Background: As interest in the use of eukaryotic microalgae as production platforms for biofuels and
bioproducts increases, it is important that species-specific tools are made available. Species-specific
molecular toolboxes will allow the engineering of improved performance to drive down manufacturing
costs to overcome the economic thresholds for production of commercially competitive products.
Historically, most genetic manipulation of eukaryotic microalgae focused on model algal strains such
as Chlamydomonas reinhardtii, Chlorella sp., and Phaeodactylum tricornutum. The strong foundation
built using model algal species now allows more rapid development of species-specific molecular tool
boxes as novel microalgae-based products are identified in new algal strains. Customized tools for
engineering improved production in strains that hold promise for fuels, foods, and high value
chemicals will be a focus of future research. A snapshot of the current status of eukaryotic algal molecular engineering
tools is provided plus data generated in our laboratory as we developed tools for manipulation of Auxenochlorella
Methods: Current methods for manipulation of eukaryotic microalgae are reviewed and manipulation of Auxenochlorella
protothecoides done in our laboratory is used to provide examples of some useful approaches. This paper broadly reviews
all the methods currently in use or being developed to generate transgenic eukaryotic microalgae.
Results: Provide original data on Auxenochlorella protothecoides for manipulation of the chloroplastic and nuclear
genomes. Design of vectors and different approaches are discussed.
Conclusion: Engineering improved strains of eukaryotic microalgae for economical production of biofuels and
bioproducts is becoming a reality. Traditional insertional random mutagenesis is being augmented with new tools for
directed strain improvement. The development and application of state of the art genome editing approaches to algal
production strains has a real potential to help overcome economic thresholds and to enable microalgae as biorefineries to
enter the marketplace alongside fossil fuels. The increased availability of sequenced genomes in combination with the
reduced cost of generating proprietary genome sequences positions genetic engineering of eukaryotic microalgae at the
threshold for rapid commercial application.