Characterising the genes involved in the triacylglyceride biosynthesis pathway of Chlamydomonas reinhardtii
Jit Ern Chen and Alison G. Smith
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
Plant-based biofuels have been suggested as one of the major solutions to our energy-environmental problems. Historically, considerable effort has gone into the study of lipid metabolism in higher plants to elucidate the identity of genes encoding components of lipid biosynthesis pathways, and the ways in which to modify these components to produce industrially useful lipids. Algal lipid metabolism on the other hand has been relatively ignored until very recently. In the green alga Chlamydomonas reinhardtii, I have identified candidate genes for the each of the steps in the two known triacylglyceride biosynthesis pathways. While in silico identification of genes is a first step, in order to begin intelligent metabolic engineering of the TAG synthesis pathway we will need to confirm the functionality of putative genes and to understand how these gene products interact in the pathway that we want to modify. I have now begun to clone out these genes and plan to use them to rescue yeast mutants which have deficiencies in their TAG synthesis pathways: this will verify their function inferred from bioinformatics. I will then determine the enzymatic characteristics of these gene products via liposome and radioactive substrate studies, with the intention of using the data to construct a simple mathematical model to predict the flux of TAG intermediates through the pathway. Gene silencing and constitutive expression of selected genes in Chlamydomonas will be carried out and the lipid profile of these transformed mutants will be analysed to identify changes in lipid composition, giving us a clearer picture about what the in vivo function of these genes are. These mathematical and lipid composition information will be used to identify points of interest in the pathway for directed metabolic engineering, which the aim of increasing the production of industrially valuable lipids.
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