Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast
View Patent ↗Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.
1. A method of expressing a gene comprising:
transforming an algal cell by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence, a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site; and
cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.
2. The method of claim 1 , wherein the heterologous coding sequence is driven by a cognate promoter of the endogenous chloroplast gene.
3. The method of claim 1 , wherein the chimeric construct comprises at least one non-cognate UTR to the endogenous chloroplast gene.
4. The method of claim 1 , wherein the endogenous chloroplast gene exhibits autoregulation of translation.
5. The method of claim 4 , wherein the endogenous chloroplast gene encodes a photosystem II (PSII) reaction center protein.
6. The method of claim 2 , wherein the endogenous chloroplast gene re-inserted at the distal site is driven by a non-cognate promoter.
7. The method of claim 2 , wherein the endogenous chloroplast gene re-inserted at the distal site is driven by a cognate promoter.
8. The method of claim 7 , wherein a gene product of the heterologous coding sequence is repressed.
9. The method of claim 6 , wherein expression of a gene product encoded by the heterologous coding sequence is not repressed.
10. The method of claim 9 , wherein the expression of the gene product is modulated by light.
11. The method of claim 1 , wherein a gene product encoded by the heterologous coding sequence is constitutively expressed.
12. The method of claim 5 , wherein the transformed cell is cultivated in media for photosynthetic growth.
13. The method of claim 1 , wherein the cells are homoplasmic for the integration.
14. The method of claim 1 , wherein codons of the heterologous coding sequence are optimized to reflect abundantly expressed genes of the algal cell.
15. The method of claim 1 , wherein the endogenous chloroplast gene is psbA.
16. The method of claim 2 , wherein the cognate promoter is a psbA promoter.
17. The method of claim 6 , wherein the non-cognate promoter is a psbD promoter.
18. The method of claim 1 , wherein the heterologous coding sequence comprises SEQ ID NO:1.
19. The method of claim 1 , wherein one UTR is a psbD 5′ UTR.
20. The method of claim 1 , wherein expression of the chimeric construct is determined by accumulation of mRNA encoded by the heterologous coding sequence.
21. The method of claim 1 , wherein expression of the chimeric construct is determined by accumulation of protein encoded by the heterologous coding sequence.
22. The method of claim 1 , wherein the heterologous coding sequence encodes a peptide, a protein, a protein complex, or a combination thereof.
23. The method of claim 22 , wherein the heterologous coding sequence encodes a protein.
24. The method of claim 23 , wherein the protein is a mammalian protein.
25. The method of claim 24 , wherein the mammalian protein is bioactive in the gut.
26. The method of claim 23 , wherein the protein is mammary associated serum amyloid A3 (M-SAA3).
27. The method of claim 26 , wherein the M-SAA3 is bovine M-SAA3.
28. The method of claim 1 , wherein the heterologous coding sequence is optimized for plastid codon usage.
29. The method of claim 23 , wherein the protein is a reporter protein or a mutant variant thereof.
30. The method of claim 23 , wherein the protein is a gut active protein.
31. An algal cell transformed by the method of claim 1 .
32. The algal cell of claim 31 , wherein the alga is Chlamydomonas reinhardtii.
33. A method of expressing a gene comprising:
transforming an algal cell by replacing psbA via integration of a chimeric construct comprising a nucleic acid sequence encoding SEQ ID NO:1, a promoter sequence cognate or non-cognate to psbA, and at least one UTR, wherein psbA is re-inserted at a silent distal site; and
cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.
34. The method of claim 33 , wherein the at least one UTR is psbA or psbD UTRs.
35. The method of claim 33 , wherein SEQ ID NO:1 is driven by a psbA or psbD promoter.
36. The method of claim 33 , wherein psbA at the distal site is driven by a psbA or psbD promoter.
37. The method of claim 33 , wherein the promoters driving SEQ ID NO: 1 and the re-inserted psbA are different.
38. The method of claim 37 , wherein expression of the product encoded by SEQ ID NO:1 is modulated by light.
39. An algal cell transformed by the method of claim 33 .
40. The algal cell of claim 39 , wherein the alga is Chlamydomonas reinhardtii.
41. An algal extract obtained from an algal cell transformed by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence, a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site.
42. The algal extract of claim 41 , wherein the replaced gene is psbA and a product encoded by the heterologous coding sequence is a peptide as set forth in SEQ ID NO:2.
43. The algal extract of claim 42 , wherein the peptide comprises approximately 0.25% to about 6% of the total protein in the extract.
44. The algal extract of claim 41 , wherein the alga is Chlamydomonas reinhardtii.
45. A method of expressing a gene comprising:
transforming an algal cell by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence encoding mammary associated serum amyloid A3 (M-SAA3), a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site; and
cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.
46. The method of claim 1 , wherein the heterologous coding sequence is driven by a non-cognate promoter of the endogenous chloroplast gene.
47. The method of claim 46 , wherein the endogenous chloroplast gene re-inserted at the distal site is driven by a non-cognate promoter.
48. The method of claim 46 , wherein the endogenous chloroplast gene re-inserted at the distal site is driven by a cognate promoter.
49. The method of claim 47 , wherein a gene product of the heterologous coding sequence is repressed.
50. The method of claim 48 , wherein expression of a gene product encoded by the heterologous coding sequence is not repressed.
51. A method of expressing a gene comprising:
transforming an algal cell by replacing an endogenous chloroplast gene via integration of a chimeric construct having a heterologous coding sequence, a promoter sequence cognate or non-cognate to the endogenous chloroplast gene, and at least one UTR, wherein the endogenous chloroplast gene is re-inserted at a silent distal site and driven by a non-cognate promoter; and
cultivating the transformed algal cell under conditions to allow for expression of the chimeric construct.