Dataset Information

Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols.

ABSTRACT: The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT) are essential actors for triglycerides (TG) biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In this report, we expressed in Escherichia coli and purified two soluble and active forms of Y. lipolytica Dga1p as fusion proteins: the first one lacking the N-terminal hydrophilic segment (Dga1p?19), the second one also devoid of the N-terminal putative transmembrane domain (Dga1p?85). Most DGAT assays are performed on membrane fractions or microsomes, using radiolabeled substrates. We implemented a fluorescent assay in order to decipher the substrate specificity of purified Dga1p enzymes. Both enzyme versions prefer acyl-CoA saturated substrates to unsaturated ones. Dga1p?85 preferentially uses long-chain saturated substrates. Dga1p activities are inhibited by niacin, a specific DGAT2 inhibitor. The N-terminal transmembrane domain appears important, but not essential, for TG biosynthesis. The soluble and active proteins described here could be useful tools for future functional and structural studies in order to better understand and optimize DGAT enzymes for biotechnological applications.

SUBMITTER: Haili N

PROVIDER: S-EPMC5079557 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols.

Haïli Nawel N Louap Julien J Canonge Michel M Jagic Franjo F Louis-Mondésir Christelle C Chardot Thierry T Briozzo Pierre P

PloS one 20161025 10

The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT) are essential actors for triglycerides (TG) biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In t ...[more]

PMID: 27780240

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Project description:Plants and microalgae have the ability to harness sunlight into energy dense molecules such as triacylglycerols (TAGs). TAGs hold great importance for human and animal nutrition, and are also a source for renewable energy. Acyl-CoA-dependent TAG synthesis by diacylglycerol acyltransferases (DGATs) in oilseeds has been well characterized, but how the ectopic introduction of TAG synthesis in vegetative tissues of plants affects metabolism is largely unknown. Here we report the identification and characterization of several Chlamydomonas reinhardtii Diacylglycerol Acyltransferase Type Two (DGTT) enzymes and their use in engineering TAG biosynthesis in plant vegetative tissues. Focusing on DGTT2 we demonstrate that it can accept a broad range of substrates. Production of DGTT2 in Arabidopsis results in distinct seedling phenotypes and accumulation of TAG with very long chain fatty acids (VLCFA), in both seedlings (23.4-fold increase) and in the leaves of soil-grown plants (13.3-fold increase). Levels of surface lipids, such as waxes and cutins, are decreased in DGTT2-producing lines, consistent with a decreased carbon/acyl flux to the surface lipid pathway. Global transcript analysis showed that very few genes had altered expression. Little to no change in the transcripts of wax/cutin pathway genes was observed in response to altered carbon partitioning into TAGs in the leaves suggesting that the supply of acyl groups for surface lipid biosynthesis is not transcriptionally adjusted in the transgenic lines. As an indication that the DGTT2-producing plants are richer in feed value, the generalist herbivore Spodoptera exigua reared on the transgenic plants gained more weight. Apparently, the substrate specificity of DGTT2 from Chlamydomonas when produced in the Arabidopsis cells leads to diversion of carbon from surface compounds in TAGs and enhances the nutritional value of leaves. 4 biological replicates DGTT2 transgenic plants are compared to 4 biological replicates of wild type

Project description:Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

Dataset Information

Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols.

Publications

Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols.

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