Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil

Abstract

SummarySeed oil composed of wax esters with long-chain monoenoic acyl moieties represents a highvalue commodity for industry. Such plant-derived sperm oil-like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low-input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl-CoA substrates. To produce plant-derived sperm oillike liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1Δc/Oleo3:EYFP:MmWS, Oleo3:mCherry: MmFAR1Δc/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/ 18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.

Expression of Camelina WRINKLED1 Isoforms Rescue the Seed Phenotype of the Arabidopsis wri1 Mutant and Increase the Triacylglycerol Content in Tobacco Leaves

“…Metabolic engineering in Camelina is underway to modify the fatty acid composition by blocking the endogenous fatty acid desaturase 2 (FAD) and fatty acid elongase (FAE) 1 pathways, which would make it more suitable for biofuel (Nguyen et al, 2013), and to incorporate new pathways to produce unusual fatty acids for industrial uses (Snapp et al, 2014; Liu et al, 2015; Nguyen et al, 2015), and omega-3 long-chain polyunsaturated fatty acids for human consumption (Petrie et al, 2014). Wax esters replaced 21% of the seed oil TAGs in Camelina transformed with two genes encoding a fatty alcohol-forming acyl-CoA reductase (FAR) and a wax ester synthase (WS) (Iven et al, 2016). Currently only two key genes that are involved in fatty acid and TAG biosynthesis metabolism, FAD2 (Kang et al, 2011) and DGAT1 (Kim et al, 2016), respectively, have been identified to exist in three copies in Camelina .…”

Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis

“…In contrast to TAG consisting of a glycerol backbone esterified by three fatty acyl chains, WE is composed of fatty acid esterified to fatty alcohol, which would not cause the generation of glycerol as an undesirable by-product during the refinement of WEs to biodiesel [11]. Because WEs have a higher energy density than TAG-derived lipids, they could represent a superior feedstock for biodiesel production [12]. Currently, WEs have multiple important technical applications in a variety of areas such as medicine, cosmetics, and food industries, and have the potential for cost-effective and sustainable production of lubricants [12, 13].…”

“…Because WEs have a higher energy density than TAG-derived lipids, they could represent a superior feedstock for biodiesel production [12]. Currently, WEs have multiple important technical applications in a variety of areas such as medicine, cosmetics, and food industries, and have the potential for cost-effective and sustainable production of lubricants [12, 13]. Depending on the chain lengths and the degree of unsaturation of fatty acids and fatty alcohol components, WEs have different physical and chemical properties such as melting temperature, oxidation stability, and pressure stability [14].…”

“…Unlike other oil storing plant seeds that accumulate large quantities of TAGs as energy reserves, the desert shrub jojoba’s seeds accumulate liquid WEs up to 60% of the seed dry weight, and these WEs are composed mainly C 20:1 fatty acids and C 20:1 /C 22:1 fatty alcohols, which generates a carbon chain length of C 38 –C 44 [15]. However, considering the high price and limited supply of jojoba oil due to the low yield and high cost for cultivating jojoba plant, there is still a strong need for an alternative cost-effective solution for sustainable production of WEs [12]. Recent engineering efforts show that other oilseed plants such as Camelina sativa have attracted attention as a promising biological platform form cost-effective production of WEs [12–14, 16, 17].…”

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil

“…Moreover, the seed yield is adequate to facilitate the rapid production of oil in quantities large enough for small-scale testing and product development. In recent years, several different oil traits have been developed in C. sativa seeds, such as high 18:1n-9 (Kang et al, 2011;, high 16:0 , and high v-7 fatty acids (Nguyen et al, 2015), as well as the production of diverse extrinsic lipid compounds, such as wax esters (Iven et al, 2016), fish oils (Petrie et al, 2014;Ruiz-Lopez et al, 2014), nervonic acid (Huai et al, 2015), and acetyl glyceride oils (Liu et al, 2015).…”