High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient wax ester synthesis enzymes

Abstract

BackgroundBiotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived wax esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of wax esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a wax synthase (WS) in seeds are sufficient.ResultsFor optimization of the enzymatic activity and subcellular localization of wax ester synthesis enzymes, two fusion proteins were created, which showed wax ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for wax ester production. The tested seven enzyme combinations resulted in different yields and compositions of wax esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in wax esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into wax esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total wax esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed−1 of wax esters, containing 27–34 mol% oleyl oleate.ConclusionsThe overall yields and the compositions of wax esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of wax ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate wax ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1057-4) contains supplementary material, which is available to authorized users.

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

“…There are two studies published within the last years investigating the usage of bacterial WSD to produce industrial lubricants in plants (Ruiz‐Lopez et al, 2017; Yu et al, 2018). Researchers are especially interested to identify combinations of WE synthesizing enzymes that produce WE consisting of long chain monounsaturated fatty acid and fatty alcohol moieties.…”

“…A promising approach to push the synthesis of WE to the almost exclusive formation of one WE species, namely 18:1/18:1 WE, is achieved by providing only a narrow pool of substrates. It was successfully shown by three different studies that expressing FAR/WS(D) combinations in the A. thaliana fad2fae1 mutant (Smith et al, 2003) almost all WE produced were 18:1/18:1, no matter which FAR/WS (D) combination was analyzed (Heilmann et al, 2012;Iven et al, 2016;Yu et al, 2018). FAE1 elongates fatty acids beyond 18 carbon chain length, and FAD2 introduces double bonds to monounsaturated fatty acids (Kunst et al, 1992;Okuley et al, 1994).…”

“…Since the banning of whale hunting, WE are industrially produced from fossil fuel or plant‐derived TAG or are extracted from the expensive seeds of the slow‐growing desert plant jojoba ( Simmondsia chinensis , S. chinensis ) (Sturtevant et al, 2020). Nowadays, attempts to produce WE in transgenic plants in an industrial scale are steadily increasing (Heilmann et al, 2012; Iven et al, 2016; Lardizabal et al, 2000; Ruiz‐Lopez et al, 2017; Yu et al, 2018).…”

“…In order to avoid the production of industrial WE from limiting fossil fuel resources, attempts to synthesize WE in plant seeds are steadily increasing (Heilmann et al, 2012; Iven et al, 2016; Lardizabal et al, 2000;Ruiz‐Lopez et al, 2017; Yu et al, 2018). In the beginning eukaryotic FAR (jojoba FAR (ScFAR) and mouse FAR (MmFAR)) in combination with eukaryotic DGAT1‐like or DGAT2‐like WS (jojoba WS (ScWS) and mouse WS (MmWS)) were expressed in the seeds of the model plant Arabidopsis thaliana ( A. thaliana ) and revealed a successful WE production (Heilmann et al, 2012; Lardizabal et al, 2000).…”

“…Due to that, MaFAR was also tested in combination with ScWS in A. thaliana and Camelina sativa ( C. sativa ) (Iven et al, 2016). In 2017 and 2018, two studies were published, successfully synthesizing WE in A. thaliana and C. sativa seeds when using the soluble bacterial WSD AbWSD1, MaWSD2, and MhWS2 in combination with different FAR enzymes (Ruiz‐Lopez et al, 2017; Yu et al, 2018).…”