Abstract
Phaeodactylum tricornutum is a well-developed model diatom for both marine ecology and microalgal biotechnology, which has been enabled by the sequenced genome and the availability of gene delivery tools, such as biolistic transformation and E. coli-mediated conjugation. Till now, these tools have mainly relied on two selectable markers of bacterial origin which confer resistance to antibiotics Zeocin and nourseothricin. An alternative cost-effective and preferably endogenous selectable marker would facilitate gene stacking efforts through successive transformation or conjugation. We performed UV-mutagenesis of P. tricornutum to obtain mutations in the phytoene desaturase (PDS) gene, conferring resistance to the bleaching herbicide norflurazon. Two mutants displaying high tolerance to norflurazon and carrying unique mutations in PtPDS1 (PHATRDRAFT_45735) were selected. These mutants revealed novel point mutations at a conserved residue Gly290 to Ser/Arg. Homology-based structural modeling of mutated PDS1, over a resolved crystallographic model of rice PDS1 complexed with norflurazon, suggests steric hindrance by bulkier residue substitution may confer herbicide resistance. We report the characterization of PtPDS1 mutants and the development of the first endogenous selectable marker in diatoms suitable for industrial strain development, with the added benefit of biocontainment. The plasmid carrying the mutated PDS1 as a selection marker and eGFP as a reporter was created. An optimized biolistic transformation system is reported which allowed the isolation of positive transgenic events at the rate of 96.7%. Additionally, the ease of in vivo UV-mutagenesis may be employed as a strategy to create PDS-norflurazon-based selectable markers for other diatoms.
Highlights
Diatoms are an extremely morphologically diverse group of photosynthetic microalgae that inhabit most aquatic environments and contribute approximately 20 to 40% of atmospheric O21,2
For the development of an endogenous selection marker, suitable for genetic engineering in P. tricornutum, we focused on the resistance to the herbicide norflurazon
Uniform Aniline Blue permeabilization and staining of treated wild type (WT) P. tricornutum cells was observed at 1.5 μM and higher norflurazon concentrations, which was associated with a breakdown of cellular membranes, a desintegration of the chloroplast and a loss of cell viability (Supplementary Fig. S1)
Summary
Diatoms are an extremely morphologically diverse group of photosynthetic microalgae that inhabit most aquatic environments and contribute approximately 20 to 40% of atmospheric O21,2. Numerous genetic engineering tools, such as gene delivery[21,22,23,24], genome editing[15,25,26,27,28,29] and gene silencing[30], have been established to explore the genome information and empower the biotechnological exploitation of P. tricornutum These tools depend on an efficient selectable marker for the selection of transgenic events. Disruption of native genes leading to loss of function, such as PtUMPS and PtAPT, have been reported as counter selectable markers in P. tricornutum for genome editing applications[29] Their utility in generating transgenic events is limited due to the availability of a functional copy in the recipient cells which metabolizes the synthetic metabolite analog into a suicidal product leading to mortality of all cells. The PtPDS1 sequence from the most tolerant mutant was used to create the first endogenous selectable marker in P. tricornutum that is suitable for efficient selection of transgenic events and use in the production of industrial strains ensuring biocontainment
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