# US 2016/0257965 A1 · Regulatory elements from labyrinthulomycetes microorganisms

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- Number: US 2016/0257965 A1
- Document ID: US20160257965A1
- Status: Published application
- Kind: A1
- Application number: 15/056,857
- Filed: 2016-02-29
- Published: 2016-09-08
- Earliest claimed: 2015-03-02
- Assignees: Synthetic Genomics, Inc.
- Applicants: Synthetic Genomics, Inc.
- Inventors: NICKY C. CAIAZZA; MAUNG NYAN WIN; JUN URANO
- Web page: https://patentgrep.com/patent/US20160257965A1

## CPC

- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/80: for fungi
- 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 disclosure generally relates to novel polynucleotide molecules for use in regulating gene expression in recombinant cells, such as labyrinthulomycetes cells. The disclosure further relates to nucleic acid constructs, such as vectors and expression cassettes, containing a regulatory element operably linked to a heterologous nucleotide sequence. The disclosure further relates to methods for stably transforming a host cell, such as a labyrinthulomycetes cell with transgenes. Stably transformed recombinant cells, progeny, biomaterials derived therefrom, and methods for preparing and using the same are also provided.

## Claims

### Claim 1

A nucleic acid construct comprising a nucleic acid sequence that comprises a promoter, wherein the nucleic acid sequence:
a. hybridizes under high stringency conditions to at least 50 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of any one or more of SEQ ID NOs:1-70, 180-202, and complements thereof; or
b. exhibits at least 80% sequence identity to at least 50 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of any one or more of SEQ ID NOs:1-70, 180-202, and complements thereof;
c. wherein the promoter is operably linked to a heterologous nucleic acid sequence.

### Claim 2 (depends on 1)

The nucleic acid construct of claim 1, wherein said nucleic acid molecule comprises at least 50 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of any one or more of SEQ ID NOs:1-70, 180-202, and complements thereof.

### Claim 3 (depends on 1)

The nucleic acid construct of claim 1, wherein the nucleic acid sequence exhibits at least 80% sequence identity to at least 50 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:196, SEQ ID NO:183, SEQ ID NO:191, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:190, and SEQ ID NO:192.

### Claim 4 (depends on 3)

The nucleic acid construct of claim 3, wherein the nucleic acid sequence exhibits at least 80% sequence identity to at least 50 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:196, SEQ ID NO:183, or SEQ ID NO:191.

### Claim 5 (depends on 1)

The nucleic acid construct of claim 1, wherein the nucleic acid sequence exhibits at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:196, SEQ ID NO:198, and SEQ ID NO:199.

### Claim 6 (depends on 1)

The nucleic acid construct of claim 1, wherein the nucleic acid sequence exhibits at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:59, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:186, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:196, SEQ ID NO:198, and SEQ ID NO:199.

### Claim 7 (depends on 1)

The nucleic acid construct of claim 1, wherein said nucleic acid molecule is a promoter.

### Claim 8 (depends on 1)

The nucleic acid construct of claim 1, wherein said nucleic acid sequence exhibits at least 80% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:198, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:190, and SEQ ID NO:191.

### Claim 9 (depends on 8)

The nucleic acid construct of claim 8, wherein the promoter is functional in a Schizochytrium or Aurantiochytrium cell.

### Claim 10 (depends on 1)

The nucleic acid construct of claim 1, wherein said heterologous nucleic acid sequence encodes a polypeptide or a functional RNA.

### Claim 11 (depends on 10)

The nucleic acid construct of claim 10, wherein said heterologous nucleic acid sequence encodes a functional RNA selected from: a ribosomal RNA, a tRNA, a ribozyme, a transactivating (tr) RNA of a CRISPR system, a crispr (cr) RNA of a CRISPR system, a chimeric guide RNA of a CRISPR system, a micro RNA, an interfering RNA (RNAi) molecule, a short hairpin (sh) RNA, or an antisense RNA molecule.

### Claim 12 (depends on 9)

The nucleic acid construct of claim 9, wherein said heterologous nucleic acid sequence is operably linked to a terminator.

### Claim 13 (depends on 12)

The nucleic acid construct of claim 12, wherein the terminator comprises a sequence having at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID NOs: 71-78.

### Claim 14 (depends on 10)

The nucleic acid construct of claim 10, that is functional in a labyrinthulomycetes cell.

### Claim 15 (depends on 10)

The nucleic acid construct of claim 10, wherein said construct is further defined as an expression cassette or a vector.

### Claim 16 (depends on 10)

A nucleic acid construct of claim 10, wherein the heterologous nucleic acid sequence encodes a transcription factor, DNA binding protein, splicing factor, nuclease (including, without limitation, an RNA-guided endonuclease such as a cas protein of a CRISPR system), a recombinase (e.g., a cre or flp recombinase), a G protein, a nucleotide cyclase, a phosphodiesterase, a kinase, a polypeptide of that participates in protein secretion or protein trafficking, a structural protein, a hormone, a cytokine, an antibody, a transporter, an enzyme having lypolytic activity, a thioesterase, an amidase, a lipase, a fatty acid synthase or a component of a fatty acid synthase complex, a pfaA, pfaB, pfaC, pfaD, or pfaE polypeptide, an acyl-CoA synthetase, an acyl-ACP synthetase, an acyl carrier protein, an acyl-CoA carboxylase, an acyl transferase, an enzyme that participates in glycolysis, a dehydrogenase, an enzyme of the TCA cycle, a fatty acid desaturase, or a fatty acid elongase.

### Claim 17 (depends on 10)

The nucleic acid construct of claim 10, wherein said heterologous nucleic acid sequence encodes a selectable marker or a reporter gene.

### Claim 18 (depends on 17)

The nucleic construct of claim 17, wherein said 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 acetyl 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, xylA, 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 19 (depends on 10)

A method of transforming a eukaryotic cell, comprising:
(i) introducing into a eukaryotic cell a nucleic acid molecule according to claim 10; and
(ii) selecting or screening for a transformed eukaryotic cell.

### Claim 20 (depends on 19)

A method according to claim 19, wherein the nucleic acid molecule is introduced by a biolistic procedure or electroporation.

### Claim 21 (depends on 19)

A recombinant eukaryotic cell produced by a method according to claim 19.

### Claim 22 (depends on 1)

A recombinant cell comprising a nucleic acid molecule of claim 1.

### Claim 23 (depends on 22)

The recombinant cell of claim 22, wherein said nucleic acid molecule is stably integrated into the genome of said recombinant cell.

### Claim 24 (depends on 22)

The recombinant cell of claim 22, wherein the recombinant cell belongs to the class labyrinthulomycetes.

### Claim 25 (depends on 24)

The recombinant cell of claim 24, wherein said labyrinthulomycetes microorganism is an Aplanochytrium, an Aurantiochytrium, a Diplophrys, a Japonochytrium, an Oblongichytrium, a Schizochytrium, a Thraustochytrium, or an Ulkenia microorganism.

### Claim 26

An amplification reaction mixture comprising primers adapted for amplifying a nucleic acid comprising at least 50 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID Nos:1-70, SEQ ID Nos:180-202, complements thereof, and nucleic acids exhibiting at least 80% sequence identity thereto.

### Claim 27

A ligation reaction mixture comprising a nucleic acid comprising at least 50 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID Nos:1-70, SEQ ID Nos:180-202, complements thereof, and nucleic acids exhibiting at least 80% sequence identity thereto.