Regulatory elements from labyrinthulomycetes microorganisms

Patent metadata

Field	Value
Publication number	US-10633665-B2
Application number	US-201815916171-A
Country	US
Kind code	B2
Filing date	Mar 8, 2018
Priority date	Mar 2, 2015
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

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.

First claim

What is claimed is:

1. A nucleic acid construct comprising a nucleic acid sequence that comprises a promoter, wherein the nucleic acid sequence:
exhibits at least 90% sequence identity to at least 650 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:193, and SEQ ID NO:198;
wherein the promoter is operably linked to a heterologous nucleic acid sequence.

2. The nucleic acid construct of claim 1 , wherein said nucleic acid sequence exhibits at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO:193, and SEQ ID NO:198.

3. The nucleic acid construct of claim 1 , wherein said nucleic acid sequence exhibits at least 95% sequence identity to at least 650 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:193, and SEQ ID NO:198.

4. The nucleic acid construct of claim 3 , wherein the nucleic acid sequence exhibits at least 98% sequence identity to at least 650 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:191, SEQ ID NO:193, and SEQ ID NO:198.

5. The nucleic acid construct of claim 1 , wherein the nucleic acid sequence exhibits at least 98% sequence identity to is selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:182, SEQ ID NO:186, SEQ ID NO: 193, and SEQ ID NO: 198.

6. The nucleic acid construct of claim 1 , wherein the promoter is functional in a Schizochytrium or Aurantiochytrium cell.

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

8. The nucleic acid construct of claim 7 , wherein said heterologous nucleic acid sequence encodes a functional RNA selected from the group consisting of 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, and an antisense RNA molecule.

9. The nucleic acid construct of claim 1 , wherein said heterologous nucleic acid sequence is operably linked to a terminator.

10. The nucleic acid construct of claim 9 , 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.

11. The nucleic acid construct of claim 1 , wherein the promoter is functional in a Labyrinthulomycetes cell.

12. The nucleic acid construct of claim 7 , wherein said construct is an expression cassette or a vector.

13. The nucleic acid construct of claim 7 , wherein the heterologous nucleic acid sequence encodes a transcription factor, DNA binding protein, splicing factor, nuclease, a cas protein, a recombinase, a G protein, a nucleotide cyclase, a phosphodiesterase, a kinase, a polypeptide 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.

14. The nucleic acid construct of claim 7 , wherein said heterologous nucleic acid sequence comprises a selectable marker or a reporter gene.

15. The nucleic construct of claim 14 , 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.

16. A method of transforming a eukaryotic cell, comprising:
(i) introducing into a eukaryotic cell the nucleic acid construct of claim 9 ; and
(ii) selecting or screening for a transformed eukaryotic cell.

17. The method according to claim 16 , wherein the nucleic acid construct is introduced by a biolistic procedure or electroporation.

18. A recombinant eukaryotic cell produced by the method of claim 16 .

19. A recombinant cell comprising the nucleic acid construct of claim 1 .

20. The recombinant cell of claim 19 , wherein said nucleic acid construct is stably integrated into the genome of said recombinant cell.

21. The recombinant cell of claim 19 , wherein the recombinant cell is a Labyrinthulomycetes cell.

22. The recombinant cell of claim 21 , wherein said Labyrinthulomycetes cell is of a microorganism selected from the group consisting of an Aplanochytrium , an Aurantiochytrium , a Diplophrys , a Japonochytrium , an Oblongichytrium , a Schizochytrium , a Thraustochytrium , and an Ulkenia microorganism.

Assignees

• Conagen Inc

Inventors

• Caiazza Nicky C
• Win Maung Nyan
• Urano Jun

Classifications

• C12N15/79 Primary

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/80

  for fungi · CPC title

• C12N5/00

  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) · CPC title

• C12N15/00

  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) · CPC title