Plant biomass is a promising renewable starting material for chemical and fuel derivations. Complete consumption of sugar components in the plant cell wall is essential for a cost effective process design. The yeast Saccharomyces cerevisiae can be engineered to co-consume cellobiose and xylose, a scheme that allows simultaneous consumption of sugar content from cellulose and hemicellulose polysaccharides, decreasing overall process time. As the reactions are expected to occur in anaerobic conditions given the large-scale nature of the process, cellobiose phosphorylase (CBP), an enzyme that catalyzes intracellular cellobiose breakdown in anaerobic bacteria was studied and showed energetic benefits in anaerobic conditions.
However, xylose was identified as a mixed-inhibitor and a substrate forming a byproduct of the CBP reaction. Protein engineering of CBP partially relieved the negative impact of xylose. A single distal mutation on CBP altered its susceptibility to xylose, potentially by modifying spatial conformation of CBP. In parallel, a novel xylose synthetic pathway involving xylulose-1-phosphate was tested, in an attempt to increase the xylose consumption rate, decrease cellular xylose and its negative impact on CBP. However, the existing xylose utilization pathway outperformed the synthetic pathway, likely because exogenous gene integration interfered with the existing regulatory network. Finally, cellobiose consumption was used as a minimal 2-gene synthetic biology pathway to explore cell physiology. This pathway induced drastic changes in cellular metabolites, allowing for the identification of novel regulation controlling ATP levels in cells. Uncoupling extracellular glucose sensors Snf3/Rgt2 from carbon utilization revealed a novel role of Snf3/Rgt2 in ATP regulation at the transcriptional level, potentially via the master regulator Gcn4. This regulation acted independently of proton pumping by Pma1, which is a known to be a major source of ATP consumption in yeast cells, but whose regulation remains to be determined. Together, these two pathways set an upper bound on ATP concentrations needed for optimal fermentation.