The concept of adaptive evolution implies underlying genetic mutations conferring a selective advantage to an organism under particular environmental conditions. Thus, a flow cytometry-based strategy was used to study the adaptive evolution in Chlamydomonas reinhardtii wild-type strain CC124 and starchless mutant sta6-1 cells, with respect to lipid metabolism under
nitrogen-(N) depleted and -replete conditions.
RESULTS: The successive sorting and regeneration of the top 25,000 high-
lipid content cells of CC124 and sta6-1, combined with
nitrogen starvation, led to the generation of a new population with an improved
lipid content when compared to the original populations (approximately 175% and 50%
lipid increase in sta6-1 and CC124, respectively). During the adaptive evolution period, the major
fatty acid components observed in cells were C16:0, C16:1, C18:0, and C18:1-3, and elemental analysis revealed that cellular
carbon to
nitrogen ratio increased at the end of adaptive evolution period In order to gain an insight into highly stimulated intracellular
lipid accumulation in CC124 and sta6-1 resulting from the adaptive evolution, proteomics analyses of newly generated artificial high-
lipid content populations were performed. Functional classifications showed the heightened regulation of the major
chlorophyll enzymes, and the
enzymes involved in carbon fixation and uptake, including
chlorophyll-ab-
binding proteins and
Rubisco activase. The key control
protein (periplasmic
L-amino acid oxidase (LAO1)) of
carbon-
nitrogen integration was specifically overexpressed.
Glutathione-S-
transferases and
esterase, the
enzymes involved in lipid-metabolism and lipid-body associated
proteins, were also induced during adaptive evolution.
CONCLUSIONS: Adaptive evolution results demonstrate the potential role of photosynthesis in terms of
carbon partitioning, flux, and fixation and
carbon-
nitrogen metabolism during
lipid accumulation in microalgae. This strategy can be used as a new tool to develop C. reinhardtii strains and other microalgal strains with desired phenotypes such as high
lipid accumulation.