Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis

Xiahui Hao,Yangmin Gong,Fabrice Rébeillé,Zhuo Chen,Eric Maréchal,Xiaoming Tan,Hanhua Hu,Hong Chen,Lingjie You,Ling Luo,Juliette Jouhet,Fang Wei,Yufang Pan

doi:10.1186/s13068-018-1309-3

Abstract

BackgroundIn photosynthetic oleaginous microalgae, acyl-CoA molecules are used as substrates for the biosynthesis of membrane glycerolipids, triacylglycerol (TAG) and other acylated molecules. Acyl-CoA can also be directed to beta-oxidative catabolism. They can be utilized by a number of lipid metabolic enzymes including endogenous thioesterases, which catalyze their hydrolysis to release free fatty acids. Acyl-CoA availability thus plays fundamental roles in determining the quantity and composition of membrane lipids and storage lipids.ResultsHere, we have engineered the model diatom Phaeodactylum tricornutum to produce significantly increased TAGs by disruption of the gene encoding a Hotdog-fold thioesterase involved in acyl-CoA hydrolysis (ptTES1). This plastidial thioesterase can hydrolyze both medium- and long-chain fatty acyl-CoAs, but has the highest activity toward long-chain saturated and monounsaturated fatty acyl-CoAs. The maximum rate was found with oleoyl-CoA, which is hydrolyzed at 50 nmol/min/mg protein. The stable and targeted interruption of acyl-CoA thioesterase gene was achieved using a genome editing technique, transcription activator-like effector nucleases (TALENs). Disruption of native ptTES1 gene resulted in a 1.7-fold increase in TAG content when algal strains were grown in nitrogen-replete media for 8 days, whereas the content of other lipid classes, including phosphoglycerolipids and galactoglycerolipids, remained almost unchanged. The engineered algal strain also exhibited a marked change in fatty acid profile, including a remarkable increase in 16:0 and 16:1 and a decrease in 20:5. Nitrogen deprivation for 72 h further increased TAG content and titer of the engineered strain, reaching 478 μg/109 cells and 4.8 mg/L, respectively. Quantitative determination of in vivo acyl-CoAs showed that the total acyl-CoA pool size was significantly higher in the engineered algal strain than that in the wild type.ConclusionsThis study supports the role of ptTES1 in free fatty acid homeostasis in the plastid of Phaeodactylum and demonstrates the potential of TALEN-based genome editing technique to generate an enhanced lipid-producing algal strain through blocking acyl-CoA catabolism.

Highlights

In photosynthetic oleaginous microalgae, acyl-CoA molecules are used as substrates for the biosynthesis of membrane glycerolipids, triacylglycerol (TAG) and other acylated molecules
Intracellular acyl-CoAs may be subject to desaturation, elongation, hydrolysis, and acyl transfer to various acyl acceptors, all of which contribute to acyl-CoA availability
Our previous study showed that heterologous expression of the 4-HBT-II subfamily thioesterase in E. coli fadD88 strain induced a higher level of accumulation of both stearic (C18:0) and oleic (C18:1) acids when compared to the control strain harboring the empty plasmid [21]

Summary

Introduction

Acyl-CoA molecules are used as substrates for the biosynthesis of membrane glycerolipids, triacylglycerol (TAG) and other acylated molecules. Enhanced lipid production was achieved via heterologous co-expression of a yeast diacylglycerol acyltransferase and a plant oleosin [6], overexpression of malic enzyme [7], glycerol-3-phosphate acyltransferase [8, 9], targeted knockdown of phosphoenolpyruvate carboxykinase [10], disruption of the UDP-glucose pyrophosphorylase gene [11], overexpression of a gene involved in nitric oxide emission via the by-production of fumaric acid [12] and inhibition of acetyl-CoA utilization for sterol metabolism [13, 14] These studies for enhancing lipid production in P. tricornutum can be classified into two different approaches: (1) overexpressing enzymes, especially acyltransferases, to increase acyl flux being channeled to TAG synthesis pathway or enhance reductant (NADPH) supply for lipogenic enzymes; (2) inhibition of or blocking off competing pathway to decrease lipid catabolism or increase substrate supply. This study demonstrates the potential of TALEN-mediated knockout of target genes as an effective strategy to enhance TAG production in oleaginous diatoms

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Discussion

Conclusion