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Revision as of 13:31, 22 July 2011
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Project Abstract: Y’s multi-guard
This year our project is aimed at increasing the tolerance of Saccharomyces cerevisiae to inhibiting compounds in lignocellulosic hydrolysates, such as weak acids, furans and phenolic compounds formed or released during pretreatment and hydrolysis.
The core of our project is directional modification of TOR (The target of rapamycin), a highly conserved Ser/Thr protein kinase which is the central component of a major regulatory signalling network that controls cell growth in diverse eukaryotic organisms, ranging from yeast to man. Former research shows that amino acid starvation could strongly inhibit the phosphorylation of TOR, which would induce the entry of some transcription factors to the nucleus and cause dramatic phenotypic changes such as some cell cycle arrest and entry into G0, general downregulation of protein synthesis, accumulation of the reserve carbohydrate glycogen, upregulation of stress response genes, autophagy and alterations in nitrogen and carbon metabolism. While the inhibitors in lignocellulosic hydrolysates, especially acetate, would also cause similar phenotypic changes. Based on the similarity between the cellular responses caused by nutrient starvation and inhibitors, we are seeking for ways to mutate the TOR to make it ignorant of the inhibiting signal keep on normal physiological activity.
Noticeable work has been done on mTORC1 (mammalian TOR complex 1) that modulation in certain amino acid sites could promote the activity in response to adverse nutrient status and inhibitors, while same work has not been done on Saccharomyces cerevisiae. However, the high homogeneity between TOR and mTOR makes it possible to transplant the modulation into Saccharomyces cerevisiae. We seperated the TOR into three parts and picked up 6 amino acid sites. Gibson Assembly and overlap PCR have been used for the introduction of site-directed mutagenesis.
Besides the mutation of TOR, a complete genetic circuit is needed to achieve the function. We tested different transcriptional factors and promoters which used to be regulated by the unmutated TOR and found out the optimum combination.
Finally, all the modules will be integrated into a YAC (Yeast Artificial Chromosome). We hope to make it become the 17th chromosome of the yeast (normally 16). With this additional chromosome, the yeast will gain stable growth, high yield and productivity of ethanol and an extension in life span regardless of the existence of inhibitors in lignocellulosic hydrolysates. Furthermore, we also want to apply this integrated YAC with different functions and into others kinds of organism. That is to say, with this universal and standard module, we can put it into any organism needs to be remodeled, just like an USB.
