Abstract Detail

Biochemistry, metabolism, carbon flux

Xu, Yang [1], Chen, Guanqun [1], Weselake, Randall J. [1].

Recombinant variants of Brassica napus type-1 diacylglycerol acyltransferase increase oil accumulation in baker’s yeast.

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acylation of sn-1,2-diacylglycerol to form triacylglycerol (TAG). During Brassica napus seed development, the level of DGAT activity may have a substantial effect on the flow of carbon into seed oil; thus, this reaction may represent a bottleneck in seed oil accumulation. Over-expression of DGAT1 has been shown to boost seed oil content in canola-type B. napus and other oleaginous plants including Arabidopsis thaliana and soybean (Glycine max). Previous research has shown that embryo-specific over-expression of wild type B. napus DGAT1 in B. napus leads to increased seed oil content. Further increases in B. napus seed oil content may be obtained through the introduction of DGAT1 improved through protein engineering. Previously, we used directed evolution to successfully generate B. napus DGAT1 (BnaC.DGAT1.a) mutated libraries. During the screening process, several variants resulting in increased TAG accumulation in Saccharomyces cerevisiae strain H1246 (MATα are1-Δ::HIS3, are2- Δ::LEU2, dga1- Δ::KanMX4, lro1- Δ::TRP1 ADE2) were identified. In order to develop insight into the underlying causes for increased TAG production in yeast brought about through expression of the BnaC.DGAT1.a variants, seven recombinant BnaC.DGAT1.a variants were chosen for characterization. Wild type recombinant BnaC.DGAT1.a and empty vector (lac Z) were included as controls. The various microsomal fractions containing these variant enzymes were prepared from cells during the mid-log phase and then analyzed for DGAT activity using radiolabeled substrate. BnaC.DGAT1.a variants were designed to accumulate with a V5-tag at the C-terminus and thus their production could be detected by Western blotting. The results of these assays indicated that there were three explanations for increased TAG accumulation in yeast transformed with cDNAs encoding the variant enzymes: 1) BnaC.DGAT1.a with increased specific activity; 2) increased production of enzyme polypeptide; and 3) a combination of increased specific activity and polypeptide accumulation.

1 - University of Alberta, Department of Agricultural, Food and Nutritional Science, 410 Agriculture Forestry Centre, edmonton, alberta, T6G2p5, Canada

Keywords:
DGAT1
oilseed rape
Saccharomyces cerevisiae H1246
triacylglycerol biosynthesis
mutagenesis.

Presentation Type: Oral Paper:Papers for Topics
Session: 27
Location: Hall A/The Shaw Conference Centre
Date: Tuesday, July 28th, 2015
Time: 9:15 AM
Number: 27006
Abstract ID:1159
Candidate for Awards:None