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. 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 bacterial neomycin phosphotransferase protein that confers resistance to kanamycin or a chloramphenicol acetyl transferase protein that confers resistance to chloramphenicol;
wherein the polynucleotide comprises a transgene encoding the protein of interest that is codon-optimized for expression in cyanobacteria and is joined to the 3′ end of a nucleic acid encoding the bacterial neomycin phosphotransferase (NPT) protein or the chloramphenicol acetyltransferase protein.
2. The expression construct of claim 1 , wherein the bacterial neomycin phosphotransferase polypeptide is NPT1.
3. The expression construct of claim 1 , wherein the transgene encodes a terpene synthase.
4. The expression construct of claim 3 , wherein the terpene synthase is an isoprene synthase.
5. The expression construct of claim 3 , wherein the terpene synthase is a monoterpene synthase.
6. The expression construct of claim 5 , wherein the monoterpene synthase is beta-phellandrene synthase.
7. The expression construct of claim 6 , wherein the beta-phellandrene synthase is lavender, tomato, grand fir, pine, or spruce beta-phellandrene synthase.
8. The expression construct of claim 3 , wherein the terpene synthase is a sesquiterpene synthase.
9. The expression construct 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. A host cell comprising the expression construct of claim 1 .
11. The host cell of claim 10 , wherein the host cell is a cyanobacteria host cell.
12. The host cell of claim 11 , wherein the cyanobacteria host cell is a single celled cyanobacteria.
13. The host cell of claim 12 , where the single-celled cyanobacteria is a Synechococcus sp., a Thermosynechococcus elongatus , a Synechocystis sp., or a Cyanothece sp.
14. The host cell of claim 11 , wherein the cyanobacteria host cell is a micro-colonial cyanobacteria.
15. The host cell of claim 14 , wherein the micro-colonial cyanobacteria is a Gloeocapsa magma, Gloeocapsa phylum, Gloeocapsa alpicola, Gloeocpasa atrata, Chroococcus spp., or Aphanothece sp.
16. The host cell of claim 11 , wherein the cyanobacteria host cell is a filamentous cyanobacteria.
17. The host cell of claim 16 , wherein the filamentous cyanobacteria is an Oscillatoria spp., a Nostoc sp., an Anabaena sp., or an Arthrospira sp.
18. A cyanobacterial cell culture comprising cyanobacteria of claim 11 .
19. A method of expressing a transgene encoding a protein of interest in cyanobacteria, the method comprising culturing a cyanobacterial cell culture of claim 18 under conditions in which the transgene is expressed.
20. A method of producing a terpenoid in cyanobacteria, the method comprising culturing a cell culture of claim 18 under conditions in which the transgene is expressed.
21. 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.
22. The expression construct of claim 2 , wherein the bacterial antibiotic resistance protein is the neomycin phosphotransferase polypeptide and the transgene encodes a terpene synthase.
23. A host cell comprising the expression construct of claim 22 .
24. The host cell of claim 23 , wherein the host cell is a cyanobacterial host cell.