Template:Groningen/Demonstrate

Still under construction, used to demonstrate ideas -JM

The goal of our project is threefold. First, we want to degrade cellulose to glucose using Saccharomyces cerevisiae expressing an artificial cellulosome. Secondly, we want to produce styrene from glucose using S. cerevisiae. The final goal is to produce styrene directly from cellulose in a consolidated bioprocess.
We have designed and constructed S. cerevisiae strains that meet these goals. A number of experiments were performed to prove the functionality of these strains.

• Cellulose degradation

  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.

  

  Figure 1. Row A to D contain pure ReCell, ball-milled ReCell, pure cellulose and phosphorylated cellulose, respectively. Lanes 1 to 6 contain BglI, EgA, CbhI, a mix of 1-3, the positive control and negative control, respectively. Intensity of pink colour corresponds to the degree degradation. Ball milling of ReCell and phosphorylation of cellulose increase degradability of cellulose.

  

  Figure 2. Row A to D contain pure ReCell, ball-milled ReCell, pure cellulose and phosphorylated cellulose, respectively. Lanes 1 to 6 contain EgA, EgII, EgII 1:1000, EgII 1:10.000, positive control and negative control, respectively.Intensity of pink colour corresponds to the degree degradation. EgII is able to degrade ball milled ReCell, pure cellulose and phosphorylated cellulose; therefore, ball milling and phosphorylation are effective pretreatments.

  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 3. Growth curves of BJ1991 strains containing the artificial cellulosome, with start OD600 of 0.1, with different galactose induction durations.

  	OD600 at T=0h	OD600 at T=12h	D600 at T=36h
  YPH499++	0.51	0.55	1.36
  negative control	0.1	0.06	0.1

  Table 1. Growth on phosphorylated cellulose using YPH499 containing PAL2 and the cellulosome (YPH499++). OD600 was measured at t=0, t=12h and t=36h. Initial OD600 value of YPH499++ is high due to absorbance from the phosphorylated cellulose.

  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.

• Styrene production

  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.

  

  Figure 4: HPLC 254 nm intensity plotted against time. Peaks at 17,5 min retention time can be seen, corresponding to styrene.

  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.

• Consolidated bioprocessing

  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.

  

  Figure 5. Trans-cinnamate production from cellobiose in yeast. HPLC intensity at 254 nm is plotted against the retention time.

  

  Figure 6. Styrene production from cellobiose in yeast. HPLC intensity at 254 nm is plotted against the retention time.

  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.

In conclusion, we have demonstrated that we can grow our S. cerevisiae strains on cellulose, and that it can produce styrene from glucose and cellobiose. This leads to the reasonable assumption that this strain is able to break down and grow on cellulose and meanwhile produce styrene.