(322f) Transgenic Expression of a Bacterial Exo-Acting Intracellular α-Amylase in the Chlamydomonas Reinhardtii Chloroplast

Microalgae with their relatively high starch and lipid contents are one of the most promising non-food-crop-based feedstocks for biofuels production. To date, most studies have focused on accumulating algal lipids for biodiesel production and some have focused on hydrogen generation. Very little attention has been paid to simultaneously converting algal carbohydrate and lipids into biofuels. Although some algal species are able to accumulate significant amounts of carbohydrates (mainly starch) and lipid, their rigid cell wall retards the extraction of those carbon-rich compounds. In order to overcome this barrier, this study constructed an amylase-producing strain of the unicellular green alga Chlamydomonas reinhardtii, with the goal of converting algal starch into soluble sugars in vivo, which would facilitate their recovery. An exo-acting intracellular thermophilic α-amylase AmyB from the hyperthermophilic bacterium Thermotoga neapolitana was used as the amylase gene of choice. The AmyB gene was synthesized with codon usage optimized for the C. reinhardtii chloroplast; it was cloned into chloroplast expression vector p666 to yield plasmid p666AmyB. The vector p666AmyB was inserted into the site between the psaB and atpA genes on the chloroplast genome of C. reinhardtii by micro-particle bombardment, without interruption of chloroplast gene function. PCR analysis indicated that AmyB was integrated into the chloroplast genome of the transformants, and western blotting demonstrated the AmyB protein accumulation. The activity of AmyB can reach 17.4 U/g dry biomass. Thus, α-amylase was successfully expressed within the chloroplast, accomplishing an important step towards improving the ability to harness and harvest the carbon-compounds synthesized by micoalgae.