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

Abstract

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 sn-1 and sn-3 positions of triacylglycerol (TAG) in these engineered seeds, leaving only a minor amount (∼10%) at sn-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 sn-2 position of glycerol backbone. In order to increase the level of DHA at sn-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 sn-2 position of the TAG glycerol backbone were investigated for substrate preference for DHA. In transiently expressing these LPAATs in Nicotiana benthamiana, a Mortierella alpina 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 M. alpina and Emiliania huxleyi showed a high preference for DHA-CoA acylation onto oleoyl-LPA. When docosahexaenoyl-LPA was used as the acyl receiver, M. alpina LPAAT also showed a high preference for DHA-CoA. Stable overexpression of M. alpina 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 sn-2 position of seed TAG. LC-MS analysis of the seed TAG species also confirmed that overexpression of M. alpina LPAAT increased di-DHA and tri-DHA TAGs, suggesting that the M. alpina LPAAT could enrich DHA at the TAG sn-2 position, leading to a metabolic engineering of oil seed for channeling DHA into the sn-2 position of TAG and to a higher DHA level.

Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil

“…These fatty acids are intermediates in the metabolic pathway we have used to produce DHA in this study. For example, the use of different versions of the GA7 construct may yield fish oil-like levels of EPA and even higher levels of the relatively rare fatty acid DPA, or possibly further increase the amount of DHA that can be produced in canola seed (Shrestha et al, 2018).…”

Ultra‐high α‐linolenic acid accumulating developmental defective embryo was rescued by lysophosphatidic acid acyltransferase 2

“…Previously, acyltransferases such as diacylglycerol acyltransferase and phospholipid:diacylglycerol acyltransferase have been successfully coexpressed with FA modification enzymes and have rescued defective embryos (Li et al ., 2012; Bates et al ., 2014; Lunn et al ., 2018). LPAT is known as a valuable acyltransferase that exhibits various abundant substrate preferences in different species (Knutzon et al ., 1999; Nath et al ., 2009; Yu et al ., 2014; Kim et al ., 2015; Shrestha et al ., 2018). In the present study we found that CsLPAT2 could rescue our defective embryo phenotype, a result that will provide more options for the production of UFAs in crops in the future.…”

Plant and algal lysophosphatidic acid acyltransferases increase docosahexaenoic acid accumulation at the sn-2 position of triacylglycerol in transgenic Arabidopsis seed oil

“…Expression of the M . alpina LPAAT increases sn -2 DHA in seed oil, which was confirmed by both lipase digestion and 13 C NMR [ 26 ]. Nevertheless, a more sensitive and convenient analytical method to quickly screen for and quantify sn -2 DHA will greatly aid characterization of DHA-containing glycerolipids and functional analysis of LPAATs that can increase sn -2 DHA in seed oils.…”

“…In vitro measurement of LPAAT enzyme activity is challenging; eukaryote LPAATs are bound to the ER membrane or to the thylakoid membrane of the plastid [ 44 ]. Typically, measurements of LPAAT activity are performed with a heterogeneous microsomal preparation, containing either heterologously expressed protein [ 26 , 45 – 47 ] or natively expressed protein [ 21 , 27 , 46 , 48 ]. To mitigate background acyltransferase activity, the slc1Δ or ale1Δ (also referred to as slc4Δ ) yeast mutants lacking LPAAT have been used to heterologously express plant acyltransferase proteins [ 49 ].…”