Characterization of a broad substrates specificity acyl-CoA:diacylglycerol acyltransferase 1 from the green tide alga Ulva prolifera

Triacylglycerols(triglycerides,TAGs)are the major carbon and energy storage forms in various organisms,and important components of cellular membranes and signaling molecules;they have essential functions in multiple physiological processes and stress regulation.Acyl-CoA:diacylglycerol acyltransferase(DGAT)catalyzes the final and only committed acylation step in the synthesis of TAGs in eukaryotes.The present work identified and isolated a novel gene,UpDGAT1,from the green tide alga Ulva prolifera.The activity of UpDGAT1 was confirmed by heterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruple mutant.Results of thin-layer chromatography and BODIPY staining indicated that UpDGAT1 was able to restore TAG synthesis and lipid body formation in the yeast.Lipid analysis of yeast cells revealed that UpDGAT1 showed broad substrate specificity,accepting saturated as well as mono-and polyunsaturated acyl-CoAs as substrates.High salinity and high temperature stresses increased UpDGAT1 expression and TAG accumulation in U.prolifera.The present study provides clues to the functions of UpDGAT1 in TAG accumulation in,and stress adaptation of,U.prolifera.

Producing Designer Oils in Industrial Microalgae by Rational Modulation of Co-evolving Type-2 Diacylglycerol Acyltransferases

Microalgal oils, depending on their degree of unsaturation, can be utilized as either nutritional supplements or fuels; thus, a feedstock with genetically designed and tunable degree of unsaturation is desirable to maximize process efficiency and product versatility. Systematic profiling of ex vivo （in yeast）, in vitro, and in vivo activities of type-2 diacylglycerol acyltransferases in Nannochloropsis oceanica （NoDGAT2s or NoDGTTs）, via reverse genetics, revealed that NoDGAT2A prefers saturated fatty acids （SFAs）, NoDGAT2D prefers monounsaturated fatty acids （MUFAs）, and NoDGAT2C exhibits the strongest activity toward polyunsaturated fatty acids （PUFAs）. As NoDGAT2A, 2C, and 2D originated from the green alga, red alga, and eukaryotic host ancestral participants of secondary endosymbiosis, respectively, a mecha- nistic model of oleaginousness was unveiled, in which the indigenous and adopted NoDGAT2s formulated functional complementarity and specific transcript abundance ratio that underlie a rigid SFA：MUFA：PUFA hierarchy in triacylglycerol （TAG）. By rationally modulating the ratio of NoDGAT2A＇：2C^D transcripts, a bank of N. oceanica strains optimized for nutritional supplement or fuel production with a wide range of degree of unsaturation were created, in which proportion of SFAs, MUFAs, and PUFAs in TAG varied by 1.3-, 3.7-, and 11.2-fold, respectively. This established a novel strategy to simultaneously improve productivity and quality of oils from industrial microalgae.

Cloning, Characterization and Functional Analysis of Two Type 1 Diacylglycerol Acyltransferases (DGAT1s) from Tetraena mongolica

Two cDNAs encoding putative type 1 acyl-CoA： diacylglycerol acyltransferases （DGAT1, EC 2.3.1.20）, were cloned from Tetraena mongolica Maxim., an extreme xerophyte with high oil content in the stems. The 1,488-bp and 1,485-bp of the open reading frame （ORF） of the two cDNAs, designated as TmDGAT1a and TmDGAT1b, were both predicted to encode proteins of 495 and 494 amino acids, respectively. Southern blot analysis revealed that TmDGAT1a and TmDGAT1b both had low copy numbers in the T. mongolica genome. In addition to ubiquitous expression with different intensity in different tissues, including stems, leaves and roots, TmDGAT1a and TmDGAT1b, were found to be strongly induced by high salinity, drought and osmotic stress, resulting in a remarkable increase of triacylglycerol （TAG） accumulation in T. mongolica plantlets. TmDGAT1a and TmDGAT1b activities were confirmed in the yeast H1246 quadruple mutant （DGA1, LRO1, ARE1, ARE2） by restoring DGAT activity of the mutant host to produce TAG. Overexpression of TmDGAT1a and TmDGAT1b in soybean hairy roots as well as in T. mongolica calli both resulted in an increase in oil content （ranging from 37% to 108%）, accompanied by altered fatty acid profiles.

