An integrated system approach to engineer yeast as a cell factory for lignocellulose-based biorefineries

Abstract

The global energy matrix continues to rely heavily on fossil fuels-based energy sources whose combustion played a strongly negative impact in GHG emission levels. The increasing climate concerns guided the development of environmentally friendly technologies towards a more sustainable development in the world. In this context, microbial cellular metabolisms can be rewired, creating efficient platforms to produce biofuels and biochemicals from biomass in lignocellulosic biorefineries. In our former Fapesp grant, we applied systems metabolic engineering strategies to develop high-performance Saccharomyces cerevisiae strains and established a set of mutations to reshape cellular metabolism and improve fermentation fitness to produce 2G biofuels. However, several limitations still need to be addressed to produce a robust microbial platform for future biorefineries. An integrated system approach will be applied to: i. engineering membrane transporter proteins for improved xylose uptake and alleviated catabolic repression for C5/C6 co-fermentation (Module Yeast MCF.A). In this part, an automated HTS assembly of synthetic protein libraries for directed evolution of C5-transporters will be screened with genetically encoded xylose biosensors and cell sorting by flow cytometry; ii. Module MCF.B will investigate the signaling network that regulates the xylose assimilation and fermentation in S. cerevisiae. A panel of in vivo fluorescent biosensors for single-cell real-time monitoring of the three main sugar sensing pathways in yeast will be used to study the effect of a set of mutations identified in our former project that boosts xylose utilization. The data will be used to further engineering xylose sensing and signaling pathways towards faster xylose assimilation; iii. The last MCF.C module aims to integrate biophysical, biochemical and multi-omics data to explore cell wall integrity and stress-induced by 2G inhibitors in yeast, focusing on phenolic compounds. The project will have international collaborations, establishing a collaborative network and exchange of students. The combined approaches will solve key bottlenecks towards engineering an efficient and robust microbial cell factory to be used in the bio-based industry. (AU)