5-05: Algal starch levels typically found in nutrient deprived cultures produced under replete conditions in an engineered strain of Chlamydomonas reinhardtii

The saccharification of starch coupled with fermentation to ethanol is a mature technology that currently provides the majority of biofuels in the United States. However, traditional food-based starch feedstocks, such as corn, cannot meet our current fuel demands and their use remains controversial. Microalgae have been of recent interest for use as a biofuel feedstock because they can produce large quantities of carbohydrates, contain little recalcitrant biomass, and do not impact the food supply. The primary carbohydrate found in the green alga Chlamydomonas reinhardtii is starch, but high yields of this bioenergy carrier are usually only found under conditions of nutrient stress (N, S, P).  We have introduced a native enzyme key to starch biosynthesis, isoamylase 1 (Isa1), into the wildtype and Isa1 knock out (sta7) strains of C. reinhardtii, which has resulted in a starch excess phenotype. These mutant strains accumulate 3 to 4 fold more total glucan under nutrient-replete conditions by diverting metabolic flux into starch biosynthesis at the expense of cell division and protein synthesis. We have characterized isoamlyase 1 transcript expression and protein abundance and activity using q-RT-PCR, proteomics, and zymograms. Changes in the whole cell proteome and starch granule sub-proteome were determined. Starch granule size, morphology, crystallinity, and branching frequency were characterized, with differences detected between granules produced by wildtype C. reinhardtii and starch excess strains. The role of isoamylase in starch granule biosynthesis, possible mechanisms responsible for this phenotype, and implications for biofuel production will be discussed.