Fusion constructs as protein over-expression vectors
View Patent ↗This invention provides compositions and methods for providing high product yield of transgenes expressed in cyanobacteria and microalgae.
1. A cyanobacterial host cell that comprises an expression construct comprising a polynucleotide encoding a fusion protein, said fusion protein comprising (i) a protein of interest to be expressed in cyanobacteria joined to (ii) a polypeptide comprising a β-subunit of phycocyanin (cpcB) or a polypeptide comprising an α- subunit of phycocyanin (cpcA),
wherein the polynucleotide encoding said fusion protein comprises a transgene encoding the protein of interest that is codon-optimized for expression in cyanobacteria joined to the 3′ end of a nucleic acid encoding the polypeptide comprising cpcB or the polypeptide comprising cpcA.
2. The cyanobacterial host cell of claim 1 , wherein the transgene encodes a terpene synthase.
3. The cyanobacterial host cell of claim 2 , wherein the terpene synthase is an isoprene synthase, a monoterpene synthase, or a sesquiterpene synthase.
4. The cyanobacterial host cell of claim 3 , wherein the terpene synthase is an isoprene synthase.
5. The cyanobacterial host cell of claim 3 , wherein the terpene synthase is a monoterpene synthase.
6. The cyanobacterial host cell of claim 5 , wherein the monoterpene synthase is beta-phellandrene synthase.
7. The cyanobacterial host cell of claim 6 , wherein the beta- phellandrene synthase is lavender, tomato, grand fir, pine, or spruce beta-phellandrene synthase.
8. The cyanobacterial host cell of claim 3 , wherein the terpene synthase is a sesquiterpene synthase.
9. The cyanobacterial host cell of claim 8 , wherein the sesquiterpene synthase is a farnesene synthase, a zingiberene synthase, a caryophellene synthase, a longifolene synthase, a taxadiene synthase, or a dictyophorine synthase.
10. The cyanobacterial host cell of claim 1 , wherein the cyanobacteria host cell is a single celled cyanobacteria.
11. The cyanobacterial host cell of claim 10 , where the single-celled cyanobacteria is a Synechococcus sp., a Thermosynechococcus elongatus , a Synechocystis sp., or a Cyanothece sp.
12. The cyanobacterial host cell of claim 1 , wherein the cyanobacteria host cell is a micro-colonial cyanobacteria.
13. The cyanobacterial host cell of claim 12 , wherein the micro-colonial cyanobacteria is a Gloeocapsa magma, Gloeocapsa phylum, Gloeocapsa alpicola, Gloeocpasa atrata, Chroococcus spp., or Aphanothece sp.
14. The cyanobacterial host cell of claim 1 , wherein the cyanobacteria host cell is a filamentous cyanobacteria.
15. The cyanobacterial host cell of claim 14 , wherein the filamentous cyanobacteria is an Oscillatoria spp., a Nostoc sp., an Anabaena sp., or an Arthrospira sp.
16. A cyanobacterial cell culture comprising the cyanobacterial host cell of claim 1 .
17. A method of expressing a transgene at high levels, the method comprising culturing a cyanobacterial cell culture of claim 16 under conditions in which the transgene is expressed.
18. The method of claim 17 , wherein the transgene encodes a terpene synthase.
19. An expression construct comprising a polynucleotide encoding a fusion protein, said fusion protein comprising a protein of interest to be expressed in cyanobacteria joined to a polypeptide comprising a β-subunit of phycocyanin (cpcB) or comprising an α-subunit of phycocyanin (cpcA),
wherein the polynucleotide comprises a transgene encoding the protein of interest that is codon-optimized for expression in cyanobacteria that is joined to the 3′ end of a nucleic acid encoding the cpcB or cpcA.
20. A method of modifying a cyanobacterial cell to express a transgene, the method comprising introducing an expression construct of claim 1 into the cyanobacterial cell.