Difference between revisions of "Team:Calgary/Cellobiohydrolase"
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Revision as of 09:24, 27 October 2020
SYNOPSIS
For developing our modified CBH-1 we utilized a hybridized catalytic domain combining the sequence from P. funiculosum and T. reesei. These modifications allow the protein to operate at more moderate conditions than the wild type. The linker sequence and cellulose-binding module were then added from the P. funiculosum wild type. These modifications were shown to increase the productivity of the protein and widen the operating parameters for the protein.
Structural models were generated to get a starting point for the protein. The predicted structure was then protonated in silico at numerous pHs. After protonation, the structures were solvated in water and underwent molecular dynamic simulation. Metrics around the simulations were taken and modelled to show protein stability within the pH 3 to 7 range.
WHAT IS CELLOBIOHYDROLASE
What impact does it have in our project
Cellobiohydrolase(CBH) is the second cellulase in our cellulose degradation efforts. After the endoglucanase has finished with the cellulose, CBH cleaves units off of the end. This is accomplished by breaking the celluloses 1,4-beta-D-glycosidic bonds. This then leaves the substrate primed for beta-glucosidase.
CBHs are commonly comprised of three different components. The first is a catalytic domain responsible for the enzymatic activity of the cellulase. Next, there is the cellulose-binding module that anchors the cellulase to the substrate. The final component is a flexible linker region that connects the two. All three of these components contribute to the efficiency, and therefore the components and their interactions with each other will be modelled for better understanding.
PRIMARY STRUCTURE
Getting the Sequence Right
For our modified CBH we looked at organisms that would best be able to provide us with a blend of high efficiency and broad operating conditions. We found the CBHs of T. reesei and P. funiculosum particularly intriguing. After discovering 'Engineering enhanced cellobiohydrolase activity,' a paper blending the two's catalytic domains and comparing the other components of CBH. The resulting domain was proven to be more effective than CBH from T. reesei and more resilient than the CBH from P funiculosum. This hybridized catalytic domain has the following sequence.
QSAGTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCALDGAAYASTYGVTTSG
NSLSIGFVTQSNVGARLYLMASDTTYQEFTLLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLK
FINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNRYGGTCDPDGCDWNPYRLGN
TSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMV
LVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPSG
Next came the challenge of deciding on what linker and CBM to use. Luckily, this was also included in 'Engineering enhanced cellobiohydrolase activity'. We were able to see the proven improvements that accompanied these components from the P funiculosum wildtype.
STRUCTURAL PREDICTION AND INTEROGATION
What are the loops doing?
Using the Chimera and MODELLER software, we generated a starting structure file via homology modelling. This starting structure is shown below and was the cornerstone from which all of our models were built.
RESULTS
What we accomplished
In order to provide a sustainable, community-based solution, we plan to genetically modify Rhodosporidium toruloides, an oleaginous yeast that naturally produces beta-carotene and lipids, to be more robust and resource-efficient. By modifying the yeast to produce cellulase, it can then use common agricultural waste products as an energy source for synthesizing its oil. It can then be eaten as a vitamin A supplement. The yeast strain, while naturally safe and non-pathogenic, will also be genetically modified to include a kill switch for bio-containment, and optimized for oil production.
REFERENCES
2. Berendsen, et al. (1995) Comp. Phys. Comm. 91: 43-56.
