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Team:Tianjin/Project
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Project Abstract: Y’s multi-guard
Decades of years have passed since the concept of "Synthetic Biology" being introduced, it is now essential to view cells as true ‘programmable’ entities, and develop effective strategies for assembling devices and modules into intricate, customizable larger scale systems rather than creation and perfection of genetic devices and small modules based on them. Meanwhile, regulations on gene expression have evolved from transcription, to translation, and finally signal transduction level.
This year we are aimed at increasing the tolerance of Saccharomyces cerevisiae to inhibitors in lignocellulosic hydrolysates, such as weak acids, furans and phenolic compounds formed 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 signaling transduction network that controls cell growth in diverse eukaryotic organisms, ranging from yeast to man. Former research such as transcriptomics, proteomic and metabonomics results show that inhibitors, especially acetate, would a series of phenotypic and intracellular changes induced by TOR complex, including the entry of some transcription factors to the nucleus, 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. Thus we are seeking for ways to mutate the TOR to make it ignorant of the inhibiting signal to maintain normal physiological activity and keep the overall signaling pathway on the right direction.
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 mammalian TOR makes it possible to transplant the modulation into Saccharomyces cerevisiae. We separated TOR1, the key part of TORC1, into three parts and picked up 4 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.
