No abstract

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

The fatty acid and sterol compositions of five species of marine dinoflagellates (Scrippsiella sp. Symbiodinium microadriaticum Freud, Gymnodinium sp., Gymnodinium sanguineum Hirasaki, and Fragilidium sp.) are reported. All contained the major fatty acids that are considered common in dinoflagellates, but the proportions were quite variable, and some species contained low contents of some polyunsaturated fatty acids. Concentration ranges for the major fatty acids were: 16:0 (9.0%-24.8%), 18:4(n-3) (2.5%-11.5%), 18:5(n-3) (7.0%-43.1%), 20:5(n-3) (EPA) (1.8%-20.9%), and 22:6(n-3) (DHA) (9.9%-26.3%). Small amounts of novel very-long-chain highly unsaturated C 28 fatty acids occurred in all species. Each dinoflagellate contained a complex mixture of 4-methyl sterols and 4-desmethyl sterols. Four species contained cholesterol, although the amounts were highly variable (from 0.2% of total sterols in Scrippsiella sp. to 45.6% in Fragilidium sp.). All but G. sanguineum contained the 4-methyl sterol dinosterol, and all species contained sterols lacking a double bond in the ring system (i.e. stanols); in Scrippsiella sp. cholestanol composed 24.3% of the total sterols. Other common features of the 4-methylsterol profiles were the presence of 23,24-dimethyl alkylation and unsaturation at ⌬ 22 in the side chain. In Scrippsiella sp., four steroidal ketones were identified: cholestanone, dinosterone, 4␣,23,24-trimethyl-5␣-cholest-8(14)-en-3-one, and dinostanone. The structures of these corresponded to the major sterols in this species, suggesting that the sterols and steroidal ketones are biosynthetically linked. Steroidal ketones were not detected in the other species. Although fatty acid profiles can be used to distinguish among algal classes, they were not useful for differentiating among dinoflagellate species. In contrast, whereas some taxonomic groupings of dinoflagellates display similar sterol patterns, others, such as the gymnodinoids studied here, clearly do not. The combination of fatty acid, sterol, and steroidal ketone profiles may be useful complementary chemotaxonomic tools for distinguishing morphologically similar species. The identification of steroidal ketones supports earlier suggestions that certain dinoflagellates might be a significant source of such components in marine environments.

BackgroundOmega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) such as eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) are critical for human health and development. Numerous studies have indicated that deficiencies in these fatty acids can increase the risk or severity of cardiovascular, inflammatory and other diseases or disorders. EPA and DHA are predominantly sourced from marine fish although the primary producers are microalgae. Much work has been done to engineer a sustainable land-based source of EPA and DHA to reduce pressure on fish stocks in meeting future demand, with previous studies describing the production of fish oil-like levels of DHA in the model plant species, Arabidopsis thaliana.Principal FindingsIn this study we describe the production of fish oil-like levels (>12%) of DHA in the oilseed crop species Camelina sativa achieving a high ω3/ω6 ratio. The construct previously transformed in Arabidopsis as well as two modified construct versions designed to increase DHA production were used. DHA was found to be stable to at least the T5 generation and the EPA and DHA were found to be predominantly at the sn-1,3 positions of triacylglycerols. Transgenic and parental lines did not have different germination or seedling establishment rates.ConclusionsDHA can be produced at fish oil-like levels in industrially-relevant oilseed crop species using multi-gene construct designs which are stable over multiple generations. This study has implications for the future of sustainable EPA and DHA production from land-based sources.

SummaryThe assembly of multistep recombinant pathways in stably transformed plants is a cornerstone of crops producing new products yet can be a laborious and timeconsuming process. Any heterologous expression platform capable of providing a rapid estimation of the functional assembly of an entire pathway would guide the design of such transgenic traits. In this study, we use a Nicotiana benthamiana transient leaf expression system to simultaneously express five genes, from five independent T DNA binary vectors, to assemble a complete recombinant pathway in five days. In this study, we demonstrate the production of long-chain polyunsaturated fatty acids (LC-PUFA) requiring five transgene-encoded reactions to convert endogenous fatty acids to LC-PUFA. The addition of a triacylglycerol assembly enzyme, Arabidopsis thaliana diacylglyceride-O-acyltransferase, and fractionation of the total lipid profile demonstrated that leaf oils contained 37% newly synthesised LC-PUFA, including 7% arachidonic acid (AA), 6% eicosopentaenoic acid and 3% docosahexaenoic acid. The calculation of enzymatic conversion efficiencies at each step of LC-PUFA synthesis suggests that this transient assembly of a complicated multistep pathway is highly efficient. Unlike experiments using stably transformed plants our assembly of an intricate pathway maintained full gene-for-gene interchangeability and required a fraction of the time and glasshouse space. Furthermore, an exogenous LC-PUFA fatty acid substrate, AA, was fed and metabolised by a transiently expressed D17-desaturase enzyme, and provided results similar to those obtained in yeast feeding experiments. Although the assay was ideal for LC-PUFA pathways, this assay format may become a powerful tool for the characterisation and step-wise improvement of other recombinant pathways and multigenic traits.