Engineering Saccharomyces cerevisiae strains to improve acid- and lignocellulosic inhibitor-tolerance

Abstract

Plant biomass degradation has been studied for years due to the incredible amount of byproducts generated during this process - ranging from biofuels to other noteworthy chemicals. The candidate current PhD's project is dedicated on developing and applying new tools for improving functional metagenomic screenings, more specifically for mining cellulases. We've generated metagenomic libraries in a broad-host range vector in order to expand the enzyme recovery rate in metagenomic screenings by using alternative hosts. Thus, we found and characterized a new ²-glucosidase which, among other interesting features, displayed tolerance to high amounts of ethanol and 5-hydroxymethyl furfural (5-HMF), one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, this enzyme showed synergistic effect on Bacillus subtilis GH5-CBM3 endoglucanase activity, improving the amount of glucose released when using CMC as substrate. While in this continuous pursuit of better yields for biomass degradation, the idea of using a consortium of engineered microorganisms emerged. For this, we've came up with the idea of engineering S. cerevisae, that is already a good fermenter organism, to produce this ethanol- and 5-HMF- tolerant ²-glucosidase in combination with the Bacillus subtilis GH5-CBM3 endoglucanase (which we demonstrated acts in synergy) together with some genes that convey stress tolerance (for instance, tolerance to 5-HMF or to acidic conditions). In this sense, in our group we've worked with some genes (also recovered from metagenomics screenings) that confer acidic resistance in E. coli and other gram-positive and gram-negative bacteria (clpX, hu and rbp genes, which display putative functions of protease, histone-like protein and RNA-binding protein, respectively). Moreover, these genes were successfully transferred to Arabidopsis thaliana and allowed it to grow at pH of 3.5. Mastering the manipulation of yeast genomes, we can create a community of several different strains of the same organism, each producing different genes, such as the enzymes of interest and/or resistance genes. Therefore, the main goals of the present project are: (i) to apply the previous data obtained in the PhD's project (metagenomic genes and enzymes of interest) to engineer S. cerevisiae; (ii) to learn how to use genetic tools to manipulate yeast genomes; (iii) to use these tools to create engineered acid- and lignocellulosic inhibitor-resistant yeast strains producing enzymes of interest; (iv) to learn about biomass hydrolysis and fermentation processes and apply this knowledge to evaluate the efficiency of the engineered strains generated. Our ultimate goal is obtaining the mutant strains and establishing the methodologies of yeast engineering and fermentation process, thus, we can bring this know-how to apply in our laboratory at FFCLRP-USP. Additionally, all the technology and expertise regarding yeast manipulation will generate significant advances in metabolic modeling and in our country, as first steps in the aspiration of, one day, creating a community of yeast strains that can resist to harsh environmental conditions and produce high yields of lignocellulose-degrading enzymes.