AUTHOR=Shrestha Pushkar , Hussain Dawar , Mulder Roger J. , Taylor Matthew C. , Singh Surinder P. , Petrie James R. , Zhou Xue-Rong 

TITLE=Increased DHA Production in Seed Oil Using a Selective Lysophosphatidic Acid Acyltransferase

JOURNAL=Frontiers in Plant Science

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2018.01234

DOI=10.3389/fpls.2018.01234

ISSN=1664-462X

ABSTRACT=<p>Metabolic engineering of the omega-3 (ω3) long chain polyunsaturated fatty acid biosynthesis pathway has generated fish oil-like levels of pharmaceutically and nutritionally important docosahexaenoic acid (DHA) in plant seeds. However, the majority of DHA has been accumulated at the <italic>sn</italic>-1 and <italic>sn</italic>-3 positions of triacylglycerol (TAG) in these engineered seeds, leaving only a minor amount (∼10%) at <italic>sn</italic>-2 position and indicating a strong discrimination (or, a very poor specificity) for DHA by seed lysophosphatidic acid acyltransferases (LPAATs), which mediate the acylation of <italic>sn</italic>-2 position of glycerol backbone. In order to increase the level of DHA at <italic>sn</italic>-2 position of TAG and to increase overall DHA level in seeds, we attempted to discover DHA-preferring LPAATs. Several LPAATs for acylation of the <italic>sn</italic>-2 position of the TAG glycerol backbone were investigated for substrate preference for DHA. In transiently expressing these LPAATs in <italic>Nicotiana benthamiana</italic>, a <italic>Mortierella alpina</italic> LPAAT had the highest substrate specificity for accumulating DHA onto oleoyl-lysophosphatidic acid (oleoyl-LPA), while the plant LPAATs tested showed lower preference for DHA. In a competition assay with a pool of four ω3 acyl-Coenzyme A (CoA) substrates involved in the DHA biosynthesis pathway, LPAATs from both <italic>M. alpina</italic> and <italic>Emiliania huxleyi</italic> showed a high preference for DHA-CoA acylation onto oleoyl-LPA. When docosahexaenoyl-LPA was used as the acyl receiver, <italic>M. alpina</italic> LPAAT also showed a high preference for DHA-CoA. Stable overexpression of <italic>M. alpina</italic> LPAAT in an Arabidopsis line that expressed the DHA biosynthesis pathway significantly increased both the total DHA levels and the distribution of DHA onto the <italic>sn</italic>-2 position of seed TAG. LC-MS analysis of the seed TAG species also confirmed that overexpression of <italic>M. alpina</italic> LPAAT increased di-DHA and tri-DHA TAGs, suggesting that the <italic>M. alpina</italic> LPAAT could enrich DHA at the TAG <italic>sn</italic>-2 position, leading to a metabolic engineering of oil seed for channeling DHA into the <italic>sn</italic>-2 position of TAG and to a higher DHA level.</p>