A transporter engineering system approach for expanding the sugar uptake repertoire of a Saccharomyces cerevisiae Industrial Strain employing CRISPR/Cas and direct evolution technologies.

Abstract

In the second-generation (2G) ethanol production process, the enzymatic hydrolysis of lignocellulosic biomass releases a mixture of sugars, including hexoses (e.g., glucose), pentoses (e.g., xylose), and cellodextrins (e.g., cellobiose). Over the fermentation stage, the transport of these sugars into the yeast cell is the first and critical step to enable the microbial production of ethanol. One of the main limitations of this process is that Saccharomyces cerevisiae lacks an efficient sugar transporter system (STS) able to internalize other sugars cited above than glucose, also cannot metabolize them. To overcome these drawbacks, this proposal aims to develop yeasts capable of efficiently internalizing and co-fermenting xylose and cellobiose. As most native sugar transporters derived from different microorganisms show a wide-ranging of substrates and-or low overall activity, will be employed a direct evolution approach to obtain xylose transporters (GXF1 from Candida intermedia and Cs4130 from Candida sojae) and cellobiose transporters (Tr69957 from Trichoderma reesei and CDT-2 from Neurospora crassa), with improved specificity and activity. Then, CRISPR/cas9 system will be employed to perform genetic modifications into S. cerevisiae Pedra-2 (PE-2) industrial strain, including xylose and cellobiose metabolic pathways along with mutated sugar transporters. Posteriorly, a plasmid-based library coupled with a CRISPR-dCas9-based modulation system will be applied to screen target genes to improve cellobiose/xylose co-fermentation by S. cerevisiae. Finally, will be analyzed the fermentation performance of the resulting strain. In the end, this work will arise a modified PE-2 strain capable of efficiently co-ferment cellobiose and xylose, evidencing the STS engineering as a crucial strategy for industrial strains improvement purposes. (AU)