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

Abstract

The reduction of world oil supply and continued demand for new energy sources, fuels derived from renewable raw materials were considered to be of great interest . Among them stands bioethanol, which comprises an important portion of the fuel market. However, the current production is insufficient to reach such demand. This situation drives the development of new technologies. One of the most promising alternatives is obtaining ethanol from lignocellulosic biomass. Yeasts used in this process comprise a significant portion of the viability of business models. A current problem is that the fermentation time may exceed 50 hours, showing the necessity of continuous development of new strains. Considering the hypothesis that optimization of metabolic flux decrease xylose fermentation time and can be obtained by regulating the expression of genes involved in metabolism, this project proposes the characterization of terminators and promoters of S. cerevisiae and use them to optimize xylose metabolic flux in the industrial strain PE-2. mRNAs' half-life assays and flow cytometry will be used to determine the capability of terminators to stabilize the yECitrine reporter gene's mRNA. Real-Time PCR and flow cytometry will characterize strong and constitutive promoters during the fermentation of lignocellulosic hydrolysates. From the best promoters, Error-Prone PCR will create new versions with different properties. Finally, random combinations of novel promoters and terminators fused with XI, XKS and TAL1 will be transfected into strain PE-2. Medium containing xylose as a sole carbon source will be used to screen the best strains. (AU)