Codon optimization for increasing transgene expression in chloroplasts of higher seed plants

A method for increasing translation of a transgene encoding a protein of interest in a chloroplast, said method comprising

• a) replacing codons in a native sequence encoding a protein of interest with preferred codons preferentially used in psbA genes in chloroplasts in higher seed plants species;
• b) producing a synthetic, codon optimized sequence and cloning said sequence into a chloroplast transformation vector, said synthetic sequence being operably linked to 5′ and 3′ regulatory elements suitable for expression in said chloroplast;
• c) transforming a target plant with said vector, under conditions whereby said protein of interest is expressed, wherein replacing codons in said native sequence with preferred codons causes at least a two fold increase in protein expression relative to expression levels observed using the native sequence
• wherein
• each codon for phenylalanine is replaced with a preferred TTT codon;
• each codon for leucine is replaced with a preferred TTA codon;
• each codon for isoleucine is replaced with a preferred ATT codon;
• each codon for valine is replaced with a preferred GTA codon;
• each codon for serine is replaced with a preferred TCT codon;
• each codon for proline is replaced with a preferred CCT codon;
• each codon for threonine is replaced with a preferred ACT codon;
• each codon for alanine is replaced with a preferred GCT codon;
• each codon for tyrosine is replaced with a preferred TAT codon;
• each codon for histidine is replaced with a preferred CAC codon;
• each codon for glutamine is replaced with a preferred CAA codon;
• each codon for asparagine is replaced with a preferred AAC codon;
• each codon for lysine is replaced with a preferred AAA codon;
• each codon for aspartic acid is replaced with a preferred GAT codon;
• each codon for glutamic acid is replaced with a preferred GAA codon;
• each codon for cysteine is replaced with a preferred TGT codon;
• each codon for tryptophan is replaced with a preferred TGG codon;
• each codon for arginine is replaced with a preferred CGT codon;
• and
• each codon for glycine is replaced with a preferred GGT codon.