CRISPR/Cas9 disruption of glucan synthase in Nannochloropsis gaditana attenuates accumulation of β-1,3-glucose oligomers

Abstract Nannochloropsis species have garnered significant interest for biofuel production due to their ability to accumulate high levels of triacylglycerols (TAGs), especially following nitrogen-starvation. However, the first response to nutrient starvation is the synthesis of chrysolaminarin, a soluble β-1,3-glucan with β-1,6-branching. We employ CRISPR/Cas9 to knock out two key enzymes responsible for the synthesis of this oligosaccharide: a beta-glucan synthase (BGS) gene putatively responsible for the glucose β-1,3-linkages, and a transglycosylase (TGS) which putatively catalyzes β-1,6-branching. Analysis of the biomass from the generated mutants confirmed an ~5-fold decrease in the accumulation of soluble carbohydrate following nitrogen starvation, without an observed growth defect in a diel light-cycling regime compared to CRISPR-expressing controls.