Metabolic engineering and evolution of Saccharomyces cerevisiae for arabinose and xylose fermentation

Author(s)

Paulo Emílio dos Santos Costa

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2022-04-20

Examining board members

Leandro Vieira dos Santos; Thamy Lívia Ribeiro Corrêa; Ana Paula Jacobus

• Advisor: Leandro Vieira dos Santos

Abstract

The use of plant biomass for the sustainable production of bioproducts is a promising alternative to reduce the use of fossil sources and mitigate the environmental impacts resulting from their consumption. However, the metabolic limitation of the yeast Saccharomyces cerevisiae in consuming the five-carbon (C5) sugars derived from plant biomass, such as the pentoses xylose and arabinose, is one of the main bottlenecks for an economically feasible production of lignocellulose-based products. The rational microbial design associated with adaptive evolution strategies allowed the identification of novel metabolisms and genetic targets that boost the consumption of these pentoses. In this context, the goal of the present work is the construction of an industrial yeast arabinose and xylose-fermenting strain, as well as the identification of genetic bases fixed during adaptive evolution strategies and responsible for optimizing the conversion of C5. Initially, gene expression cassettes containing the arabinose metabolic pathway were designed and constructed using synthetic biology approaches, and stably integrated into the genome of an industrial xylose-fermenting strain, resulting in the yeast cell PEY-AX5. The initial strain was not able to grow on arabinose as the sole source of carbon. Adaptive evolution strategies were applied and after 65 and 190 generations, rapid growth was observed in medium containing arabinose. The population was able to completely ferment the arabinose, reaching a conversion yield of 0.42 g ethanol/g arabinose. Co-consumption of xylose and arabinose was also observed in fermentations containing the mixture of sugars. Genomic sequencing will be performed on evolved populations to identify SNPs and CNVs fixed in populations during evolution and associated with the increased arabinose fermentative capacity. In a parallel project, the effect of two beneficial mutations on xylose conversion was evaluated. The genes evaluated were ZWF1, involved in the oxidative phase of the pentose phosphate pathway, and the CLN3 gene, an important cyclin in the cell cycle, in addition to epistatic interactions with the ISU1 gene, involved in the assembling of Fe-S clusters. Gene deletions and SNPs were introduced into a xylose-fermenting strain using the CRISPR-Cas9 system. Fermentation assays showed positive effects on gene deletion, in addition to a positive interaction between the ZWF1 and ISU1 genes, with the double mutant consuming 50 g/L of xylose in less than 32 hours and yielding 0.47 g of ethanol/g of xylose. With the results produced in the present work, we were able to develop yeasts capable of efficiently consuming L-arabinose, in addition to promoting the co-consumption of C5 sugars. After genomic sequencing, we will be able to identify novel targets and metabolisms responsible for the efficient conversion of pentoses. Finally, we were able to validate the ZWF1 and CLN3 genes as new targets with biotechnological potential for xylose conversion. The set of mutations identified can be used in rational metabolic engineering approaches, designing efficient microbial platforms to convert agro-industrial residues into renewable bioproducts (AU)

• FAPESP's process: 19/06942-4 - Elucidation of the molecular bases of arabinose and xylose fermentation in Saccharomyces cerevisiae strains
• Grantee: Paulo Emílio dos Santos Costa
• Support Opportunities: Scholarships in Brazil - Master