title: 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 (Dga1pΔ19), the second one also devoid of the N-terminal putative transmembrane domain (Dga1pΔ85). 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. Dga1pΔ85 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.
authors: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France.
authors: Nawel
authors: N
authors: Haïli
authors: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France.
authors: Julien
authors: J
authors: Louap
authors: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France.
authors: Michel
authors: M
authors: Canonge
authors: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France.
authors: Franjo
authors: F
authors: Jagic
authors: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France.
authors: Christelle
authors: C
authors: Louis-Mondésir
authors: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France.
authors: Thierry
authors: T
authors: Chardot
authors: Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France.
authors: Pierre
authors: http://orcid.org/0000-0001-5212-6009
authors: P
authors: Briozzo
mesh_terms: D000595:Amino Acid Sequence; D051048:Diacylglycerol O-Acyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009525:Niacin / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology*
chemical_list: D005227:Fatty Acids; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014280:Triglycerides; D009525:Niacin; D051048:Diacylglycerol O-Acyltransferase