Team:Groningen/Demonstrate
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.
In conclusion, we have demonstrated that we can grow our S. cerevisiae strains on cellulose, and that they are able to produce styrene from glucose. Moreover, we demonstrate styrene production using the shortest cellulose strand: cellobiose.
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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. Different galactose induction times were tested. The cellulosome is able to degrade cellobiose as growth is observed.
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. The cellulosome is able to degrade phosphorylated cellulose as growth is observed.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 Figure 1 and 2 show CbhI and EgII can hydrolyze phosphorylated cellulose and ball-milled ReCell. Growth on cellobiose demonstrates BglI activity, as it degrades cellobiose. Together these results demonstrate the functionality of all 3 cellulases in our cellulosome.
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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 retention time. A peak is present at 17,5 min, corresponding to styrene. No peak is present for the control. Styrene production in yeast using glucose is achieved. -
Consolidated bioprocessing
As final proof of concept both the PAL2 gene and the cellulosome are expressed in a S. cerevisiae strain. This strain is then cultured using cellobiose.
Figure 5. Trans-cinnamate production from cellobiose in yeast. HPLC intensity at 254 nm is plotted against the retention time. Peak at 10.2 min retention time corresponds to trans-cinnamate. Peak corresponding to growth on cellobiose is higher than the control peak. This showcases that trans-cinnamate production from cellobiose is achieved. 
Figure 6. Styrene production from cellobiose in yeast. HPLC intensity at 254 nm is plotted against the retention time. Peak at 17.2 min retention time corresponds to styrene. Peak corresponding to growth on cellobiose is higher than control peak. Hence, styrene production from cellobiose is achieved. 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.