Engineering of Trichoderma reesei xylose transporters to improve the Saccharomyces cerevisiae sugar transport system

• Grant number: 22/01751-9
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): July 01, 2022
• Effective date (End): September 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Roberto do Nascimento Silva
• Grantee: Iasmin Cartaxo Taveira
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 19/11655-4 - Functional studies of gene regulatory networks in Trichoderma reesei during the cellulases formation, AP.TEM

Abstract

Whereas the production of second-generation ethanol (2G) has not yet been as consolidated as for 1G ethanol, there is much to be explored in order to optimize this process. Among the most studied possibilities, the improvement of the fermentation step may be highlighted. Glucose and xylose are the two most abundant sugars in the lignocellulosic biomass, so it is crucial for them to be efficiently metabolized. On the other hand, the most used yeast in bioprocesses is Saccharomyces cerevisiae, which is not able to ferment pentoses like xylose. Therefore, it is necessary to improve both the metabolism of S. cerevisiae pentoses and the transport of these sugars. In this sense, this project focuses on the matter of xylose transporters. Up to the present day, there have been many studies looking for specific xylose transporters, but most of them show more affinity for glucose than for xylose. An alternative in order to build yeasts that efficiently transport xylose involves the engineering of these transporters, aiming to increase their affinity for the pentose while reducing the affinity for glucose. Based on previous analyses from our research group, two sugar transporters (Tr62380 and Tr82309) from T. reesei were selected to solve this problem. From the analysis of the amino acid sequences of these transporters, motifs described in the literature as being important for xylose transportation as well as phosphorylation sites will be selected in order to make mutations in these regions of the referred proteins. Following this, in silico protein models will be built and a docking analysis will be performed to measure the potential affinity of the mutant protein for glucose and xylose, allowing the screening of mutations that may be more interesting. After the screening, expression cassettes will be constructed containing the mutant proteins to be transformed into a strain of S. cerevisiae without hexose transporters and capable of metabolizing xylose. The proteins will have their kinetic parameters measured and compared to the wild-type. At the end of this work, it is expected that these mutations will generate more efficient proteins for xylose transportation, with a lower affinity for glucose. (AU)