Reduction of UDP-glucose diphosphorylase(UGP2) gene expression does not reduce accumulation of the diatom storage sugar chrysolaminarinin Phaeodactylum tricornutum

Zhang, Yun, author
Caballero, Michael, author
Peers, Graham, author
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Diatoms are ecologically significant microalgae, responsible for 40% of the ocean's primary productivity. Diatoms distribute fixed carbon into metabolic pools such as carbohydrate, lipid, and protein. We are interested in exploring the unusual storage carbohydrate of diatoms, chrysolaminarin, which has the same composition and function as starch from plants, but a different structure (‘-1,3 and ‘-1,4 linked glucans, respectively). Decreasing carbon partitioning into chrysolaminarin may increase diatom lipid productivity for biofuels. The synthesis and degradation pathways for chrysolaminarin are unknown. Biochemical evidence suggests that making UDP glucose is the first step of chrysolaminarin synthesis. The sequenced genome of the model diatom Phaeodactylum tricornutum encodes tow predicted gene products that may make UDP glucose (UGP1, UGP2). We investigate the contribution of UGP2 to diatom carbohydrate accumulation using an RNAi approach by constructing two independent antisense knockdown vectors with unique, non-overlapping amplicons from UGP2. We identified four UGP2 knockdown strains with reduced gene expression levels by a qRT-PCR screen. These mutants did not have different growth rates of chrysolaminarin per cell compared to wild-type controls. These data suggest the UGP2 does not significantly contribute to chrysolaminarin metabolism. However, partial gene knockdowns may be adequate to reduce chrysolaminarin syntheses. Therefore, UGP2 CRISPR/Cas9 knockouts are in development to study the role of UGP2 in chrysolaminarin biology. These studies improve our understanding of diatom central carbon metabolism, which may inform bioengineering strategies to produce biofuels.
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