A simple and reproducible non-radiolabeled in vitro assay for recombinant acyltransferases involved in triacylglycerol biosynthesis

Liu, Jin; Lee, Yi-Ying; Mao, Xuemei; Li, Yantao

doi:10.1007/s10811-016-0949-6

Cited by 13 publications

(20 citation statements)

References 29 publications

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“…Although the exogenous FFA feeding experiments provided implications into the acyl-CoA substrate specificity of C. zofingiensis DGATs, solid experimental evidences are lacking, which can be addressed by in vitro assay. We have recently developed a non-radiolabeled DGAT in vitro assay [40], which allows for the measurement of activity and substrate specificity of DGAT toward a wide range of acyl-CoAs and DAGs, and has been well applied to DGATs from C. reinhardtii [25] and N. oceanica [27, 28]. Given that CzDGAT1A and CzDGTT5 showed the strongest acyltransferase activity (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2) but not their substrate specificity, which can be addressed by in vitro assay. We have recently developed a nonradiolabeled in vitro DGAT assay featured by the use of widely available and regular acyl-CoAs and DAGs [40], which has been successfully applied to examine both type I and type II DGATs from algae [25, 27, 28]. By means of this assay, CzDGAT1A and CzDGTT5, which showed the highest activity in H1246 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…H1246 cells carrying the empty vector pYES2-CT (EV control) and pYES2-CrDGTT1 (containing a type II DGAT gene from C. reinhardtii ) were from Liu et al [25]. The expression of DGAT s was induced by 2% (w/v) galactose in SD/-ura medium [40]. When necessary, free fatty acids were fed to yeast cultures as described by Siloto et al [37], with supplementation of linoleic acid (C18:2), α-linolenic acid (C18:3n3), and eicosapentaenoic acid (C20:5n3) at a concentration of 125 µM upon galactose induction.…”

Section: Methodsmentioning

confidence: 99%

“…The H1244 transformants bearing CzDGAT1A and CzDGTT5 were each grown in liquid SD/-ura medium containing 2% (w/v) galactose for 18 h at 30 °C, and then harvested for microsome preparation using a French pressure cell (Spectronics Instruments, NY, USA). The detailed procedures were described by Liu et al [40]. The resulting microsomal membrane pellets were resuspended in microsome storage buffer (50 mM Tris–HCl, pH 7.5, 10% glycerol) to give a protein concentration of 10 µg µL −1 for immediate use or stored at − 80 °C.…”

Section: Methodsmentioning

confidence: 99%

“…The in vitro DGAT assay was conducted according to our previously described procedures [40]. The acyl-CoAs tested included palmitoyl-CoA (C16:0-CoA), hexadecenoyl-CoA (C16:1-CoA), stearoyl-CoA (C18:0-CoA), oleoyl-CoA (C18:1-CoA), linoleoyl-CoA (C18:2-CoA), α-linolenoyl-CoA (C18:3n3-CoA), γ-linolenoyl-CoA (C18:3n6-CoA), arachidonyl-CoA (C20:4-CoA), eicosapentaenoyl-CoA (C20:5-CoA), and docosahexaenoyl-CoA (C22:6-CoA).…”

Section: Methodsmentioning

confidence: 99%

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Characterization of type I and type II diacylglycerol acyltransferases from the emerging model alga Chlorella zofingiensis reveals their functional complementarity and engineering potential

Mao

Kou

et al. 2019

Biotechnol Biofuels

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132

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BackgroundThe green alga Chlorella zofingiensis has been recognized as an industrially relevant strain because of its robust growth under multiple trophic conditions and the potential for simultaneous production of triacylglycerol (TAG) and the high-value keto-carotenoid astaxanthin. Nevertheless, the mechanism of TAG synthesis remains poorly understood in C. zofingiensis. Diacylglycerol acyltransferase (DGAT) is thought to catalyze the committed step of TAG assembly in the Kennedy pathway. C. zofingiensis genome is predicted to possess eleven putative DGAT-encoding genes, the greatest number ever found in green algae, pointing to the complexity of TAG assembly in the alga.ResultsThe transcription start site of C. zofingiensis DGATs was determined by 5′-rapid amplification of cDNA ends (RACE), and their coding sequences were cloned and verified by sequencing, which identified ten DGAT genes (two type I DGATs designated as CzDGAT1A and CzDGAT1B, and eight type II DGATs designated as CzDGTT1 through CzDGTT8) and revealed that the previous gene models of seven DGATs were incorrect. Function complementation in the TAG-deficient yeast strain confirmed the functionality of most DGATs, with CzDGAT1A and CzDGTT5 having the highest activity. In vitro DGAT assay revealed that CzDGAT1A and CzDGTT5 preferred eukaryotic and prokaryotic diacylglycerols (DAGs), respectively, and had overlapping yet distinctive substrate specificity for acyl-CoAs. Subcellular co-localization experiment in tobacco leaves indicated that both CzDGAT1A and CzDGTT5 were localized at endoplasmic reticulum (ER). Upon nitrogen deprivation, TAG was drastically induced in C. zofingiensis, accompanied by a considerable up-regulation of CzDGAT1A and CzDGTT5. These two genes were probably regulated by the transcription factors (TFs) bZIP3 and MYB1, as suggested by the yeast one-hybrid assay and expression correlation. Moreover, heterologous expression of CzDGAT1A and CzDGTT5 promoted TAG accumulation and TAG yield in different hosts including yeast and oleaginous alga.ConclusionsOur study represents a pioneering work on the characterization of both type I and type II C. zofingiensis DGATs by systematically integrating functional complementation, in vitro enzymatic assay, subcellular localization, yeast one-hybrid assay and overexpression in yeast and oleaginous alga. These results (1) update the gene models of C. zofingiensis DGATs, (2) shed light on the mechanism of oleaginousness in which CzDGAT1A and CzDGTT5, have functional complementarity and probably work in collaboration at ER contributing to the abundance and complexity of TAG, and (3) provide engineering targets for future trait improvement via rational manipulation of this alga as well as other industrially relevant ones.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1366-2) contains supplementary material, which is available to authorized users.

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