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

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.
Plant physiology (Impact Factor: 6.84). 10/2010; 154(4):1897-904. DOI: 10.1104/pp.110.165340
Source: PubMed


Plant oils containing ω-7 fatty acids (FAs; palmitoleic 16:1Δ(9) and cis-vaccenic 18:1Δ(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 ω-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 16:1Δ(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 ω-7 FA accumulation from <2% to 14%. Reducing competition for 16:0-ACP by down-regulating the β-ketoacyl-ACP synthase II 16:0 elongase further increased accumulation of ω-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 ω-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.

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Available from: Terence Anthony Walsh, Oct 13, 2015
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    • "We have previously developed an extensive metabolic engineering tool box of seed-specific promoters and selection markers to facilitate these studies (Horn et al., 2013; Nguyen et al., 2013). Here, we have systematically examined the use of a six transgene strategy incorporating the previously described method from Arabidopsis (Cahoon and Shanklin, 2000; Nguyen et al., 2010) that also combined RNAi silencing of FatB, encoding the 16:0-ACP thioesterase. Through this approach, camelina oil was generated with ~66% omega-7 fatty acids as well as an unexpected two-to threefold reduction in total saturated fatty acid content relative to conventional camelina oil. "
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    ABSTRACT: Seed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1∆9) and cis-vaccenic acid (18:1∆11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega-7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ∆9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to ∆9 desaturation of palmitoyl (16:0)-acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed-specific co-expression of a mutant ∆9-acyl-ACP and an acyl-CoA desaturase with high specificity for 16:0-ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega-7 monounsaturated fatty acids were obtained. Further increases in omega-7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed-specific suppression of 3-keto-acyl-ACP synthase II and the FatB 16:0-ACP thioesterase genes to increase substrate pool sizes of 16:0-ACP for the ∆9-acyl-ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.
    Plant Biotechnology Journal 07/2014; 13(1). DOI:10.1111/pbi.12233 · 5.75 Impact Factor
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    • "Addition of an extraplastidial version of the desaturase was able to raise product levels to 71% in the highest lines. This 71% accumulation is nearly equivalent to the native accumulator plant, Doxantha unguiscati which has 72% ω-7 FA (Nguyen et al., 2010). "
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    ABSTRACT: More than 300 types of modified fatty acids (mFA) are produced in triacylglycerols (TAG) by various plant species, with many of these unusual structures rendering unique physical and chemical properties that are desirable for a variety of bio-based industrial uses. Attempts to produce these mFA in crop species have thus far failed to reach the desired levels of production and highlighted the need to better understand how fatty acids are synthesized and accumulated in seed oils. In this review we discuss how some of the progress made in recent years, such as the improved TAG synthesis model to include acyl editing and new enzymes such as PDCT, may be utilized to achieve the goal of effectively modifying plant oils for industrial uses. Co-expressing several key enzymes may circumvent the bottlenecks for the accumulation of mFA in TAG through efficient removal of mFA from phosphatidylcholine. Other approaches include the prevention of feedback inhibition of fatty acid synthesis and improving primary enzyme activity in host transgenic plants. In addition, genomic approaches are providing unprecedented power to discover more factors that may facilitate engineering mFA in oilseeds. Based on the results of the last 20 years, creating a high mFA accumulating plant will not be done by simply inserting one or two genes; it is necessary to stack genes encoding enzymes with favorable kinetic activity or specificity along with additional complementary transgenes in optimized plant backgrounds to produce industrial fatty acids at desirable levels. Finally, we discuss the potential of Camelina as an industrial oilseed platform.
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    • "However , most plants that produce unusual FAs are either limited in oil yield or have undesirable agronomic features (Badami and Patil, 1980; Voelker and Kinney, 2001; Dyer et al., 2008). With very few exceptions (Knutzon et al., 1999; Nguyen et al., 2010), attempts to engineer oilseed plants to accumulate unusual FAs has resulted in low yields of the unusual FA in seed TAG, especially the engineering of unusual FAs synthesized within membrane lipids (Cahoon et al., 2007; Dyer et al., 2008; Lu et al., 2011). The limited successes in oilseed engineering highlight the fact that we still do not fully understand how plants accumulate oils with very different FA compositions, even though the enzymes, genetics, and regulation of FA and TAG synthesis have been extensively studied (Dyer et al., 2008; Snyder et al., 2009; Weselake et al., 2009; Baud and Lepiniec, 2010; Li-Beisson et al., 2010; Napier and Graham, 2010; Wallis and Browse, 2010; Lu et al., 2011; Chapman and Ohlrogge, 2012). "
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    ABSTRACT: The unique properties of vegetable oils from different plants utilized for food, industrial feedstocks, and fuel is dependent on the fatty acid (FA) composition of triacylglycerol (TAG). Plants can use two main pathways to produce diacylglycerol (DAG), the immediate precursor molecule to TAG synthesis: (1) De novo DAG synthesis, and (2) conversion of the membrane lipid phosphatidylcholine (PC) to DAG. The FA esterified to PC are also the substrate for FA modification (e.g., desaturation, hydroxylation, etc.), such that the FA composition of PC-derived DAG can be substantially different than that of de novo DAG. Since DAG provides two of the three FA in TAG, the relative flux of TAG synthesis from de novo DAG or PC-derived DAG can greatly affect the final oil FA composition. Here we review how the fluxes through these two alternate pathways of DAG/TAG synthesis are determined and present evidence that suggests which pathway is utilized in different plants. Additionally, we present examples of how the endogenous DAG synthesis pathway in a transgenic host plant can produce bottlenecks for engineering of plant oil FA composition, and discuss alternative strategies to overcome these bottlenecks to produce crop plants with designer vegetable oil compositions.
    Frontiers in Plant Science 07/2012; 3:147. DOI:10.3389/fpls.2012.00147 · 3.95 Impact Factor
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