Metabolic Engineering of Seeds Can Achieve Levels of ω-7 Fatty Acids Comparable with the Highest Levels Found in Natural Plant Sources

Abstract

Plant oils containing v-7 fatty acids (FAs; palmitoleic 16:1D 9 and cis-vaccenic 18:1D 11 ) have potential as sustainable feedstocks for producing industrially important octene via metathesis chemistry. Engineering plants to produce seeds that accumulate high levels of any unusual FA has been an elusive goal. We achieved high levels of v-7 FA accumulation by systematic metabolic engineering of Arabidopsis (Arabidopsis thaliana). A plastidial 16:0-ACP desaturase has been engineered to convert 16:0 to 16:1D 9 with specificity .100-fold than that of naturally occurring paralogs, such as that from cat's claw vine (Doxantha unguis-cati). Expressing this engineered enzyme (Com25) in seeds increased v-7 FA accumulation from ,2% to 14%. Reducing competition for 16:0-ACP by down-regulating the b-ketoacyl-ACP synthase II 16:0 elongase further increased accumulation of v-7 FA to 56%. The level of 16:0 exiting the plastid without desaturation also increased to 21%. Coexpression of a pair of fungal 16:0 desaturases in the cytosol reduced the 16:0 level to 11% and increased v-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.There is increasing interest in the use of plant oils as renewable sources of industrial chemical feedstocks Carlsson, 2009). Recent developments in olefin metathesis have demonstrated that long-chain monoene FA from vegetable oils can be efficiently split into the corresponding short-chain a-olefin and v-alkenoic acids (Rybak et al., 2008;Meier, 2009). Thus, ethenolytic metathesis of v-7 fatty acids (FAs), such as palmitoleic or cis-vaccenic acids, yields 1-octene and 9-decenoate. 1-Octene is a high-demand feedstock with a global consumption of over half a million tons per year that is primarily used as a comonomer in the expanding production of linear low density polyethylene. It is mainly synthesized from petroleumderived ethylene via oligomerization to yield a complex mixture of a-olefins or from coal-derived syngas (Anonymous, 2007).A plant oil containing high (.70%) content of v-7 FA would represent a new and sustainable feedstock for 1-octene production. Several natural plant oil sources of v-7 FA have been reported, e.g. milkweed (Asclepias syriaca) accumulates approximately 25% v-7 FA (comprising approximately 10% 16:1D 9 and approximately 15% 18:1D 11 ) and cat's claw vine (Doxantha unguis-cati) accumulates approximately 72% v-7 FA (comprising approximately 55% 16:1D 9 and approximately 17% 18:1D 11 ), but the low yields and poor agronomic properties of these plants preclude their commercial use for v-7 FA production. Isolation of the D 9 -16:0-ACP desaturases genes responsible for the production of v-7 FA from milkweed and Doxantha (Cahoon et al., 1997 presented an opportunity for their heterologous expression and v-7 FA production in transgenic crops. However, heterologous expression of the milkweed desaturase in Arabidopsis (Arabidopsis thaliana) failed to produce detectable v-7 FA, and when the Doxantha desaturase was expressed in Brassica napus, it resulted in the accumul...

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

“…al. (17), for ω-7 fatty acids, to produce designer fatty acids that could constitute improved feedstocks for industry. These would be renewable and could serve as alternatives to feedstocks currently obtained from petrochemicals.…”

Heterotrophy of filamentous oleaginous microalgae Tribonema minus for potential production of lipid and palmitoleic acid

“…Moreover, palmitoleic acid is thought to be better to provide low temperature property than other saturated fatty acid when it was converted into biodiesel because of its carbon chain length and unsaturated double bond number (Knothe, 2010;Cao et al, 2014). Besides, palmitoleic acid could be used to produce 1-octene which is highly consumed in the expanding production of linear low density polyethylene (Rybak et al, 2008;Nguyen et al, 2010).…”

Ectopic overexpression of MucACP- and #916;9 desaturase leads to and #969;-7 fatty acid accumulation in tobacco leaves

“…Transgenic Arabidopsis seeds that expressed the Asclepias syriaca ACP-Δ9D did not accumulate ω-7 fatty acid. However, in Doxantha ACP-Δ9D-expressing transgenic Arabidopsis seeds, newly synthesized ω-7 fatty acid was detected (Nguyen et al, 2010). In wild cat's claw, the ω-7 fatty acid composition of seed oil could be up to 79% (64% C16:1Δ9, 15% C18:1Δ11).…”

“…The aim of this strategy is to improve the stability, quality, and nutritional value of vegetable oils for the purpose of wider utilization. One strategy to enhance the synthesis of ω-7 fatty acids and improve their accumulation is to overexpress ACP-Δ9D and/or CoA-Δ9D in target plant seeds (Grayburn et al, 1992;Nguyen et al, 2010;Xue et al, 2013;Nguyen et al, 2015). Here, the MucACP-Δ9D gene was isolated from the ω-7 fatty acid-rich wild cat's claw and inserted into an overexpression vector for the transformation of tobacco plants.…”