Targeting Nuclear-Encoded Recombinant Proteins to the Chloroplast in Microalgae

Abstract

The application generally relates to chloroplast targeting of nuclear-encoded proteins of interest in microalgae. Provided herein are expression cassettes comprising a nucleotide sequence encoding a chloroplast targeting peptide operably linked to a nucleotide sequence encoding a protein of interest, wherein said chloroplast targeting peptide comprises the bipartite targeting sequence of the phosphoribulokinase of Nannochloropsis gaditana (NgPRK BTS). The invention further provides vectors comprising the expression cassettes, and microalgae having stably incorporated or transiently expressed into their nuclear genomes an expression cassette described above. Methods are also provided for the production of a protein of interest in the chloroplast of a microalga, as well as methods for modulating chloroplast pathways.

1 .- 15 . (canceled) 
     
     
         16 . An isolated nucleic acid comprising:
 (a) a first nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 9, wherein said first nucleotide sequence encodes a chloroplast targeting peptide; and, operably linked to the first nucleotide sequence,   (b) a second nucleotide sequence encoding a protein of interest, wherein the protein of interest is not  Nannochloropsis gaditana  phoshoribulokinase (PRK), wherein the protein of interest is selected from the group comprising a chloroplast transporter, a protein of transcription or translation machinery, a transcription factors/enhancer/silencer, a nuclease, and a chaperone.   
     
     
         17 . A nucleic acid expression cassette comprising (a) a first nucleotide sequence encoding a chloroplast targeting peptide operably linked to a second nucleotide sequence encoding a protein of interest that is not  Nannochloropsis gaditana  PRK and (b) a promoter and a terminator operably linked to the first and second nucleotide sequences,
 wherein the chloroplast targeting peptide comprises SEQ ID NO: 8, and wherein the expression cassette ensures expression of the first and second nucleotide sequences in the nucleus of a host cell transformed with said expression cassette. wherein the protein of interest is selected from the group comprising a chloroplast transporter, a protein of transcription or translation machinery, a transcription factors/enhancer/silencer, a nuclease, and a chaperone.   
     
     
         18 . A vector comprising the expression cassette according to  claim 17 . 
     
     
         19 . A recombinant host cell which has been transformed with the expression cassette according to  claim 17  or the vector according to  claim 18 , wherein said host cell is a microalga. 
     
     
         20 . The host cell according to  claim 19 , wherein said microalga is a heterokont microalga selected from the group consisting of a  Nannochloropsis  species, a  Phaeodactylum  species, and combinations thereof. 
     
     
         21 . The host cell according to  claim 19 , wherein said microalga is the diatom  Nannochloropsis gaditana.