# US 9,518,102 B2 · Polypeptide for improving protein production in microorganisms of the phylum Labyrinthulomycota > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,518,102 B2 - Document ID: US9518102B2 - Status: Granted - Kind: B2 - Application number: 14/548,054 - Filed: 2014-11-19 - Published: 2015-05-14 - Granted: 2016-12-13 - Earliest claimed: 2009-03-16 - Assignees: Dsm IP Assets B.V. - Applicants: DSM IP Assets B.V. - Inventors: Kirk E. Apt; James Casey Lippmeier; David Simpson; Jun Wang; James P. Wynn; Ross Zirkle - Web page: https://patentgrep.com/patent/US9518102B2 ## CPC - C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00) - C07K 14/37: from fungi - C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} - C12N 1/105: Protozoal isolates - C12N 9/2431: {Beta-fructofuranosidase (3.2.1.26), i.e. invertase} - C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts - C12N 15/80: for fungi - C12P 21/005: {Glycopeptides, glycoproteins} - C12Y 302/01026: Beta-fructofuranosidase (3.2.1.26), i.e. invertase - C12R 2001/90: Protozoa {; Processes using protozoa} - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS ## Abstract The present invention relates to recombinant cells and microorganisms of the phylum Labyrinthulomycota and their use in heterologous protein production. Novel promoter, terminator, and signal sequences for efficient production and, optionally, secretion of polypeptides from recombinant host cells and microorganisms are also encompassed by the present invention. ## Claims ### Claim 1 A recombinant nucleic acid molecule encoding a fusion protein, or the complementary sequence thereof, comprising: (a) (i) a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15 or a fragment thereof, wherein the amino acid sequence or fragment thereof functions as a signal peptide, and (ii) a polynucleotide encoding a second polypeptide that is a protein, wherein the polynucleotide encoding the signal polypeptide is operably linked to the polynucleotide encoding the protein; or (b) a polynucleotide sequence that is fully complementary to the polynucleotide sequence of (a). ### Claim 2 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:15. ### Claim 3 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15. ### Claim 4 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 20 amino acid residues of SEQ ID NO:15. ### Claim 5 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 21 amino acid residues of SEQ ID NO:15. ### Claim 6 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 22 amino acid residues of SEQ ID NO:15. ### Claim 7 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 23 amino acid residues of SEQ ID NO:15. ### Claim 8 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 24 amino acid residues of SEQ ID NO:15. ### Claim 9 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 25 amino acid residues of SEQ ID NO:15. ### Claim 10 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 26 amino acid residues of SEQ ID NO:15. ### Claim 11 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 27 amino acid residues of SEQ ID NO:15. ### Claim 12 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 28 amino acid residues of SEQ ID NO:15. ### Claim 13 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 29 amino acid residues of SEQ ID NO:15. ### Claim 14 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 30 amino acid residues of SEQ ID NO:15. ### Claim 15 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 31 amino acid residues of SEQ ID NO:15. ### Claim 16 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 32 amino acid residues of SEQ ID NO:15. ### Claim 17 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 33 amino acid residues of SEQ ID NO:15. ### Claim 18 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 34 amino acid residues of SEQ ID NO:15. ### Claim 19 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence encodes a polypeptide comprising the first 35 amino acid residues of SEQ ID NO:15. ### Claim 20 (depends on 1) The recombinant nucleic acid molecule of claim 1, wherein the recombinant nucleic acid molecule is a vector. ### Claim 21 (depends on 1) A host cell comprising the recombinant nucleic acid molecule of claim 1. ### Claim 22 (depends on 21) The host cell of claim 21, wherein the host cell is a member of the order Thraustochytriales. ### Claim 23 (depends on 22) The host cell of claim 22, wherein the host cell is a Schizochytrium or a Thraustochytrium. ### Claim 24 (depends on 20) A method for production of a recombinant protein, comprising: (a) culturing a recombinant microorganism of the order Thraustochytriales in a medium, wherein the recombinant microorganism comprises the recombinant nucleic acid molecule of claim 20; and (b) isolating the protein made in step (a). ### Claim 25 (depends on 24) The method of claim 24, wherein the protein is recovered from an isolated Thraustoehytriales biomass. ### Claim 26 (depends on 24) The method of claim 24, wherein the protein accumulates in the microorganism. ### Claim 27 (depends on 24) The method of claim 24, wherein the protein accumulates in a membrane of the microorganism. ### Claim 28 (depends on 24) The method of claim 24, wherein the protein is recovered from the culture medium. ### Claim 29 (depends on 24) The method of claim 24, wherein the protein is secreted.