High-level Accumulation of Oleyl Oleate in Plant Seed Oil by Abundant Supply of Oleic Acid Substrates to Efficient Wax Ester Synthesis Enzymes

Background: Biotechnology 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.

Results: For 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 . To promote the formation of oleyl oleate in seed oil, WSs from (WSD1) and (WS2), 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 (FAR) with WSD1 or WS2 led to a high incorporation of C substrates in wax esters. The FAR/TMAWAT2-WSD1 combination resulted in the incorporation of more C alcohol and C acyl moieties into wax esters compared with FAR/WSD1. The fusion protein of a WS from (WS) with MaFAR exhibited higher specificity toward C substrates in preference to C substrates. Expression of FAR/WSD1 in the Arabidopsis 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 FAR/WSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of , lines expressing FAR/WS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed of wax esters, containing 27-34 mol% oleyl oleate.

Conclusions: The 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.