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

Abstract

Ectoine is a small molecule that confers salt tolerance to organisms. It is synthesized in bacteria by the gene products of ectA, ectB, and ectC. Ectoine is used in medical, biotechnological, and cosmetic products.

The purpose of this project was to determine whether expression of the ectA gene in the chloroplast of the model alga Chlamydomonas reinhardtii would be sufficient for ectoine production.

Non-photosynthetic Chlamydomonas was transformed by microprojectile bombardment, using a plasmid vector with a chimeric ectA gene and a photosynthesis selection marker. In addition, a transformation vector with a codon optimized version of the ectA gene was designed, and used for bombardment. Transformants were analyzed by DNA and RNA blotting techniques. SDS-PAGE, and mass spectrometry were used to evaluate EctA accumulation. HPLC, and salt tolerance experiments were used to evaluate the accumulation of ectoine.

One chloroplast transformant was isolated for the non-optimized ectA gene that was about fifty percent homoplasmic. No transformant could be isolated with the codon optimized ectA. Transcript accumulation was detected for the ectA transformant, however at low levels. EctA protein accumulation could not be detected. HPLC did not identify any ectoine accumulation, and no increased salt tolerance of the ectA transformant was observed.

It is concluded that the current chimeric ectA gene is insufficient in supporting high levels of ectoine accumulation, probably because of the low transcript levels supported, and the lack of codon optimization. Further work is needed to improve the accumulation of EctA to higher levels.