Compositions and methods for expressing genes in algae
The present application provides novel regulatory elements including promoter sequences from microorganisms. The application further discloses DNA constructs containing these novel regulatory elements, and recombinant microorganisms comprising these regulatory elements. Methods of modifying, producing, and using the regulatory elements are also disclosed. The regulatory elements and transformation methods disclosed herein are particularly suited for use in Parachlorella and other microalgae.
1. An isolated DNA molecule comprising a promoter that comprises a nucleotide sequence having at least 80% identity to at least 100 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23 operably linked to a heterologous sequence encoding a polypeptide or functional RNA.
2. The isolated DNA molecule of claim 1 , comprising a terminator.
3. The isolated DNA molecule of claim 2 , wherein the terminator is derived from the same species as the promoter.
4. The isolated DNA molecule of claim 3 wherein the terminator comprises a sequence having at least 80% identity to at least 100 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24.
5. The isolated DNA molecule of claim 1 , wherein the heterologous sequence encoding a polypeptide or functional RNA comprises at least one heterologous intron.
6. The isolated DNA molecule of claim 5 , wherein the at least one heterologous intron is derived from the same species as the promoter.
7. The isolated DNA molecule of claim 5 , comprising at least three heterologous introns.
8. The isolated DNA molecule of claim 7 , comprising at least five heterologous introns.
9. The isolated DNA molecule of claim 7 , wherein the at least three heterologous introns are derived from the same species as the promoter.
10. The isolated DNA molecule of claim 9 , wherein the at least three heterologous introns are derived from the same gene.
11. The isolated DNA molecule of claim 10 , wherein the at least three heterologous introns and the promoter are derived from the same gene.
12. The isolated DNA molecule of claim 1 , wherein the heterologous sequence encodes a functional RNA selected from the group consisting of an antisense sequence, a micro RNA, a shRNA, an siRNA, a gRNA, and a ribozyme.
13. The isolated DNA molecule of claim 1 , wherein the heterologous sequence encodes a polypeptide.
14. An expression cassette comprising the isolated DNA molecule of claim 13 , wherein the heterologous sequence encodes a polypeptide selected from the group consisting of: (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, and (l) a selectable marker.
15. A method of transforming an algal cell, comprising introducing a vector comprising an expression cassette of claim 14 into an algal cell and selecting for a transformant.
16. The method of claim 15 , wherein the expression vector is introduced by particle bombardment.
17. The method of claim 15 , wherein the expression vector is introduced by electroporation.
18. The method of claim 17 , wherein the algal cell is a Chlorophyte algal cell.
19. The method of claim 18 , wherein the algal cell is of the Trebouxiophyceae class.
20. The method of claim 17 , wherein the algal cell is of a genus selected from the group consisting of Auxenochlorella, Chlorella, Heveochlorella, Marinichlorella, Parachlorella, Pseudochlorella , and Tetrachlorella.