Auxenochlorella protothecoides populations adapted to low phosphate conditions accumulated more non-phosphorus glycerolipids and biomass than wild type progenitors

Biodiesel produced by microalgae has great potential as a portable energy that can replace traditional hydrocarbon sources. One limitation of scaling up algal cultivation is the availability of macronutrients, particularly inorganic phosphate (Pi), which is a finite and dwindling resource. Here, Auxenochlorella protothecoides was adapted to low Pi conditions by continuous cultivation in growth media containing 100 times less Pi (17.15 µM PO₄) than replete media for ∼ 40 generations. The low Pi-adapted A. protothecoides populations showed significantly higher growth rates, compared to wild type (WT) (natural, non-mutated) progenitor populations in batch experiments, with average maximum growth rates of 0.72 d⁻¹ and 0.54 d⁻¹, respectively. Total lipid profiling of the adapting A. protothecoides populations indicated a shift to non-phosphorus glycerolipids, based on UPLC/MS analyzes. The proportions of monogalactosyldiacylglycerol (MGDG) and sulfoquinovosyldiacyglycerol (SQDG) fluctuated during adaptation, accumulating 305% and 317% of the WT levels respectively by the final sampling. Time-course transcriptome profiling of A. protothecoides across all adaptation stages revealed initial increases in transcript levels, followed by global decreased expression. The short-term transcriptomic changes, prior to ∼ 11 generations, were associated with major metabolic pathways. The long-term changes indicated increased fatty acid turnover and a decrease in photosynthesis-related gene expression. Transcripts predicted to encode alternative oxidase and pyrophosphate-dependent phosphofructokinase fluctuated during adaptation. The selection of A. protothecoides under low Pi conditions resulted in a microalga variant that after only ∼40 generations utilized Pi more efficiently for growth than its wild type progenitor population, while also producing 1.22 times more biomass. The adaptive processes described herein produced commercially relevant strain material and provide avenues for future, targeted engineering of molecular pathways for increased Pi use efficiency in similar organisms.