Arabidopsis thaliana DGAT3 is a [2Fe-2S] protein involved in TAG biosynthesis.

Acyl-CoA:diacylglycerol acyltransferases 3 (DGAT3) are described as plant cytosolic enzymes synthesizing triacylglycerol. Their protein sequences exhibit a thioredoxin-like ferredoxin domain typical of a class of ferredoxins harboring a [2Fe-2S] cluster. The Arabidopsis thaliana DGAT3 (AtDGAT3; At1g48300) protein is detected in germinating seeds. The recombinant purified protein produced from Escherichia coli, although very unstable, exhibits DGAT activity in vitro. A shorter protein version devoid of its N-terminal putative chloroplast transit peptide, Δ46AtDGAT3, was more stable in vitro, allowing biochemical and spectroscopic characterization. The results obtained demonstrate the presence of a [2Fe-2S] cluster in the protein. To date, AtDGAT3 is the first metalloprotein described as a DGAT.

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

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.

Two novel Saccharomycopsis species isolated from black olive brines and a tropical plant. Description of Saccharomycopsis olivae f. a., sp. nov. and Saccharomycopsis guyanensis f. a., sp. nov. Reassignment of Candida amapae to Saccharomycopsis amapae f. a., comb. nov., Candida lassenensis to Saccharomycopsis lassenensis f. a., comb. nov. and Arthroascus babjevae to Saccharomycopsis babjevae f. a., comb. nov.

Three yeast strains related to members of the genus Saccharomycopsis were isolated. One strain (CLIB 1310) was isolated from olive brines of fermented black olives in France and two strains (CLIB 1454 and CLIB 1455) were isolated from a plant in French Guiana. Sequence analyses based on the D1/D2 domains of the nuclear large subunit rRNA gene, small-subunit rRNA gene and partial EF-1α gene revealed that the strains represented two novel taxa exhibiting extensive sequence divergence from the previously described species of the genus Saccharomycopsis. Two novel species are described to accommodate these newly isolated strains: Saccharomycopsis olivae sp. nov. (type strain CLIB 1310(T) = CBS 12701(T)) and Saccharomycopsis guyanensis sp. nov. (type strain CLIB 1455(T) = CBS 12914(T) and strain CLIB 1454). Both strains CLIB 1454 and CLIB 1455(T) displayed identical sequences but differed in their ability to metabolize sorbitol and in their morphology on agar medium. Candida amapae, Candida lassensensis and Arthroascus babjevae belonging to the Saccharomycopsis clade, are reassigned to Saccharomycopsis as novel combinations.

Citeromyces nyonsensis sp. nov., a novel yeast species isolated from black olive brine.

A yeast strain was isolated from olive brines in a fermented black olive and olive oil manufacturing plant in the town of Nyons (France). On the basis of domains 1 and 2 (D1/D2) large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) region sequence analyses, the strain CLIB 1303(T) was found to be closely related, but clearly distinct, from the three existing species of the genus Citeromyces: Citeromyces matritensis, Citeromyces siamensis and Citeromyces haiwaiiensis. Strain CLIB 1303(T) exhibited 6 bp, 7 bp and 12 bp divergences in the D1/D2 LSU rRNA gene with C. siamensis, C. matritensis and C. hawaiiensis, respectively. ITS region divergence amounted to more than 8 %, 4 % and 4.5 % with C. siamensis, C. matritensis and C. hawaiiensis, respectively, in addition to several indels. Like C. matritensis and C. siamensis strains, strain CLIB 1303(T) was shown to be halotolerant and osmotolerant. Phenotypically, strain CLIB 1303(T) can be distinguished from other species of the genus Citeromyces by its inability to assimilate trehalose. The strain CLIB 1303(T) (= CBS 12700(T)) was assigned to a novel species, Citeromyces nyonsensis sp. nov.