Difference between revisions of "Template:Groningen/Demonstrate"

As cellulose is difficult to degrade, we first investigated different cellulose pretreatment methods. We used commercially available cellulases BglI, CbhI, and EgII (EgA) and the EgII we express in yeast. Using these enzymes we investigated ball milling of ReCell (toilet paper purified from waste water obtained from a sponsor) and phosphorylation of pure cellulose.

Subsequently, we transformed S. cerevisiae with an artificial cellulosome containing 3 different cellulases and a scaffold. The cellulosome activity was tested by growing the strains on cellobiose (𝛃-1,4 linked dimer of glucose) and cellulose. Sufficient cellulosome activity should enable the yeast to grow on cellulose or cellobiose.

Figure 1 shows that CbhI can hydrolyze phosphorylated cellulose and (ball-milled) ReCell, and figure 2 shows the same for EgII. The growth on cellobiose as shown in figure 3 demonstrates that BglI is active, as this is the only cellulase present that can hydrolyze cellobiose into glucose required for growth. These results demonstrate that the 3 cellulases we plan on using each function as expected with our cellulose sources. Table 1 shows that the S. cerevisiae strain containing the cellulosome can grow on phosphorylated cellulose. This confirms that we made the goal of constructing a strain that can degrade cellulose and grow on the created glucose.

To produce styrene in S. cerevisiae, the PAL2 gene from Arabidopsis thaliana is expressed. The Pal2 enzyme catalyzes the reaction of phenylalanine to trans-cinnamate. Natively present Fdc1 then converts trans-cinnamate into styrene. PAL2-containing strains are cultured on glucose medium, and HPLC is performed.

As shown in figure 4, styrene is present in the PAL2-containing strain, but not in the control strain. This suggests that Pal2 indeed converts phenylalanine to trans-cinnamate, which can be natively converted to styrene. It thus demonstrates that we meet our second goal, of creating styrene using S. cerevisiae.

After demonstrating that both original goals of the project have been met, we decided to take it further. The PAL2 gene is expressed in a S. cerevisiae strain that is also expressing the artificial cellulosome. This strain is then cultured with cellobiose.

As shown in figure 5, the trans-cinnamate levels are higher in the strain grown on cellobiose compared to the strain grown without cellobiose. Figure 6 shows styrene production in the strain grown on cellobiose, whereas nothing is visible in the control. This demonstrates that the consolidated strain is able to convert cellobiose into trans-cinnamate and styrene.