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

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

doi:10.3389/fpls.2018.01234

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.

Highlights

Omega-3 long chain polyunsaturated fatty acids (ω3 LCPUFAs), such as eicosapentaenoic acid (EPA) (20:5ω3), docosapentaenoic acid (DPA) (22:5ω3), and docosahexaenoic acid (DHA) (22:6ω3) are important essential fatty acids for human health
Our previous metabolic engineering of the DHA biosynthesis pathway in Arabidopsis or camelina seeds led to 10–15% DHA accumulation (Petrie et al, 2012, 2014), which are similar to levels in fish oil
The distribution of the DHA was mainly at the TAG sn-1/sn-3 positions, suggesting that Arabidopsis or camelina enzymes might have a low preference for DHA compared to other fatty acids for acylation of the sn-2 position of the glycerol backbone, mediated by Lysophosphatidic acid acyltransferase (LPAAT)

Summary

Introduction

Omega-3 long chain polyunsaturated fatty acids (ω3 LCPUFAs), such as EPA (20:5ω3), DPA (22:5ω3), and DHA (22:6ω3) are important essential fatty acids for human health. Several investigations have indicated their health benefits, which range from their positive roles in fetal development through to osteoporosis, as well as preventing cardiovascular disease, diabetes, and Alzheimer’s disease (Swanson et al, 2012; Haslam et al, 2013). The demand for these fatty acids is increasing for dietary purposes, medical treatments, and livestock and aquaculture feed. Cheng et al (2010) reported the production of 25% EPA in Brassica carinata seeds. The majority of DHA is located at the sn-1 and sn-3 positions (∼90%) of TAG, with only a small proportion found at the sn-2 position (∼10%) (Petrie et al, 2012, 2014)

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Results

Conclusion