Functional characterization of acyltransferases from Salvia hispanica that can selectively catalyze the formation of trilinolenin.

Salvia hispanica (chia) is an important oilseed crop cultivated commercially in South America, Australia, and
India. It is the richest terrestrial natural source of α-linolenic acid (ALA), an ω-3 polyunsaturated fatty acid with
varied health benefits. In this study, we have measured the total lipid content, fatty acid composition in four
phases of seed development and analyzed the major triacylglycerol (TAG) molecular species present in Indian
chia seed oil. We found that the mature seeds produced 28% oil, 65% of ALA, and trilinolenin as the major TAG
species. To make TAG rich in ALA, there should be specialized enzymes that can efficiently transfer ALA to TAG.
To study this hypothesis, we performed a characterization of TAG synthesizing enzymes present in chia. We have
identified two acyl CoA:diacylglycerol acyltransferases (ShDGAT1 and ShDGAT2) and one phospholipid:diacylglycerol
acyltransferase (ShPDAT1) from the chia transcriptome data. Functional characterization of these
enzymes was conducted by heterologous expression in a TAG deficient mutant of Saccharomyces cerevisiae.
Substrate specificity studies showed that ShDGAT2-1 and ShPDAT1 exhibited a strong preference towards
substrates containing ALA and could incorporate 45% and 80% ALA into TAG, respectively. Both enzymes
incorporated ALA in a concentration-dependent manner into TAG and were able to form trilinolenin in yeast. Our
results provide a first insight into the high ALA accumulation in chia and the first demonstration of trilinolenin
formation by DGAT2. The two identified enzymes (ShDGAT2-1 and ShPDAT1) can be used to metabolically
engineer other oilseed crops to produce high levels of ALA.