Engenharia genética de Saccharomyces cerevisiae para cofermentação de xilose e glicose e produção de etanol de segunda geração

Author(s)

João Gabriel Ribeiro Bueno

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-02-17

Examining board members

Leandro Vieira dos Santos; Elizabeth Bilsland; Jeferson Gross

• Advisor: Leandro Vieira dos Santos

Abstract

The global energy matrix is based on fossil fuels, such as oil, gas and coal. In recent decades, the world community has expressed an intense concern related to the consequences caused by the use of these compounds, such as the increase in greenhouse gas emissions. Second-generation (2G) ethanol, produced from lignocellulosic biomass, emerges as promise sustainable advanced biofuel. However, several biotechnological challenges need to be overcome in order to produce economically viable 2G ethanol. The development of engineered Saccharomyces cerevisiae yeast strains, capable of converting xylose, the second most abundant sugar on lignocellulosic biomass, into ethanol, is an essential step to a feasible 2G process. A major bottleneck in the conversion of bioproducts from these biomass-derived sugars is the low xylose uptake by S. cerevisiae membrane transporters and catabolic repression, leading to glucose inhibition (C6). Membrane proteins capable to transport xylose are strongly inhibited on high concentrations of this sugar, in addition to presenting low affinity to xylose compared to glucose, leading to competition between substrates and extending the fermentation time. Xylose is only assimilated by S. cerevisiae cells when glucose concentrations are low in the extracellular environment. Therefore, this project aimed to identify novel and efficient heterologous xylose transporters and to develop a strain capable of co-fermenting xylose and glucose. In this work, we identified and characterized four new xylose transporters Cs186, Cs2608, Cs3894 and Cs4130, evaluated in EBY.VW4000 strain modified with the xylose consumption pathway integrated into the genome. Curiously, the Cs4130 transporter demonstrated a superior capacity of xylose assimilation at high xylose concentrations (50 g/L) compared to the GXF1 control, considered the best heterologous transporter previously described in the literature. In the condition described, GXF1 was severely inhibited, demonstrating an opposite behavior compared to that observed by Cs4130. The structural model of Cs4130 compared to the high affinity prokaryotic transporter XylE and GXF1 points out residues in its molecular architecture that may explain the differences in behavior observed between the transporters. The novel transporter Cs4130 is presented as an excellent candidate for genetic engineering of S. cerevisiae strains for 2G biocompounds production. However, the xylose transporters Cs4130 and GXF1 show strong glucose inhibition on mixtures of both sugars. Therefore, the second part of the project aims to engineering these membrane proteins and to develop a process to increase the affinity of xylose compared to glucose. We have developed two new microbial platforms for adaptive evolution, EBY_Xyl1_hxk0, derived from EBY_Xyl1 strain, and the JBY_hxk0 constructed from an industrial high-yield pentose fermenting strain. These new platforms are unable to metabolize glucose molecules due to the disruption of glycolysis pathway by knockout of hexokinase genes, HXK1, HXK2 and GLK1. The S. cerevisiae modified strains recognized glucose molecules only as an inhibitor to xylose metabolism. Therefore, new genetic mutations present in these evolved cells, capable to co-consume xylose and glucose, will provide new data to understand the genetic basis of glucose repression. The information produced in this project will help the development of S. cerevisiae strains suitable of simultaneously fermenting xylose and glucose, reducing the total fermentation time in the 2G industry. (AU)

• FAPESP's process: 18/00888-5 - Genetic engineering of Saccharomyces cerevisiae for xylose and glucose co-fermentation and second-generation ethanol production
• Grantee: João Gabriel Ribeiro Bueno
• Support Opportunities: Scholarships in Brazil - Master