Gomparative analysis of DGAT3（diacylglycerol acyltraiisferase 3） gene from different peanut （Arachis hypogaea L.） varieties

Cultivated peanut is one of the primary sources of vegetable oil and protein in developing countries. DG〉A73 family in peanut cotyledons has no membrane-bound regions suggesting that cytosol is one of the sites for triacylglycerol （TAG） biosynthesis in oilseeds. According to functional annotation and classification of 5 cDNA libraries, 12 unigenes were found with relation of peanut DGAT3 in different organs. Three clones of unigenes, OCP- contig168t OCPcontig12101-3 and OCPcontigl2388-1 were selected for sequencing. Full length sequence of DGAT3 was obtained, showing over 98% sequence similarity with peanut DGAT3 gene AY875644 or EU183333. Upon cluster analysis, DGAT3 of 40 culti- vars were divided into 3 types, namely AhDGATS-1, AhDGAT3-2 and AhDGAT3-3. Coding regions are 1023, 1038 and 1026 base pairs which encoded proteins with 340-, 345- and 341-amino acids, respectively. DGAT3-3 might be a novel gene type among the DGAT3 family which provides great help for studying DGAT3 gene evolution in peanut.

Identification and Comparative Analysis of DGAT1 Genes among Cotton and Other Model Plants

Diacylglycerol acyltransferase （DGAT） is the key enzyme that catalyzes the triacylglycerol biosynthesis. The comparative analysis was performed on the DGAT1 genes of cotton and other model plants. Sequence analysis showed that most of the DGAT1 genes,with high variation of intron length and high conservation of intron phrase,from the cotton and other model plants had 16 exons. Additionally, 7 conserved motifs were present in these DGAT1 proteins. The core motifs were overlapped with the functional domain of DGAT1 protein. Phylogenetic analysis demonstrated that gene tree was highly consistent to species tree,suggesting that the evolutionary history of species was revealed by gene tree. There was single copy of DGAT1 gene in cotton,but at least two duplicated DGAT1 genes were iden- tified in rice,maize,poplar and moss genomes. The selective pressure analysis showed that the PtDGATla/PtDGATlb was under positive selection,but other four pairs of homologous genes were under negative selection. 17 positively selected sites were identified at subgroup level （P〉0.05）,suggesting these subgroups under relaxed functional constraint. The findings provide a basis for further studying func- tion and evolution of DGAT1 genes in cotton and other model plants.

Diacylglycerol Compounds from Barks of Betula platyphylla with Inhibitory Activity against Acyltransferase

Objective To identify the active compounds from the barks of Betula platyphylla for inhibitory on diacylglycerol acyltransferase（DGAT1）.Methods Bioassay-guided fractionation resulted in the isolation of DGAT1 inhibitory activity of lupane triterpenes.Results Ten compounds were identified as lupenone（1）,lupeol（2）,betulinic acid（3）,betulinaldehyde（4）,betulin（5）,3-deoxybetulonic acid（6）,glochidonol（7）,lup-20/29-ene-1β/3β-diol（8）,3α-hydroxy-lup-20（29）-en-23,28-dioic acid（9）,and 3α,11α-dihydroxy-23-oxo-lup-20（29）-en-28-oic acid（10）.Compounds 3-6,9,and 10 inhibited DGAT1 with IC50 values ranging from（11.2±0.3）to（38.6±1.2）μmol/L.Conclusion Compounds 6,9,and 10 are first isolated from the barks of B.platyphylla.,and compounds 3-6,9,and 10 from the barks of B.platyphylla are responsible for the inhibition on DGAT1.

Acyl-coenzyme A: cholesterol acyltransferase family

The enzymes of the acyl-coenzyme A:cholesterol acyltransferase(ACAT)family are responsible for the in vivo synthesis of neutral lipids.They are potential drug targets for the intervention of atherosclerosis,hyperlipidemia,obesity,type II diabetes and even Alzheimer’s disease.ACAT family enzymes are integral endoplasmic reticulum(ER)membrane proteins and can be divided into ACAT branch and acyl-coenzyme A:diacylglycerol acyltransferase 1(DGAT1)branch according to their substrate specificity.The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates,while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates.In this review,we mainly focus on the recent progress in the structural research of ACAT family enzymes,including their disulfide linkage,membrane topology,subunit interaction and catalysis mechanism.