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.
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.
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.
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 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.
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.