Characterization of promoters and terminators to optimize the metabolic flux of xylose in Saccharomyces cerevisiae industrial strain PE-2

Biotechnological approaches have recently introduced changes in many industry sectors to promote the use of renewable resources to produce molecules of economical relevance such as ethanol. The use of plant biomass to produce renewable fuels has attracted much attention, but the viability of such process depends on the development of genetically modified organisms able to consume 5-carbon sugars rich in plant material and to tolerate environmental and process-related stresses. To reach these goals, the engineering of metabolic pathways and adaptation of modified strains to industrial conditions will require precise transcriptional regulation of genes of interest through the use of novel and efficient combinations of promoters and terminators. Our goals were to identify promoters suitable for high gene expression during fermentation of glucose and xylose and to characterize terminators for better translational efficiency on the Saccharomyces cerevisiae industrial strain PE-2. In the study of promoters, the genes exhibiting the top ten highest expression values and the lowest variation among experiments are related to glycolysis, stress tolerance and cell wall formation. Interestingly, the qPCR analysis results showed that the transcription levels in the majority of these selected genes were differentially regulated during fermentation of glucose and xylose, suggesting good candidates for exclusive gene expression in one single carbon source or in both (PGK1p, TEF1p, TDH1p, SCW4p e CDC19p), in addition to enabling better control of heterologous gene pathways. Results showed that three terminators have higher terminator activity than CYC1t (SPG5t, IDP1t e HIS5t) and suggest that terminator efficiency is strain-dependent (AU)