Use of CRISPR / dCas9 and microfluidics to uncover gene interactions related to the conversion of xylose into ethanol by S. cerevisiae industrial strains

Abstract

Yeasts adapted to industrial conditions - industrial strains - are considered appropriate platforms for the development of commercial strains applied to the production of chemicals derived from renewable raw materials, in particular biofuels, such as the second generation ethanol (2G ethanol). The metabolic engineering of these yeast strains stands out as one of the most straightforward and time-saving approaches to obtain robust strains able to efficiently convert xylose into ethanol. In this context, the implementation of CRISPR/dCas9 multiplexed systems for the simultaneous overexpression and silencing of different genes allows the exploration of genes function in different strains as well as the uncover of possible intergenic relations, that might be decisive for the discovery of genetic targets and the design of new strains of interest. For this reason, this project proposes the implementation of a dCas9 genetic editing approach, that allows the uncover of possible gene interactions related to the xylose usage and ethanol production in the working strain. The gene targeted regulation should be followed by a droplet microfluidic screening method, that permits the selection of high xylose consuming and ethanol producing strains, implying the detection of the most suitable industrial S. cerevisiae strains for the 2G ethanol production.