Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop.

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyl-triacylglycerol-containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress' rapid domestication to meet the growing sustainable food and fuel demands.

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids.

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design-test-learn cycle, these results, which include not only the synthesis of 'designer' lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications.

Camelina sativa: An ideal platform for the metabolic engineering and field production of industrial lipids.

Triacylglycerols (TAG) containing modified fatty acids with functionality beyond those found in commercially grown oil seed crops can be used as feedstocks for biofuels and bio-based materials. Over the years, advances have been made in transgenically engineering the production of various modified fatty acids in the model plant Arabidopsis thaliana. However, the inability to produce large quantities of transgenic seed has limited the functional testing of the modified oil. In contrast, the emerging oil seed crop Camelina sativa possesses important agronomic traits that recommend it as an ideal production platform for biofuels and industrial feedstocks. Camelina possesses low water and fertilizer requirements and is capable of yields comparable to other oil seed crops, particularly under stress conditions. Importantly, its relatively short growing season enables it to be grown as part of a double cropping system. In addition to these valuable agronomic features, Camelina is amenable to rapid metabolic engineering. The development of a simple and effective transformation method, combined with the availability of abundant transcriptomic and genomic data, has allowed the generation of transgenic Camelina lines capable of synthesizing high levels of unusual lipids. In some cases these levels have surpassed what was achieved in Arabidopsis. Further, the ability to use Camelina as a crop production system has allowed for the large scale growth of transgenic oil seed crops, enabling subsequent physical property testing. The application of new techniques such as genome editing will further increase the suitability of Camelina as an ideal platform for the production of biofuels and bio-materials.

Ethanol production from banana peels using statistically optimized simultaneous saccharification and fermentation process.

Dried and ground banana peel biomass (BP) after hydrothermal sterilization pretreatment was used for ethanol production using simultaneous saccharification and fermentation (SSF). Central composite design (CCD) was used to optimize concentrations of cellulase and pectinase, temperature and time for ethanol production from BP using SSF. Analysis of variance showed a high coefficient of determination (R(2)) value of 0.92 for ethanol production. On the basis of model graphs and numerical optimization, the validation was done in a laboratory batch fermenter with cellulase, pectinase, temperature and time of nine cellulase filter paper unit/gram cellulose (FPU/g-cellulose), 72 international units/gram pectin (IU/g-pectin), 37 °C and 15 h, respectively. The experiment using optimized parameters in batch fermenter not only resulted in higher ethanol concentration than the one predicted by the model equation, but also saved fermentation time. This study demonstrated that both hydrothermal pretreatment and SSF could be successfully carried out in a single vessel, and use of optimized process parameters helped achieve significant ethanol productivity, indicating commercial potential for the process. To the best of our knowledge, ethanol concentration and ethanol productivity of 28.2 g/l and 2.3 g/l/h, respectively from banana peels have not been reported to date.