Inducible expression of genes in algae
The present application provides novel algal regulatory elements including inducible nitrate/nitrite promoter sequences and terminator sequences. The application further discloses DNA constructs comprising these novel regulatory elements, and recombinant microorganisms comprising these regulatory elements. Methods of modifying, producing, and using the regulatory elements are also disclosed. Methods disclosed in the present application are suited for inducible expressions of genes, such as a transgene or a native gene in algal species.
1. A genetically engineered alga comprising a DNA sequence comprising an algal nitrite reductase inducible promoter having at least 90% sequence identity to SEQ ID NO: 1 operably linked to a DNA of interest encoding a polypeptide or a functional RNA, wherein the DNA of interest encoding a polypeptide or a functional RNA is not regulated by the promoter in nature, and wherein the DNA molecule is integrated into the algal genome.
2. The genetically engineered alga of claim 1 , wherein the algal nitrite reductase inducible promoter is from the same algal species as the genetically engineered alga.
3. The genetically engineered alga of claim 2 , wherein the DNA of interest encodes a polypeptide, and wherein the polypeptide is heterologous to the algal species.
4. The genetically engineered alga of claim 3 , wherein the DNA of interest comprises at least one intron, wherein the intron is heterologous to the DNA of interest encoding a polypeptide or a functional RNA.
5. The genetically engineered alga of claim 2 , wherein the DNA of interest encodes a (a) a protein associated with lipid biosynthesis, (b) a lipase, (c) a protein that participates in photosynthesis, (d) a protein associated with carbon fixation, (e) a transporter protein, (f) a dehydrogenase, (g) a transcription factor, (h) a transcriptional activator, (i) a cell signaling protein, (j) an enzyme, (k) a reporter protein, (l) a selectable marker, or (m) a recombinase.
6. The genetically engineered alga of claim 5 , wherein the DNA of interest encodes Cre recombinase.
7. The genetically engineered alga of claim 1 , wherein the DNA molecule further comprises a terminator sequence operably linked to the DNA of interest encoding a polypeptide or a functional RNA.
8. The genetically engineered alga of claim 7 , wherein the terminator comprises a sequence having at least 90% identity to at least 800 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO: 2.
9. The genetically engineered alga of claim 2 , wherein the alga belongs to a genus selected from the group consisting of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Elhpsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Pelagomonas, Phaeodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Skeletonema, Spyrogyra, Stichococcus, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, and Volvox.
10. An expression cassette comprising DNA molecule comprising an algal nitrite reductase inducible promoter having at least 90% sequence identity to SEQ ID NO: 1 operably linked to a DNA of interest encoding a polypeptide or a functional RNA, wherein the DNA of interest encoding the polypeptide or a functional RNA is not regulated by the promoter in nature, wherein the DNA of interest encodes (a) a protein associated with lipid biosynthesis, (b) a lipase, (c) a protein that participates in photosynthesis, (d) a protein associated with carbon fixation, (e) a transporter protein, (f) a dehydrogenase, (g) a transcription factor, (h) a transcriptional activator, (i) a cell signaling protein, (j) a metabolic enzyme, (k) a reporter protein, (l) a selectable marker, (m) a recombinase, n) an antisense sequence, (o) a shRNA, (p) an siRNA, (q) a gRNA, or (r) a ribozyme.
11. The expression cassette of claim 10 , further comprising a terminator sequence operably linked to the DNA of interest encoding the polypeptide or a functional RNA.
12. The expression cassette of claim 11 , wherein the terminator comprises a sequence having at least 90% identity to SEQ ID NO: 2.
13. The expression cassette of claim 10 , wherein the DNA molecule encoding a polypeptide or functional RNA comprises at least one intron, wherein the intron is heterologous to the DNA of interest encoding a polypeptide or functional RNA.
14. A method of selectively expressing a DNA of interest in an algal cell comprising: a) transforming an algal cell with an isolated DNA molecule comprising an algal nitrite reductase inducible promoter having at least 90% sequence identity to SEQ ID NO: 1 operably linked to a DNA of interest encoding the DNA of interest, wherein the DNA of interest encoding the DNA of interest is not regulated by the promoter in nature to generate transformed algal cells;
b) growing the transformed algal cells in a media that selectively permits the expression of the DNA of interest in the algal cell.
15. The method according to claim 14 , wherein the isolated DNA molecule is transformed by particle bombardment.
16. The method according to claim 14 , wherein the isolated DNA molecule is transformed by electroporation.
17. The method of claim 14 , wherein the promoter sequence is a nitrite reductase, and wherein the algal cells are grown in a media comprising Nitrate, wherein the expression of the DNA of interest is induced.
18. The method of claim 14 , wherein the algal cells are grown in a media comprising ammonium salt, wherein the expression of the DNA of interest is repressed.
19. The method of claim 14 , wherein the DNA of interest encodes (a) a protein associated with lipid biosynthesis, (b) a lipase, (c) a protein that participates in photosynthesis, (d) a protein associated with carbon fixation, (e) a transporter protein, (f) a dehydrogenase, (g) a transcription factor, (h) a transcriptional activator, (i) a cell signaling protein, (j) a metabolic enzyme, (k) a reporter protein, (l) a selectable marker, (m) a recombinase, n) an antisense sequence, (o) a shRNA, (p) an siRNA, (q) a gRNA, or (r) a ribozyme.
20. The method of claim 14 , wherein the DNA molecule comprises a terminator sequence operably linked to the DNA of interest.
21. The method of claim 20 , wherein the terminator comprises a sequence having at least 90% identity to SEQ ID NO: 2.
22. The method of claim 14 , wherein the DNA molecule encoding the DNA of interest comprises at least one intron, wherein the intron is heterologous to the DNA of interest.
23. A genetically engineered alga of claim 1 , wherein the alga is a Trebouxiophyte alga.
24. A genetically engineered alga of claim 23 , wherein the alga is from the genus Parachlorella.