Phenotypic screening identifies Brefeldin A/Ascotoxin as an inducer of lipid storage in the algae Chlamydomonas reinhardtii

Abstract

a b s t r a c tThe use of microalgae as a biofuel feedstock is highly desired, but current methods to induce lipid accumulation cause severe stress responses that limit biomass and, thus oil yield. To address these issues, a high throughput screening (HTS) method was devised to identify chemical inducers of growth and lipid accumulation. Optimization was performed to determine the most effective cell density, DMSO and Nile Red (NR) concentrations to monitor growth and lipid accumulation. The method was tested using 1717 compounds from National Cancer Institute (NCI) Diversity Set III and Natural Products Set II in Chlamydomonas reinhardtii. Cells were inoculated at low density and 10 μM of the test compound was added. After 72 h, cell density was measured at OD 550 and lipid accumulation assessed using NR fluorescence. Primary screening identified 8 compounds with a hit rate of 0.47% and a robust Z′ discrimination factor (0.68 ± 0.1). Of these, Brefeldin A (BFA) was the most successful at inducing lipid accumulation and was used to evaluate secondary screens including measuring levels of fatty acids, photosynthetic pigments, proteins and carbohydrates. The effectiveness of BFA was confirmed in Chlorella sorokiniana UTEX 1230. This study demonstrates the power of chemical genomics approaches in biofuel research.

Identification and Metabolite Profiling of Chemical Activators of Lipid Accumulation in Green Algae

“…Importantly, for four compounds, galactolipids were essentially equivalent to the wild type, indicating that these membrane lipids were not a major source of acyl chains in the TAGs, as occurs in many stress conditions (Hu et al, 2008;Urzica et al, 2013;Allen et al, 2017) or with treatment with the small compounds brefeldin A (Wase et al, 2015) or fenpropimorph . In contrast, treatment with WD20067 decreased galactolipid content to about 25% of control values, which is more similar to nutrient deprivation (Goncalves et al, 2013;Urzica et al, 2013;Allen et al, 2017).…”

“…The screening protocol used was devised previously and tested using a small-compound library designed for this purpose (Wase et al, 2015) and is outlined in Supplemental Figure S1. The primary selection required two phenotypic tests: (1) the lipophilic dye Nile Red was used to identify treated cells that accumulated neutral lipids; and (2) the assessment of growth and accumulated cellular mass using spectrophotometric measurements at OD 600 .…”

“…Analysis of photosynthetic pigments, including chlorophylls a and b and total carotenoids, was conducted as reported previously (Wase et al, 2015). Briefly, cultures (50 mL) were grown with or without compound in triplicate at the specified final concentrations for 72 h at 25°C with shaking in a New Brunswick Innova 43 incubator under a photon flux density of 54 mmol m 22 s 21 .…”

“…Several of these lipid activators were employed to probe pathways permissive to both algal growth and TAG accumulation (McCourt and Desveaux, 2010;Wase et al, 2015). For screening, low-density algal cultures were treated with compounds from a large chemical library for 72 h, and both growth and lipid accumulation were assessed (Wase et al, 2015).…”

Current advances in molecular, biochemical, and computational modeling analysis of microalgal triacylglycerol biosynthesis

“…It has been proposed that during early N deficit, membrane lipids are possibly converted to TAG (Jia et al, 2015). Apart from N deficit, several other stress factors such as pH, temperature, salinity, and phosphorous and carbon limitation also induce TAG accumulation in microalgae (Wase et al, 2015). Labeling experiments in microalgae demonstrated that TAG acts as a reservoir for PUFAs and can be used for rapid formation of plastidic lipids (Cohen et al, 2000).…”