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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    • "It has been noted that newly evolved enzymes tend to exhibit lower catalytic activity and stability than the archetypes from which they evolved (Shanklin, 2000). These properties have hindered attempts to accumulate high levels of unusual fatty acids in crop plants upon the expression of variant acyl-ACP desaturases (Nguyen et al., 2010). It is possible that coexpression of a variant desaturase along with its progenitor (or a T104K/ S202E mutant thereof) from the same species might facilitate heterodimer formation that could stabilize the variant and thereby increase unusual fatty acid production. "
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    ABSTRACT: Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast, animals and bacteria. Both classes exhibit a dimeric quaternary structure. Here we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-ACP desaturase, specifically the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified and its properties validated by X-ray crystallography, size exclusion chromatography and activity assay. Heterodimers comprising distinctly tagged wild type (WT) and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only half the number of active sites, purified heterodimers exhibit equivalent activity to WT homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA Ole1p from Saccharomyces which requires two catalytically-competent subunits for activity. Copyright © 2015, Plant Physiology.
    Plant physiology 07/2015; 169(1). DOI:10.1104/pp.15.00622 · 6.84 Impact Factor
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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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