Development of flow cytometric procedures for the efficient isolation of improved lipid accumulation mutants in a sp. microalga.

The successful development of microalgae-based biofuel production will rely on improvements in the amount and rate of fuel molecule precursor accumulation. Mutagenesis and selection is an attractive approach to improve fuel molecule productivity. Previous screening methods have been laborious, the numbers of mutants isolated have been small, and overall performance of mutants may have been compromised by the presence of deleterious secondary mutations generated by random mutagenesis that affect other cellular processes and growth. We report an improved method of isolating high triacylglycerol (TAG) accumulating mutants of a sp., KAS603, using flow cytometric-based selection. In addition to selection for high TAG accumulating strains, the method requires that growth of mutants be competitive with other cells in the population. Not only is growth competitive, but there is improved performance in TAG accumulation with repeated selection, suggesting purification from deleterious secondary mutations. The procedure resulted in the isolation of mutants with far higher efficiency (thousands of fold) that outperformed wild type substantially better (1.8-2.5-fold) than with previous methods. This opens the door to new approaches to the characterization of genes involved in TAG accumulation and other cellular processes.