Homologous recombination in Nannochloropsis: a powerful tool in an industrially relevant alga.

T he availability of facile methods for targeted gene knockout and gene replacement based on homologous recombination in bacteria and yeast systems has driven rapid progress in understanding many of the complex metabolic and regulatory networks in prokaryotic and eukaryotic cells. The lack of such tools in other organisms is a major impediment to progress both in fundamental research and in research that is directed toward practical economic and societal outcomes. For example, the current goal to replace fossil fuels with renewable energy sources produced by plants and algae is hampered in both the short term and the long term by our limited knowledge of their metabolic systems and how they can be modified to create organisms that produce more and better energy-rich molecules. Thus, the report in PNAS by Kilian et al. (1) of efficient and reliable genetic transformation of the commercially important alga, Nannochloropsis, via homologous recombination is a significant step forward. Nannochloropsis species have been used for several decades to produce nutraceuticals and feed supplements (2–5). Several Nannochloropsis species have received attention more recently as oil-rich organisms with promise as a source of algal biofuels (6–9). Nonetheless, relatively little is known about the biology of this algal species, including the genes and enzymes involved in lipid biosynthesis. This situation is changing rapidly with the advent of important new tools, not the least of which is the ability to perform targeted gene knockouts and gene replacements using the methods of Kilian et al. (1). Coupled with the availability of genome sequences from at least a dozen independent genome sequencing and transcriptomics projects (www.ncbi.nlm.nih. gov/bioproject?term=nannochloropsis), a rapidly growing community of academic and industrial Nannochloropsis researchers (over 100 publications and patents thus far in 2011), and new DNA delivery methods (10), one or more Nannochloropsis species may emerge as significant new model alga systems that can build on progress made with Chlamydomonas reinhardtii, the most thoroughly studied alga to date (11). To demonstrate gene replacement by homologous recombination in Nannochloropsis sp. (strain W2J3B), Kilian et al. (1) target the nitrate reductase (NR) and nitrite reductase (NiR) genes. WT cells can grow using ammonium (NH4 ), nitrate (NO3 ), or nitrite (NO2 ) as a nitrogen source. Mutants lacking a functional NR gene grow in the presence of NH4 +