Auxenochlorella protothecoides adapted to grow efficiently in low phosphate conditions display altered lipid profiles
Renewable biodiesel produced by microalgae has great potential as a portable source of high-density energy that can replace certain traditional hydrocarbon sources. One limitation of scaling up algal cultivation to industrial levels is the availability of key macronutrients, particularly inorganic phosphorus (Pi), which is a finite and dwindling resource. Here, Auxenochlorella protothecoides was adapted to low Pi conditions through continuous cultivation in 100 times less Pi media for 285 days, or ~41 generations. The adapted populations demonstrated significantly higher growth rates than wild type (WT) cells in low Pi, with average maximum growth rates of 0.72 d-1 and 0.54 d-1, respectively in batch-culture experiments. Based on UPLC/MS analyses, the total lipid content of the adapting A. protothecoides populations showed a shift from their typical profile in nutrient replete media to the accumulation of non-phosphorus glycerolipids, with 306% MGDG and 189% SQDG, relative to WT at initial exposure, followed by a decline, and a steady increase until the final time point was reached. Transcriptome profiling by Illumina RNA-seq collected at 5 time points throughout the experiment, and results of the lipid analyses revealed a trend of increased transcript levels during the first ~11 generations of adaptation, followed by an overall decrease in gene expression after ~34 generations. The short-term changes in gene expression were associated with shifts in major metabolic pathways including carbon metabolism, oxidative phosphorylation, glycolysis, and gluconeogenesis. By comparison, certain transcripts showing decreased expression, reflected increased fatty acid turnover, and a stable decrease in photosynthesis-related gene expression. These results illustrate the utility of laboratory-directed evolution for the selection of microalgae populations with altered cultivation traits, revealing distinct phases of adaptation, based on expression profiles.  The results further demonstrate the use of metabolic engineering to select a microalga variant after only ~40 generations of growth in low-phosphate conditions that utilizes Pi more efficiently for growth than its wild type parent population and produces 1.22 times more biomass in batch growth experiments. Overall design: mRNA levels for triplicate wild type cell populations (WT1, WT2, and WT3) compared to mRNA levels of triplicate cell populations grown in low phosphate media (LP1, LP4, and LP5) collected at four time points during adaptation over >41 generations.