal cell.

### Claim 25 (depends on 1)

A method for co-transforming a eukaryotic cell comprising:
introducing an expression cassette according to claim 1 and a nucleic acid sequence encoding a selectable marker into the eukaryotic cell; and
selecting for the presence of the selectable marker in a transformed eukaryotic cell to provide a eukaryotic cell transformed with the expression cassette.

### Claim 26 (depends on 25)

The method according to claim 25, wherein the eukaryotic cell is an algal cell.

### Claim 27 (depends on 25)

The method according to claim 25, wherein the selectable marker gene is selected from the group consisting of a gene conferring resistance to an antibiotic, a gene conferring resistance to an herbicide, a gene encoding acetyl CoA carboxylase (ACCase), a gene encoding acetohydroxy acid synthase (ahas), a gene encoding acetolactate synthase, a gene encoding aminoglycoside phosphotransferase, a gene encoding anthranilate synthase, a gene encoding bromoxynil nitrilase, a gene encoding cytochrome P450-NADH-cytochrome P450 oxidoreductase, a gene encoding dalapon dehalogenase, a gene encoding dihydropteroate synthase, a gene encoding a class I 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a gene encoding a class II EPSPS (aroA), a gene encoding a non-class I/II EPSPS, a gene encoding glutathione reductase, a gene encoding glyphosate acetyltransferase, a gene encoding glyphosate oxidoreductase, a gene encoding hydroxyphenylpyruvate dehydrogenase, a gene encoding hydroxy-phenylpyruvate dioxygenase, a gene encoding isoprenyl pyrophosphate isomerase, a gene encoding lycopene cyclase, a gene encoding phosphinothricin acteyl transferase, a gene encoding phytoene desaturase, a gene encoding prenyl transferase, a gene encoding protoporphyrin oxidase, a gene encoding superoxide dismutase, arg7, his3, hisD, hisG, manA, nit1, trpB, uidA, xy1A, a dihydrofolate reductase gene, a mannose-6-phosphate isomerase gene, a nitrate reductase gene, an ornithine decarboxylase gene, a thymidine kinase gene, a 2-deoxyglucose resistance gene; and an R-locus gene.

### Claim 28 (depends on 25)

The method according to claim 25, wherein the selectable marker gene is operably linked to a promoter active in the eukaryotic cell.

### Claim 29 (depends on 25)

The method according to claim 25, wherein the expression cassette and the nucleic acid sequence encoding a selectable marker are present in the same vector.

### Claim 30 (depends on 26)

The method according to claim 26, wherein the algal cell is from a species selected from the group consisting of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, and Volvox.

### Claim 31 (depends on 30)

The method according to claim 30, wherein the algal cell is a Tetraselmis algal cell.

### Claim 32 (depends on 1)

A eukaryotic host cell comprising an expression cassette according to claim 1.

### Claim 33 (depends on 32)

The eukaryotic host cell according to claim 32, wherein the eukaryotic host cell is an algal cell.

### Claim 34

A nucleic acid molecule comprising a nucleotide sequence encoding a peptide with at least 80% sequence identity to SEQ ID NO:36 or a functional fragment thereof, operably linked to a heterologous nucleic acid sequence encoding a protein.

### Claim 35 (depends on 34)

The nucleic acid molecule according to claim 34, wherein the protein is selected from the group consisting of: (a) a protein associated with lipid biosynthesis; (b) a protein having lipolytic activity; (c) a protein that participates in photosynthesis; (d) a protein associated with carbon fixation; (e) a transporter protein; (f) a dehydrogenase; (g) a transcription factor; and (h) a cell signaling protein.

### Claim 36 (depends on 34)

A method for targeting a protein to a chloroplast, the method comprising transforming a eukaryotic photosynthetic cell with the nucleic acid molecule of claim 34, and culturing the transformant under conditions in which the protein is expressed.

### Claim 37 (depends on 36)

The method according to claim 36, wherein the protein is selected from the group consisting of: (a) a protein associated with lipid biosynthesis; (b) a protein having lipolytic activity; (c) a protein that participates in photosynthesis; (d) a protein associated with carbon fixation; (e) a transporter protein; (f) a dehydrogenase; (g) a transcription factor; and (h) a cell signaling protein.