Transcriptome analysis reveals the genetic foundation for the dynamics of starch and lipid production in Ettlia oleoabundans

Mark H.J Sturme,Yanhai Gong,Josué Miguel Heinrich,Anne J Klok,Gerrit Eggink,Dongmei Wang,Jian Xu,Rene H Wijffels

doi:10.1016/j.algal.2018.05.004

Abstract

The oleaginous microalga Ettlia oleoabundans accumulates both starch and lipids to high levels under stress conditions such as nitrogen starvation (N−). To steer biosynthesis towards starch or lipids only, it is important to understand the regulatory mechanisms involved. Here physiological and transcriptional changes under nitrogen starvation were analysed in controlled flat-panel photobioreactors at both short and long time-scales. Starch accumulation was transient and occurred rapidly within 24 h upon starvation, while lipid accumulation was gradual and reached a maximum after 4 days. The major fraction of accumulated lipids was composed of de novo synthesized neutral lipids - triacylglycerides (TAG) - and was characterized by a decreased composition of the polyunsaturated fatty acids (PUFAs) C18:3 and C16:3 and an increased composition of the mono-unsaturated (MUFAs) and saturated (SFAs) fatty acids C18:1/C16:1 and C18:0/C16:0, respectively. RNA-sequencing revealed that starch biosynthesis and degradation genes show different expression dynamics from lipid biosynthesis ones. An immediate rapid increase in starch synthetic transcripts was followed by an increase in starch degrading transcripts and a decrease in the starch synthetic ones. In contrast, increased gene expression for fatty acid and TAG synthesis was initiated later and occurred more gradually. Expression of several fatty acid desaturase (FAD) genes was decreased upon starvation, which corresponds to the observed changes to higher levels of MUFAs and SFAs. Moreover, several homologs of transcription regulators that were implicated in controlling starch and lipid metabolism in other microalgae showed differential gene expression and might be key regulators of starch and lipid metabolism in E. oleoabundans as well. Our data provide insights into the genetic foundation of starch and lipid metabolism in E. oleoabundans under nitrogen starvation and should facilitate metabolic engineering towards tailored strains with desired storage compound composition.

Highlights

With a growing world population and declining natural oil and gas reserves, the global demand for sustainable and renewable resources becomes ever more relevant
For the pre-cultures grown in nitrogen-replete conditions (+N) samples were taken at four timepoints in the lightphase preceding nitrogen starvation, while from the onset of nitrogen starvation (−N), time-series samples were taken at the start, middle and end of the light-phase and at the end of the dark-phase over a 4-day period (Fig. 1)
For the oleaginous green microalgae E. oleoabundans this genetic information is still very limited, we set out to study the biomass and transcriptional changes that occur upon nitrogen starvation, to find genetic triggers of starch and lipid accumulation in this industrially relevant microalga

Summary

Introduction

With a growing world population and declining natural oil and gas reserves, the global demand for sustainable and renewable resources becomes ever more relevant. Microalgae can accumulate high levels of storage compounds such as carbohydrates, lipids and pigments under stress conditions and can serve as a renewable source of proteins They can be cultivated on marginal lands and in (semi-)arid regions and outdoor cultivation of marine and halotolerant microalgae will require a lower freshwater input. Thereby they compete less with agricultural crop production and have a smaller effect on drinking water supply. The market for most microalgae-derived products currently is not economically competitive with those derived from existing plant-based and petrochemical resources To overcome this gap, microalgal strain development in combination with improved biorefineries is required [4,5]. Many studies for microalgal strain development have focused on metabolic engineering by the introduction or mutagenesis of single target genes involved in lipid, starch or pigment biosynthesis [6,7] and less on engineering of

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