Expression of the bacterial ectC gene in the chloroplast of Chlamydomonas reinhardtii

Abstract

The goal of this work was to express the three enzymes that catalyse the synthesis of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) in the chloroplast of Chlamydomonas reinhardtii. Ectoine is an amino acid derivative that functions in many bacteria as compatible solute, helping the bacteria to survive and grow in highly saline environments. In addition, ectoine is used in cosmetic products, in the medical industry and for biotechnological purposes. There is also a potential relevance to agriculture, as synthesis of ectoine in cells of plants or algae could make them tolerant towards salt concentrations that would normally prohibit growth. There is an ongoing interest in developing efficient production systems for ectoine. Overexpression of ectoine in the chloroplast may lead to both an efficient and economical way to produce ectoine. There are three bacterial genes, ectA, ectB and ectC, that encode the enzymes catalysing ectoine synthesis in bacteria. We wanted to insert the three genes into a suitable vector and transform the resulting construct into the chloroplast of C. reinhardtii in order to produce ectoine in C. reinhardtii cells and increase salt tolerance. A non-photosynthetic C. reinhardtii mutant cell line was transformed by microprojectile bombardment with a plasmid vector containing a photosynthesis marker (the atpB gene) and the codon optimised transgenes OectA and OectC. Cloning of ectB was not possible in the time frame of this work. Six OectAC chloroplast transformants (out of 30) were selected and screened for the presence of the OectC gene. Two positive transformants were further analysed for ectC mRNA accumulation. Very low levels of ectC transcripts could be detected in the two transformants, but no increase of salt tolerance was observed. It is concluded that OectC mRNA levels in the analysed transformant is too low for ectoine accumulation and that more transformants should be screened for the presence of the OectAC construct. In addition, insertion of the ectB gene could be performed in order to aid ectoine synthesis.