Abstract
BackgroundPhotosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The principal activity of DGATs has been defined as a single-function enzyme catalyzing the esterification of diacylglycerol with acyl-CoA.ResultsA dual-function PtWS/DGAT associated with diatom Phaeodactylum tricornutum is discovered in the current study. Distinctive to documented microalgal DGAT types, PtWS/DGAT exhibits activities of both a wax ester synthase (WS) and a DGAT. WS/DGATs are broadly distributed in microalgae, with different topology and phylogeny from those of DGAT1s, DGAT2s, and DGAT3s. In vitro and in vivo assays revealed that PtWS/DGAT, functioning as either a WS or a DGAT, exhibited a preference on saturated FA substrate. Endogenous overexpression of PtWS/DGAT demonstrated that the DGAT activity was dominant, whereas the WS activity was condition dependent and relatively minor. Compared with the wild type (WT), overexpression of PtWS/DGAT in the diatom resulted in increased levels of total lipids (TL) and triacylglycerol (TAG) regardless of nitrogen availability. The stability and scalability of the introduced traits were further investigated at a 10-L photobioreactor, where the mutant growth resembled WT, with moderately increased productivity of TL and TAG. Furthermore, the production of wax esters increased considerably (from undetectable levels to 2.83%) under nitrogen-deplete conditions.ConclusionsPtWS/DGAT is a bifunctional enzyme and may serve as a promising target for the engineering of microalga-based oils and waxes for future industrial use.
Highlights
Photosynthetic oleaginous microalgae are promising feedstocks for biofuels
The PtDGATX was distributed separately from (Fig. 1a) and was modestly similar to characterized microalgal DGAT1s, DGAT2s, and DGAT3s (Fig. 1a). This suggested that the origin and function of PtDGATX were distinct from those of DGAT1s, DGAT2s, and DGAT3s
A relatively high identity in amino acid sequence was revealed between DGATX and wax ester synthase (WS)/diacylglycerol acyltransferases (DGATs) (20%; bacterium Acinetobacter, AF529086; Additional file 2: Figure S2), which has been characterized in bacteria as a bifunctional enzyme WS/DGAT [32]
Summary
Photosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The oleaginous microalga P. tricornutum harbors a single DGAT1 and four putative DGAT2s, of which DGAT1 [8] and DGAT2B [9] have been functionally characterized in vitro This characterization revealed a distinct substrate preference with PtDGAT1 exhibiting a preference toward saturated C 16 and C18 fatty acids (FAs) [8] and PtDGAT2B exhibiting specificity on unsaturated C16 and C18 FAs [9, 10]. In P. tricornutum, we discovered a group of acyl-CoA:diacylglycerol acyltransferases (designated as PtDGATX) with an identity high to WS/DGAT and low to DGAT1s, DGAT2s, and DGAT3s, in amino acid sequence This suggests that the function of DGATXs differs from those of the remaining types of DGATs. identification of the molecular and cellular mechanisms of PtDGATX underlying TAG or wax ester metabolism remains challenging. The engineered strains yield ( low) wax esters, a class of oleochemicals that can potentially be used for many applications in various industries [34, 35]
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