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

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.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1309-3) contains supplementary material, which is available to authorized users.

Lipid production is more than doubled by manipulating a diacylglycerol acyltransferase in algae

“…We demonstrate, using a marine alga as the cell factory, that screening proper engineering targets and manipulating a single gene can pull carbon flux to lipid production for TAG hyper‐accumulation, and the PtDGAT1 ‐overexressing P. tricornutum may serve as a promising feedstock for oil production. In addition to DGAT , there have been many reports about the manipulation of other genes for improved TAG production in P. tricornutum , such as genes involved in lipid metabolism (Balamurugan et al., 2017; Barka et al., 2016; Hamilton et al., 2014; Hao et al., 2018; Li et al., 2018; Niu et al., 2016; Wang et al., 2020), NADPH generation (Xue et al., 2015, 2017), carbon precursor production (Daboussi et al., 2014), and transcription factor (Kumar Sharma et al., 2019). The manipulation of multiple genes that provides the combination of pulling, pushing, and protection may have the potential to achieve even higher lipid level and production, which remains yet to be fully explored in the future.…”

A Review of Diatom Lipid Droplets

“…Unlike in plant leaf tissues, metabolic engineering of diatom TAG accumulation benefits from the existence of LD biogenesis and regulatory systems already in place. Various steps within the "push, pull, package, protect" paradigm have already has been accomplished in diatoms by over-expression of enzymes implicated in fatty acid and TAG biosynthesis [114,117,[240][241][242], modulating the acyl-CoA pool [243] or repression of lipid catabolism [124,125,244]. A deeper understanding of the mechanisms by which proteins are targeted to LDs could facilitate the engineering of LD-localized enzymes or metabolic pathways, which could allow for the customization of LD contents.…”

Cold adaptation in drylands: transcriptomic insights into cold‐stressed Nostoc flagelliforme and characterization of a hypothetical gene with cold and nitrogen stress tolerance

“…Lipids were extracted and determined as previously described (Jiang et al ., 2014; Hao et al ., 2018) with some modifications. In brief, 30 ml of cultures at the logarithmic phase were harvested by centrifugation at 5500 rpm for 10 min and the pellets were freeze‐dried.…”