A number of studies have been
A number of studies have been carried out to understand lipid production pathways and to increase TAGs level in higher plants. Overexpression of key tnf-a of the Kennedy pathway until now is by far the most successful strategy to increase TAG lipid content in Brassica napus, Arabidopsis thaliana and Nicotiana tabacum (Andrianov et al., 2010, Bouvier-Nave et al., 2000, Jako et al., 2001, Zou et al., 1997). The enzyme acyl-CoA diacylglycerol acyltransferase (DGAT) has been identified as one of the key enzymes of the Kennedy pathway. Not many reports on these enzymes are available in microalgal species so far. Recently, Wagner et al. (2010) were able to detect three putative type-2 DGAT genes in the microalga Ostreococcus tauri (class Prasinophytes) by database searches. DGAT activity was convincingly demonstrated for at least one of the corresponding proteins by heterologous expression in yeast (Wagner et al., 2010), indicating a role in TAG synthesis in the native cells.
In recent years it has been suggested numerous times that the genetic engineering of key factors of the lipid production pathways, like the DGAT genes, could be a promising approach to increase the cellular lipid content in microalgae, e.g. for more efficient biofuels production (Chisti, 2008, Courchesne et al., 2009, Dunahay et al., 1995, Dunahay et al., 1996, Durrett et al., 2008, Leon-Banares et al., 2004, Work et al., 2011, Yu et al., 2011). Therefore with this work we aimed to determine the effects of overexpression of three potential type-2 DGAT genes in the green microalga Chlamydomonas reinhardtii (class Chlorophyceae). Overexpression strains were generated and characterized in detail with regards to growth parameters and lipid profiles.
Material and methods
Results and discussion
Conclusion Overexpression of DGAT genes in microalgae in order to increase their lipid content has been proposed numerous times since the same strategy worked well for some other plant organisms. In this work, we identified five putative type-2 DGAT candidates in the green microalga C. reinhardtii in silico and generated cell lines efficiently over-expressing the three most promising candidate genes, designated CrDGAT2a, b and c. These lines were characterized in terms of growth, lipid content and fatty acid composition in standard growth conditions and during nutrient (nitrogen or sulfur) starvation. From our data we conclude that the comparably simple biotechnological strategy to overexpress DGAT candidate genes was not applicable for C. reinhardtii to increase the lipid content or to alter the lipid composition. In fact, genetic and metabolic data during nutrient stress conditions presented in this and earlier work (Bolling and Fiehn, 2005, Doebbe et al., 2010, Gonzalez-Ballester et al., 2010, Miller et al., 2010, Msanne et al., 2012, Nguyen et al., 2011a, Siaut et al., 2011, Sugimoto et al., 2007, Sugimoto et al., 2008, Timmins et al., 2009) indicates that lipid accumulation is based on a complicated interplay of cellular pathways. To successfully adopt biotechnology to optimize the lipid production in microalgae like Chlamydomonas it seems necessary to obtain a more complete picture of the cellular networks regulating lipid production, storage and degradation in the future.
Acknowledgments We would like to acknowledge Neste Oil Oyj, the CLIB Graduate Cluster Industrial Biotechnology and EU-FP7 collaborative project SUNBIOPATH (03SF0361G) for funding and the Consortium Bioenergy OWL with the Stadtwerke Bielefeld and Biogas Nord GmbH for supporting our work.
Inhibition of diacylglycerol acyl transferase-1 (DGAT-1) is of increasing interest as a mechanism for therapeutic treatment of diabetes, obesity and other diseases which together constitute metabolic syndrome., , DGAT-1 deficient (Dgat) mice are viable, resistant to weight gain when fed a high-fat diet and show increased insulin and leptin sensitivity. Ourselves, and others, , , , , , , , have recently reported small molecule inhibitors of DGAT-1 from different chemical series and the progress of some compounds into clinical development supports the potential for therapeutic use., , In this Letter we report our discovery and optimisation of oxadiazole amide inhibitors, leading to the clinical candidate .