# US 2014/0154740 A1 · Nannochloropsis kozak consensus sequence

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- Number: US 2014/0154740 A1
- Document ID: US20140154740A1
- Status: Published application
- Kind: A1
- Application number: 13/705,284
- Filed: 2012-12-05
- Published: 2014-06-05
- Earliest claimed: 2012-12-05
- Assignees: ExxonMobil Research and Engineering Company
- Inventors: Peter DeHoff; Leah Soriaga
- Web page: https://patentgrep.com/patent/US20140154740A1

## CPC

- 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)

## Abstract

The present invention provides Nannochloropsis consensus Kozak sequences for use in protein expression in eukaryotic cells, such as algal cells. The invention further provides expression constructs, expression cassettes, cloning or expression vectors and host eukaryotic cells, such as algal cells, and methods for expressing proteins of interest which take advantage of the consensus Kozak sequences as described herein.

## Claims

### Claim 1

A recombinant DNA molecule comprising a Kozak sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, operably linked to a heterologous nucleotide sequence encoding a polypeptide, wherein positions 1, 2, and 3 of the Kozak sequence comprise the initiating methionine codon of the polypeptide.

### Claim 2 (depends on 1)

The recombinant DNA molecule according to claim 1, wherein the Kozak sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

### Claim 3 (depends on 1)

An expression cassette comprising the recombinant DNA molecule comprising a Kozak sequence according to claim 1, wherein the Kozak sequence is further operably linked to a eukaryotic promoter positioned upstream of the Kozak sequence.

### Claim 4 (depends on 3)

The expression cassette according to claim 3, wherein the eukaryotic promoter is active in a heterokont species or a eukaryotic microalgal species.

### Claim 5 (depends on 3)

The expression cassette according to claim 3, wherein the eukaryotic promoter is a Simian vacuolating virus (SV40) promoter, a cauliflower mosaic virus (CaMV) promoter, a cytomegalovirus (CMV) promoter, a promoter derived from a heterokont species, or a promoter derived from a eukaryotic microalgal species.

### Claim 6 (depends on 5)

The expression cassette according to claim 5, wherein the eukaryotic promoter is a Nannochloropsis promoter.

### Claim 7 (depends on 3)

The expression cassette according to claim 3, wherein the gene encoding a polypeptide is codon optimized for expression in an algal or heterokont species.

### Claim 8 (depends on 3)

A vector comprising an expression cassette according to claim 3.

### Claim 9 (depends on 8)

The vector according to claim 8, further comprising a selectable marker.

### Claim 10 (depends on 9)

A method for transforming a eukaryotic cell comprising:
introducing a vector according to claim 9 into the eukaryotic cell; and
selecting for a transformed eukaryotic cell.

### Claim 11 (depends on 1)

A recombinant eukaryotic microorganism comprising:
a recombinant DNA molecule that comprises a recombinant DNA molecule according to claim 1,
wherein the recombinant eukaryotic microorganism produces a greater amount of the polypeptide than a control eukaryotic microorganism that is substantially identical to the recombinant eukaryotic microorganism in all respects except that the control eukaryotic microorganism comprises a recombinant DNA molecule that does not include a heterologous Kozak sequence selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:14.

### Claim 12 (depends on 11)

The recombinant eukaryotic microorganism according to claim 11, wherein the Kozak sequence is part of a translational initiation consensus sequence selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:16.

### Claim 13 (depends on 11)

The recombinant eukaryotic microorganism according to claim 11, wherein the eukaryotic microorganism is selected from the group consisting of a fungal cell, a heterokont cell, an algal cell, and a plant cell.

### Claim 14 (depends on 13)

The recombinant eukaryotic microorganism according to claim 13, wherein the eukaryotic microorganism is an algal cell.

### Claim 15 (depends on 14)

The recombinant eukaryotic microorganism according to claim 14, wherein the algal cell is a species of genera 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 16 (depends on 15)

The recombinant eukaryotic microorganism according to claim 15, wherein the algal cell is a Nannochloropsis algal cell.

### Claim 17 (depends on 4)

A method for expressing a protein of interest in a eukaryotic cell, the method comprising transfecting the eukaryotic cell with a vector comprising the expression cassette of claim 4, and culturing the eukaryotic cell under conditions in which the nucleotide sequence encoding the polypeptide is expressed.

### Claim 18 (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 19 (depends on 18)

The method according to claim 18, wherein the eukaryotic cell is an algal cell.

### Claim 20 (depends on 19)

The method according to claim 19, wherein the algal cell is a species of genera 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 21 (depends on 20)

The method according to claim 20, wherein the algal cell is a Nannochloropsis algal cell.

### Claim 22 (depends on 17)

The method according to claim 17, wherein the protein of interest is (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 or (h) a cell signaling protein.

### Claim 23 (depends on 4, 17)

The method according to claim 17, wherein expression of the protein of interest is enhanced at least 5% relative to expression achieved from a substantially identical eukaryotic cell lacking a vector comprising the expression cassette of claim 4.

### Claim 24 (depends on 4, 23)

The method according to claim 23, wherein expression of the protein of interest is enhanced at least 10% relative to expression achieved from a substantially identical eukaryotic cell lacking a vector comprising the expression cassette of claim 4.

### Claim 25 (depends on 4, 24)

The method according to claim 24, wherein expression of the protein of interest is enhanced at least 20% relative to expression achieved from a substantially identical eukaryotic cell lacking a vector comprising the expression cassette of claim 4.