Development of New Genetic Manipulation Tools for Metabolic Engineering of Diatoms
Final rept. 1 Mar 2007-31 May 2008
CALIFORNIA UNIV SAN DIEGO LA JOLLA
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This projects goal was to develop genetic manipulation tools for metabolic engineering of diatoms for biodiesel lipid production and other purposes. New diatom selectable markers were tested, relying on mutations to antibiotic resistance in two ribosomal protein genes. Three new diatom transformation vectors were constructed, using the nat1 gene as selectable marker placed under control of either the Thalassiosira pseudonana ACCase promoter, Nitzschia alba rpL41 promoter, or SV40 promoter. An available vector using the fcp promoter was also evaluated. Successful transformation was achieved in T. pseudonana, T. oceanica, and T. weissflogii with the fcp promoter, with T. pseudonana using the ACCase and rpL41 promoters, and N. alba using the rpL4l promoter. A procedure for the enrichment of protoplasts from N. alba was developed, as well as a method to generate auxospores from T. pseudonana. Constructs were made to test for homologous gene replacement in T. pseudonana. A direct-selection RNAi vector was constructed using an inverted repeat from the T. pseudonana tryptophan synthase Beta subunit fused to nitrate reductase expression control elements. Initial attempts at transformation and selection were encouraging, with phenotypic results consistent with functional RNAi. In summary, this project has developed several new tools for diatom genetic manipulation.