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

• Published in: Algal research (Amsterdam) Vol. 58; no. C; p. 102385
• Main Authors: Vogler, Brian W., Ashford, Amy, Posewitz, Matthew C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier B.V 01.10.2021; Elsevier
• Subjects: Carbohydrate; CRISPR/Cas9; Glucan synthase; Nannochloropsis; Transglycosylase; Nannochloropsis; Glucan synthase; Carbohydrate; Transglycosylase; CRISPR/Cas9
• ISSN: 2211-9264; 2211-9264
• DOI: 10.1016/j.algal.2021.102385

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. •Cas9 mediated knockout of β-glucan synthase or a putative branching enzyme.•Replicate genetically distinct knockouts of each gene demonstrated significantly reduced carbohydrate accumulation.•Total fatty acid content increases without significant effects on cellular growth