Dataset Information

Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds.

ABSTRACT: Seeds of members of the genus Cuphea accumulate medium-chain fatty acids (MCFAs; 8:0-14:0). MCFA- and palmitic acid- (16:0) rich vegetable oils have received attention for jet fuel production, given their similarity in chain length to Jet A fuel hydrocarbons. Studies were conducted to test genes, including those from Cuphea, for their ability to confer jet fuel-type fatty acid accumulation in seed oil of the emerging biofuel crop Camelina sativa. Transcriptomes from Cuphea viscosissima and Cuphea pulcherrima developing seeds that accumulate >90% of C8 and C10 fatty acids revealed three FatB cDNAs (CpuFatB3, CvFatB1, and CpuFatB4) expressed predominantly in seeds and structurally divergent from typical FatB thioesterases that release 16:0 from acyl carrier protein (ACP). Expression of CpuFatB3 and CvFatB1 resulted in Camelina oil with capric acid (10:0), and CpuFatB4 expression conferred myristic acid (14:0) production and increased 16:0. Co-expression of combinations of previously characterized Cuphea and California bay FatBs produced Camelina oils with mixtures of C8-C16 fatty acids, but amounts of each fatty acid were less than obtained by expression of individual FatB cDNAs. Increases in lauric acid (12:0) and 14:0, but not 10:0, in Camelina oil and at the sn-2 position of triacylglycerols resulted from inclusion of a coconut lysophosphatidic acid acyltransferase specialized for MCFAs. RNA interference (RNAi) suppression of Camelina ?-ketoacyl-ACP synthase II, however, reduced 12:0 in seeds expressing a 12:0-ACP-specific FatB. Camelina lines presented here provide platforms for additional metabolic engineering targeting fatty acid synthase and specialized acyltransferases for achieving oils with high levels of jet fuel-type fatty acids.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC4493788 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds.

Kim Hae Jin HJ Silva Jillian E JE Vu Hieu Sy HS Mockaitis Keithanne K Nam Jeong-Won JW Cahoon Edgar B EB

Journal of experimental botany 20150511 14

Seeds of members of the genus Cuphea accumulate medium-chain fatty acids (MCFAs; 8:0-14:0). MCFA- and palmitic acid- (16:0) rich vegetable oils have received attention for jet fuel production, given their similarity in chain length to Jet A fuel hydrocarbons. Studies were conducted to test genes, including those from Cuphea, for their ability to confer jet fuel-type fatty acid accumulation in seed oil of the emerging biofuel crop Camelina sativa. Transcriptomes from Cuphea viscosissima and Cuphe ...[more]

PMID: 25969557

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Project description:Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 (CpFatB1) (C8 specificity, lower activity) and CpFatB2 (C14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify CpFatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a "positive patch" within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from CpFatB1 into CpFatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control.IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Similarly, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing.

Project description:BackgroundAlcohol-to-jet (ATJ) is one of the technical feasible biofuel technologies. It produces jet fuel from sugary, starchy, and lignocellulosic biomass, such as sugarcane, corn grain, and switchgrass, via fermentation of sugars to ethanol or other alcohols. This study assesses the ATJ biofuel production pathway for these three biomass feedstocks, and advances existing techno-economic analyses of biofuels in three ways. First, we incorporate technical uncertainty for all by-products and co-products though statistical linkages between conversion efficiencies and input and output levels. Second, future price uncertainty is based on case-by-case time-series estimation, and a local sensitivity analysis is conducted with respect to each uncertain variable. Third, breakeven price distributions are developed to communicate the inherent uncertainty in breakeven price. This research also considers uncertainties in utility input requirements, fuel and by-product outputs, as well as price uncertainties for all major inputs, products, and co-products. All analyses are done from the perspective of a private firm.ResultsThe stochastic dominance results of net present values (NPV) and breakeven price distributions show that sugarcane is the lowest cost feedstock over the entire range of uncertainty with the least risks, followed by corn grain and switchgrass, with the mean breakeven jet fuel prices being $0.96/L ($3.65/gal), $1.01/L ($3.84/gal), and $1.38/L ($5.21/gal), respectively. The variation of revenues from by-products in corn grain pathway can significantly impact its profitability. Sensitivity analyses show that technical uncertainty significantly impacts breakeven price and NPV distributions.ConclusionsTechnical uncertainty is critical in determining the economic performance of the ATJ fuel pathway. Technical uncertainty needs to be considered in future economic analyses. The variation of revenues from by-products plays a significant role in profitability. With the distribution of breakeven prices, potential investors can apply whatever risk preferences they like to determine an appropriate bid or breakeven price that matches their risk profile.

Dataset Information

Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds.

Publications

Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds.

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