Metabolic engineering and systems biology for increasing biofuel production in microalgae

Abstract

The saccharification of starch coupled with fermentation to ethanol and the transesterification of vegetable oils to produce biodiesel are mature technologies that currently provide the majority of biofuels in the United States. However, traditional food-based starch and oil feedstocks, such as corn and soybean, 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 and lipids, contain little recalcitrant biomass, and do not impact the food supply. In the green alga Chlamydomonas reinhardtii, the primary carbohydrate produced is starch and the primary storage lipid produced is triacylglyceride (TAG), but high yields of these bioenergy carriers are usually only found under conditions of nutrient stress (N, S, P). As a strategy for increasing TAG yields, starchless mutants that divert carbon away from starch biosynthesis and into TAG production were investigated. These starchless mutants produce more TAG than wildtype, but at the expense of total productivity and lower overall anabolic activity. To increase starch levels, a native enzyme key to starch biosynthesis, isoamylase 1 (ISA1), was introduced into these algae which resulted in starch excess phenotypes. 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. The starches produced by these algae are more crystalline and larger in size than wildtype. None of the current genetically-tractable model algal species are competitive TAG production strains. To alleviate this the commercially cultivated, oleaginous alga Nannochloropsis gaditana CCMP526 was developed into a new model algal species for investigating TAG production. The genome and transcriptome was sequence and assembled, gene models developed, and metabolic pathways in this organism were reconstructed. Phylogenomic analysis identified genetic attributes of this organism, including gene expansions and unique stramenopile photosynthesis genes, that may explain the distinguishing oleaginous phenotypes observed. The availability of a genome sequence and transformation method are facilitating investigations into N. gaditana lipid biosynthesis and will permit genetic engineering strategies to further improve this naturally productive algal strain.