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 nucleic acid sequence encoding a fusion protein comprising (a) a protein of interest encoded by a transgene and (b) a polypeptide leader sequence, wherein the transgene is codon-optimized for expression in cyanobacteria, and is fused to the 3′ end of a nucleic acid sequence encoding the polypeptide leader sequence, wherein the polypeptide leader sequence is a cyanobacteria protein that is expressed in cyanobacteria at a level of at least 1% of the total cellular protein.
2. The expression construct of claim 1 , wherein the transgene fused to the 3′ end of the leader nucleic acid sequence encoding the polypeptide leader sequence is a β-subunit of phycocyanin (cpcB), an α-subunit of phycocyanin (cpcA), a phycoerythrin subunit (cpeA or cpeB), an allophycocyanin subunit (apcA or apcB), a large subunit of Rubisco (rbcL), a small subunit of Rubisco (rbcS), a D1/32 kD reaction center protein (psbA) of photosystem-II, a D2/34 kD reaction center protein (psbD) of photosystem-II, a CP47 (psbB) or CP43 (psbC) reaction center protein of photosystem-II, a psaA or psaB reaction center protein of photosystem-I, a psaC or psaD reaction center protein of photosystem-I, an rpl ribosomal RNA protein, or an rps ribosomal RNA protein.
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, a monoterpene synthase, or a sesquiterpene synthase.
5. The expression construct of claim 3 , wherein the terpene synthase is a beta-phellandrene synthase.
6. The expression construct of claim 3 , wherein the terpene synthase is lavender, tomato, grand fir, pine, or spruce beta-phellandrene synthase; a farnesene synthase, a zingiberene synthase, a caryophellene synthase, a longifolene synthase, a taxadiene synthase, or a dictyophorine synthase.
7. A host cell comprising an expression construct of claim 1 .
8. The host cell of claim 7 , wherein the host cell is a cyanobacteria host cell.
9. The host cell of claim 8 , wherein the cyanobacteria is single celled cyanobacteria, micro-colonial cyanobacteria, or filamentous cyanobacteria.
10. The host cell of claim 8 , where the cyanobacteria is a Synechococcus sp., a Thermosynechococcus elongatus , a Synechocystis sp., a Cyanothece sp., a Gloeocapsa magma , a Gloeocapsa phylum , a Gloeocapsa alpicola , a Gloeocpasa atrata , a Chroococcus spp., an Aphanothece sp., an Oscillatoria spp., a Nostoc sp., an Anabaena sp., or an Arthrospira sp.
11. A cyanobacterial cell culture comprising a cyanobacteria host cell of claim 8 .
12. A method of expressing a transgene at high levels, the method comprising culturing a cyanobacterial cell culture of claim 11 under conditions in which the transgene is expressed.
13. A method of producing a terpenoid in cyanobacteria, the method comprising culturing a cell culture of claim 11 under conditions in which the transgene is expressed, wherein the transgene encodes a terpene synthase.
14. A method of modifying a cyanobacterial cell to express a transgene at high levels, the method comprising introducing an expression construct of claim 1 into the cell.