Inducible expression of genes in algae

Abstract

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.

What is claimed is: 
     
         1 . An isolated DNA molecule comprising an algal nitrate reductase or nitrite reductase inducible promoter operably linked to a DNA of interest encoding a polypeptide or functional RNA, wherein the DNA of interest encoding a polypeptide or functional RNA is not regulated by the promoter in nature. 
     
     
         2 . The isolated DNA molecule according to  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 according to  claim 3  wherein the terminator comprises a sequence having at least 80% identity to at least 75 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. 
     
     
         5 . The isolated DNA molecule of  claim 1 , wherein the promoter 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 NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, and 49. 
     
     
         6 . The isolated DNA molecule of  claim 1 , wherein the DNA of interest encoding a polypeptide or functional RNA is heterologous to the promoter sequence. 
     
     
         7 . The isolated DNA molecule of  claim 1 , wherein the DNA of interest encoding a polypeptide or functional RNA and the promoter are from the same algal species, wherein the DNA of interest and the promoter are not juxtaposed to each other in nature. 
     
     
         8 . The isolated DNA molecule according to  claim 6 , wherein the DNA of interest 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. 
     
     
         9 . The isolated DNA molecule according to  claim 8 , wherein the at least one heterologous intron is derived from the same species as the promoter. 
     
     
         10 . The isolated DNA molecule of  claim 8 , comprising at least three heterologous introns. 
     
     
         11 . The isolated DNA molecule according to  claim 10 , comprising at least five heterologous introns. 
     
     
         12 . The isolated DNA molecule according to  claim 10 , wherein the at least three heterologous introns are derived from the same species as the promoter. 
     
     
         13 . The isolated DNA molecule according to  claim 12 , wherein the at least three heterologous introns are derived from the same gene. 
     
     
         14 . The isolated DNA molecule according to  claim 13 , wherein the at least three heterologous introns and the promoter are derived from the same gene. 
     
     
         15 . The isolated DNA molecule of  claim 8 , wherein the DNA of interest 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. 
     
     
         16 . The isolated DNA molecule of  claim 1 , wherein the promoter and the terminator are from the same gene. 
     
     
         17 . The isolated DNA molecule of  claim 1 , wherein the promoter and the terminator are from different genes. 
     
     
         18 . The isolated DNA molecule of  claim 16 , 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) an enzyme, (k) a reporter protein, (l) a selectable marker, or (m) a recombinase. 
     
     
         19 . The isolated DNA molecule of  claim 18 , wherein the DNA of interest encodes Cre recombinase.