Intervention of triethylamine on Dunaliella tertiolecta reveals metabolic insights into triacylglycerol accumulation

Highlights

• The single cell lipid of algae increased by 1.7 times under 100 ppm triethylamine.

• The formation of triacylglycerides under triethylamine stress was mainly through bypass pathway.

• Triosephosphate translocator was down-regulated under triethylamine.

• Three DNA Binding With One Finger Protein genes were up-regulated to varying degrees.

Abstract

Dunaliella tertiolecta is a highly oil-producing microalgae. Here, we report its de novo transcriptome assembly and quantitative gene expression analysis of the intervention of triethylamine, with a focus on the complex interactions of pathways involved in the production of triacylglycerol (TAG) biofuel precursors. After growing under 100 ppm triethylamine, we quantified the cellular content of the major biomolecules, including total lipids, single cell oil content, and lipid components. The transcriptome was assembled de novo and the main functional classes, related pathways, and expression of important genes were quantified by the mapping of reads to the transcriptome. Almost 65,925 high-quality readings were produced on the Illumina HiSeq sequencing platform. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during triethylamine was not through the Kennedy pathway, but the bypass pathway by high expression of phosphate: diacylglycerol acyltransferase. The glycolytic pathway has also been down-regulated, not only in the cytoplasm but also in the chloroplast. Triosephosphate translocator was downregulated, and the DNA binding with One Finger protein gene was upregulated. The quantitative transcriptome study reveals a broad overview of how triethylamine stress leads to excessive TAG production in Dunaliella, and provides various genetic engineering goals and strategies to improve biofuel precursors in oleaginous microalgae.

Introduction

Under the adverse environments, some plants or algae will accumulate triacylglycerols (TAGs). TAGs or oils can be converted to fatty acid methyl esters, which can be used in biodiesel production directly [1]. Compared to land plants, microalgae-based biofuels have important advantages over plant-based biofuels including its greater solar energy conversion efficiency, non-arable land utilization and high content of neutral lipids with energy-dense [2,3]. Besides, microalgae are single-cell photosynthetic organisms with a clear genetic background, which makes it possible to engineer microalgae genetically. Recently, D. tertiolecta has received considerable attention in recent years because of its efficient lipids production (up to 67% of the organism dry weight). Under environmental stimuli such as high light, salt stress, and nutrient deprivation, the lipid can further massively be accumulated in D. tertiolecta [4]. Another important advantage is that it lacks a rigid cell wall, which eases genetic manipulation and product extraction [5]. Furthermore, its cultivation process could eliminate contaminations from the clean cultures, utilize inorganic nutrients presented in brackish water, wastewater, or saltwater, along with the sunshine to generate biofuels by absorbing CO₂ as a carbon source through photosynthesis.

Adding chemical inducers to algal culture medium is an effective way to promote the accumulation of oil in algal cells. Several chemical agents such as azide, pyridine, brefeldin A, epigallocatechin gallate, and triethylamine, were employed to trigger the biosynthesis at TAG [[6], [7], [8]]. Our previous study had found that triethylamine could boost and elevate the lipid production, resulting in a 36.8% and 33.0% increase in lipid production and lipid content per cell, respectively [9]. Triethylamine is the most effective lipid inducer for lipid content per cell ever discovered.

The de novo transcriptome assembly and quantitative gene expression analysis is a common strategy to analyze the channel metabolites and key enzymes to lipid biosynthesis in microalgae strains. There is a growing number of oleaginous microalgae from which de novo transcriptomes have been annotated and assembled but the quantitative gene expression analysis in these microalgae has not yet been conducted [[10], [11], [12], [13]]. Moreover, most of the transcriptome analysis of oil producing microalgae focused on the lipid metabolism pathway under nitrogen deficiency [14].

D. tertiolecta is known to produce large quantities of lipids in response to triethylamine stresses in our previous work [9]. But the mechanism behind it is still unclear. In the present study, we generated large-scale, highly reproducible transcript profiles of D. tertiolecta to elucidate the metabolic pathway interactions and regulatory mechanisms involved in the accumulation of TAG. D. tertiolecta was cultured under 100 ppm triethylamine and normal condition and major biomolecules including lipid productivity, lipid contents, and fatty acid methyl esters distribution were measured. The transcriptome was assembled de novo and sequenced, gene expression was quantified and analyzed, and comparative analysis of genes, pathways, and broader gene ontology categories was carried out. These findings enhance our understanding of TAG production in microalgae and will facilitate rational genetic engineering of this organism and other related species for the overproduction of TAG or other biological products.