TY - JOUR

T1 - Identification, characterization, and expression of diacylgylcerol acyltransferase type-1 from Chlorella vulgaris

AU - Kirchner, Linda

AU - Wirshing, Alison

AU - Kurt, Lutfiye

AU - Reinard, Thomas

AU - Glick, James

AU - Cram, Erin J.

AU - Jacobsen, Hans-Jörg

AU - Lee-Parsons, Carolyn W.T.

N1 - This work was supported by the Massachusetts Clean Energy Center Catalyst Program and the Northeastern Tier 1 Seed Grant to CLP and by the DAAD-ISAP Fellowship and a grant from the Graduate Academy of Leibniz University (Hanover) to LK. The authors thank Prof. Sten Stymne (Swedish University of Agricultural Sciences, Sweden) for providing the three yeast strains for this study and Prof. Christoph Benning (Michigan State University, MI, USA) for providing the pYES2 vector.

PY - 2016/1

Y1 - 2016/1

N2 - The green microalga Chlorella vulgaris can be induced to rapidly accumulate oil (40% by dry weight) with a biodiesel profile that is high in saturated and monounsaturated fatty acids. In this study, we sequenced the diacylglycerol acyltransferase type-1 from C. vulgaris (CvuDGAT1), which catalyzes the last step in triacylglycerol (TAG) biosynthesis, and characterized this protein using bioinformatics and functional expression assays in a yeast mutant. The CvuDGAT1 protein (460aa) shares motifs characteristic of other plant and microalga DGAT1s, including the binding domains for the acyl-CoA and diacylglycerol substrates and the putative active site. In addition, we report the first putative tertiary structure of DGAT1. This model shows that CvuDGAT1 is integrated into the ER membrane and predicts the proximity of the substrate-binding domains and the active site, despite being widely separated in the protein sequence. We also demonstrate CvuDGAT1's function in TAG biosynthesis by expressing the gene and restoring oil levels in a non-oil-producing yeast mutant. Interestingly, unlike Chlamydomonas reinhardtii, the CvuDGAT1 gene is constitutively expressed at high levels and may contribute to this species' ability to accumulate high levels of oil (i.e. 40% by dry weight).

AB - The green microalga Chlorella vulgaris can be induced to rapidly accumulate oil (40% by dry weight) with a biodiesel profile that is high in saturated and monounsaturated fatty acids. In this study, we sequenced the diacylglycerol acyltransferase type-1 from C. vulgaris (CvuDGAT1), which catalyzes the last step in triacylglycerol (TAG) biosynthesis, and characterized this protein using bioinformatics and functional expression assays in a yeast mutant. The CvuDGAT1 protein (460aa) shares motifs characteristic of other plant and microalga DGAT1s, including the binding domains for the acyl-CoA and diacylglycerol substrates and the putative active site. In addition, we report the first putative tertiary structure of DGAT1. This model shows that CvuDGAT1 is integrated into the ER membrane and predicts the proximity of the substrate-binding domains and the active site, despite being widely separated in the protein sequence. We also demonstrate CvuDGAT1's function in TAG biosynthesis by expressing the gene and restoring oil levels in a non-oil-producing yeast mutant. Interestingly, unlike Chlamydomonas reinhardtii, the CvuDGAT1 gene is constitutively expressed at high levels and may contribute to this species' ability to accumulate high levels of oil (i.e. 40% by dry weight).

KW - Biodiesel

KW - Chlorella vulgaris

KW - Diacylglycerol acyltransferase (DGAT1)

KW - Nitrate deprivation

KW - Oleaginous microalgae

KW - Triacylglycerol (TAG)

UR - http://www.scopus.com/inward/record.url?scp=85011844950&partnerID=8YFLogxK

U2 - 10.1016/j.algal.2015.10.017

DO - 10.1016/j.algal.2015.10.017

M3 - Article

VL - 13

SP - 167

EP - 181

JO - Algal Research

JF - Algal Research

SN - 2211-9264

ER -