A CHLOROPLAST OR ACCUMULATED LIPID PARTICLE ENRICHED WITH AN OIL-BODY PROTEIN FUSION POLYPEPTIDE AND METHOD FOR PRODUCING THE SAME IN ALGAE

• US 20200131553A1
• Filed: 03/10/2017
• Published: 04/30/2020
• Est. Priority Date: 10/03/2016
• Status: Abandoned Application

• Current Assignee: Alcantara Research Group Inc.
• Original Assignee: Alcantara Research Group Inc.
• Inventors: ALCANTARA, Joenel
• Application Number:
  US16/338,874
• Publication Number:
  US 20200131553A1
• Time in Patent Office: Days
• CPC Class Codes: A01H 13/00 Algae unicellular algae C12N1/12
• CPC Class Codes: C07K 1/14 Extraction; Separation; Purification
• CPC Class Codes: C07K 14/405 from algae
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 2500/60 Buffer, e.g. pH regulation, osmotic pressure
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

Abstract

The present invention relates to recombinant protein production in algal cells. In particular, the present invention provides methods for making recombinant polypeptides in association with accumulated lipid particles or chloroplasts. The methods involve producing the recombinant polypeptide as a fusion polypeptide with an oil body protein and the growth of the algal cells under non-homeostatic conditions to form accumulated lipid particles within the algal cells, wherein the algal lipid particles contain the fusion polypeptide.

Claims

1. A method of producing a recombinant polypeptide in an algal cell, the method comprising:

growing algal cells comprising a recombinant polypeptide under homeostatic conditions to target the recombinant polypeptide to the algal chloroplast;

wherein the recombinant polypeptide is a fusion polypeptide comprising an oleosin protein or fragment thereof and a protein of interest;

and isolating the recombinant protein.

2. The method of claim 1, comprising isolating the algal chloroplasts and wherein the recombinant protein is isolated from the isolated algal chloroplasts.

3. The method of claim 1, comprising subjecting the growing algal cells to non-homeostatic conditions to form accumulated lipid particles within the algal cells, wherein the accumulated lipid particles comprise the fusion polypeptide.

4. The method of claim 3, comprising isolating the accumulated lipid particles and wherein the recombinant protein is isolated from the isolated accumulated lipid particles.

5. The method of claim 1, comprising the step of introducing a nucleic acid encoding the recombinant polypeptide into the algal cell.

6. The method of claim 5, wherein the nucleic acid encoding the fusion polypeptide comprises one or more algal cell control elements.

7. The method of claim 1, wherein the oleosin protein or fragment thereof is a Oleo1 protein, Oleo2 protein, Oleo3 protein, Oleo 4 protein, derivatives or fragments or combinations thereof.

8. The method of claim 7, wherein the oleosin is a substantially the full length oleosin or an oleosin that excludes the hydrophobic domain, optionally selected from the group consisting of Oleo1 excluding the hydrophobic domain, Oleo2 excluding the hydrophobic domain, Oleo3 excluding the hydrophobic domain and Oleo4 excluding the hydrophobic domain.

11. The method of claim 3, wherein non-homeostatic conditions include a deficiency in one or more nutrients optionally nitrogen or phosphorus or an exogenous stress factor optionally selected from the group consisting of a non-homeostatic pH, a non-homeostatic salinity, and a non-homeostatic light intensity.

15. The method according to claim 1 wherein the oleosin is a protein encoded by a nucleic acid sequence having the sequence set forth in SEQ.ID NO:

7 to SEQ.ID NO;

12.

16. The method of claim 1 wherein the algal cell is selected form the group of algal cells consisting of green algae (Chlorophyceae), diatoms (Bacillariophyceae), yellow-green algae (Xanthophyceae), golden algae (Chrysophyceae), red algae (Rhodophyceae), brown algae (Phaeophyceae), dinoflagellates (Dinophyceae) or pico-plankton (Prasinophyceae and Eustigmatophyceae), wherein the green algae optionally belongs to the genus Clamydomonas, or Chlorella.

9-10. -10. (canceled)

12-14. -14. (canceled)

17. (canceled)

18. A method of producing algal chloroplasts enriched for recombinant polypeptide, the method comprising:

(a) introducing a nucleic acid into algal cells, the nucleic acid comprising as operably linked components

(i) a nucleic acid encoding a fusion polypeptide comprising an oleosin protein or fragment thereof to provide targeting to the algal chloroplast and a polypeptide of interest; and

(ii) a nucleic acid sequence capable of controlling expression in an algal cell;

(b) subjecting the algal cells in a growth medium to homeostatic conditions to target the fusion polypeptide to the algal chloroplast; and

(c) optionally isolating the algal chloroplasts.

19-30. -30. (canceled)

31. A method for producing accumulated lipid particles, the method comprising:

(a) introducing a nucleic acid into algal cells, the nucleic acid comprising as operably linked components

a nucleic acid encoding a fusion polypeptide comprising an oleosin protein or fragment thereof to provide targeting to the algal chloroplast and a polypeptide of interest; and

a nucleic acid sequence capable of controlling expression in an algal cell;

(b) subjecting the algal cell to non-homeostatic conditions to form accumulated lipid particles within the algal cell and optionally isolating the accumulated lipid particles.

32. The method according to claim 31 wherein the algal cell is grown in a growth medium comprising quantities of one or more nutrients which are insufficient for homeostatic algal cell growth, wherein optionally the nutrients are nitrogen or phosphorus.

33. The method according to claim 31 wherein the algal cell while grown in the growth medium is subjected to an exogenous stress factor, optionally selected from the group consisting of a non-homeostatic pH, a non-homeostatic salinity, and a non-homeostatic light intensity.

36. The method according to claims 31, wherein the algal cell during a first time period is grown under homeostatic growth conditions wherein during such first time period substantially no accumulated lipid particles are formed, and wherein the algal cell during a second time period is grown under non-homeostatic growth conditions.

37. The method according to claim 36 wherein during the first period the algal cell is grown to logarithmic growth phase, and wherein during logarithmic phase the cells are subjected to non-homeostatic growth conditions to grow the cell under non-homeostatic conditions during the second time period.

38. The method according to claim 36 wherein during the first period the algal cell is grown to early stationary phase, and wherein during early stationary phase the cells are subjected to non-homeostatic growth conditions to grow the cell under non-homeostatic conditions during the second time period.

40. The method according to claim 31 wherein the oleosin is a protein encoded by a nucleic acid sequence having the sequence set forth in SEQ.ID NO:

7 to SEQ.ID NO;

12.

41. The method according to claim 31 wherein the algal cell is selected form the group of algal cells consisting of green algae (Chlorophyceae), diatoms (Bacillariophyceae), yellow-green algae (Xanthophyceae), golden algae (Chrysophyceae), red algae (Rhodophyceae), brown algae (Phaeophyceae), dinoflagellates (Dinophyceae) or pico-plankton (Prasinophyceae and Eustigmatophyceae), wherein the green algae optionally belongs to the genus Clamydomonas, or Chlorella.

34-35. -35. (canceled)

39. (canceled)

42-47. -47. (canceled)