P17: Transgenic expression of a bacterial thermophilic amylase in the Chlamydomonas reinhardtii chloroplast

In order to investigate the feasibility of developing a  recombinant Chlamydomonas reinhardtii capable of hydrolyzing its own starch after cultivation to facilitate the starch utilization for biofuel production, a thermophilic amylase (arAmyBH) from hyperthermophilic bacterium Thermotoga neapolitana was introduced into C. reinhardtii chloroplast.  Properties of arAmyBH were studied for the application in starch hydrolysis. The optimum pH and temperature of arAmyBH were 5.5 and 65 °C, respectively. The hydrolysis performance of intracellular starch by arAmyBH was investigated. Without extraction or purification, around 20% of stored starch was hydrolyzed into oligosaccharides in vivo at 65 °C in 2 h. The dextrose equivalent (DE) value calculated from the reducing sugar was 14.9. Additionally, comparison of wild type and transformed cells demonstrated that expression of exogenous genes into chloroplast through our developed expression plasmid had no apparent negative effects on cell growth and starch accumulation. In the future study, endo-amylase such as alpha-amylase from Bacillus stearothermophilus would be introduced in the chloroplast of recombinant C. reinhardtii to achieve complete hydrolysis of microalgal starch.