Characterization of mutagenized xylose transporters for the engeneering of "Saccharomyces cerevisiae" strains

Orientador: Juliana Velasco de Castro OliveiraPrograma de Pós-Graduação em Genética e Biologia MolecularResumo: O cenário atual de constante crescimento da demanda por combustíveis traz a necessidade de se buscar por novas fontes alternativas de energia e ambientalmente mais corretas. O etanol de segunda geração, proveniente da fermentação de açúcares da biomassa vegetal, como o bagaço da cana-de-açúcar, aparece como uma alternativa sustentável para substituir, em parte, os combustíveis fósseis, mas depende da resolução de alguns entraves biotecnológicos, como o transporte e metabolização de xilose pela Saccharomyces cerevisiae. Frente a isso, este projeto teve por objetivo melhorar a eficiência de dois transportadores, o HxtB de Aspergillus niger e Gxf1 da Candida intermedia através da introdução de mutações aleatórias via PCR error prone (técnica de PCR com mutações induzidas). Como resultado, foram gerados 119 clones para o transportador Gxf1 mutado e 87 para o transportador HxtB, na cepa S. cerevisiae EBY VW.4000 que possui todos seus transportadores deletados, exceto os para maltose. Estes clones foram avaliados quanto à eficiência do transporte de xilose em diferentes concentrações deste açúcar por ensaios de drop out e, os que apresentaram melhores resultados de crescimento em relação aos controles (cepas contendo os transportadores sem mutação), foram posteriormente avaliados pela construção de curvas de crescimento em meio líquido. Para os clones com fenótipos melhorados para o transporte de xilose, observou-se que a maioria das mutações inseridas estão em domínios relacionados ao transporte de açúcar, entretanto, estudos estruturais mais aprofundados devem ser realizados para se elucidar a real influência de cada mutação. Além disso, este projeto objetivou a melhoria do consumo de xilose em cepas de S. cerevisiae PE-2 JAY291 haploides contendo os transportadores HxtB, Gxf1 e o plasmídeo pRH274 (para metabolização da xilose) por meio da mutagenização com etilmetanosulfonato e evolução dirigida. A evolução foi feita através de repiques sucessivos em meios de cultura contendo xilose em semi-anaerobiose e anaerobiose, com avaliação do crescimento de 23 clones mutados. Nesta etapa, quatro clones se destacaram no experimento, cada qual em uma condição de evolução específica, mostrando que as mutações inseridas no genoma dos mesmos conferiu características distintas pra cada um. Embora vários grupos de pesquisa no Brasil, e principalmente no exterior, vêm trabalhando nesta temática, ainda não foi obtido um microrganismo totalmente adaptado às condições brasileiras para produção de etanol 2G a partir da fermentação da xilose de forma economicamente viável. Estes transportadores, bem como a posterior identificação e validação das modificações genéticas das cepas evoluídas, contribuirão para o futuro engenheiramento genético racional de linhagens de leveduras industriaisAbstract: The current scenario of the constant growth of the demand for fuels brings with it the need to search for more sustainable and environmentally friendly energy sources. In this scenario, second generation bioethanol from the fermentation of sugar from biomass, such as sugarcane bagasse, appears as a sustainable alternative to replace fossil fuels in part, but depends on the resolution of some biotechnology barriers, such as the transport and metabolization of pentoses, mainly xylose, by the yeast Saccharomyces cerevisiae. Consequently, this project aimed to improve the efficiency of two transporters, the HxtB of Aspergillus niger and Gxf1 of Candida intermedia through the introduction of random mutations via error prone PCR. With this technique, 119 clones of Gxf1 and 87 clones of HxtB were generated in the S. cerevisiae EBY VW.4000 strain which has all of its transporters deleted except those for maltose. These clones were evaluated for the transport of xylose and other sugars at different concentrations by drop out assays, and those, which showed the best growth results in comparison with the controls (strains with the wild type transporters), was evaluated by growth curves in liquid medium. It has been observed that for clones with improved phenotypes for xylose transport, the inserted mutations are located in sugar transport domains. However, more in-depth structural studies must be performed to elucidate the real influence of each mutation. In addition, this project aimed at improving xylose consumption in haploid S. cerevisiae PE-2 JAY291 strains containing the HxtB and Gxf1 transporters as well as the plasmid pRH274 for xylose metabolism through mutagenization with ethylmethanesulfonate (EMS) and direct evolution. The evolution was made through successive culture of the mutants in culture media containing xylose in semi-anaerobiosis and anaerobiosis, with evaluation of the growth of 23 clones. In this step, four clones were distinguished in the experiment, each one in a specific evolution condition, showing that the mutations inserted in the genome of each one resulted in different growth characteristics and consequently different xylose consumption. Although several research groups in Brazil, and mainly abroad, have been working on this subject, a microorganism fully adapted to the Brazilian conditions for the production of 2G ethanol from the xylose fermentation in an economically viable manner has not been obtained yet. These transporters, as well as the subsequent identification and validation of the genetic modifications of the evolved strains, will certainly contribute to the future rational genetic engineering of industrial yeast strainsMestradoGenetica de MicroorganismosMestre em Genética e Biologia Molecular131755/2015-3CNP

Comparative transcriptome analysis reveals different strategies for degradation of steam-exploded sugarcane bagasse by Aspergillus niger and Trichoderma reesei

Abstract Background Second generation (2G) ethanol is produced by breaking down lignocellulosic biomass into fermentable sugars. In Brazil, sugarcane bagasse has been proposed as the lignocellulosic residue for this biofuel production. The enzymatic cocktails for the degradation of biomass-derived polysaccharides are mostly produced by fungi, such as Aspergillus niger and Trichoderma reesei. However, it is not yet fully understood how these microorganisms degrade plant biomass. In order to identify transcriptomic changes during steam-exploded bagasse (SEB) breakdown, we conducted a RNA-seq comparative transcriptome profiling of both fungi growing on SEB as carbon source. Results Particular attention was focused on CAZymes, sugar transporters, transcription factors (TFs) and other proteins related to lignocellulose degradation. Although genes coding for the main enzymes involved in biomass deconstruction were expressed by both fungal strains since the beginning of the growth in SEB, significant differences were found in their expression profiles. The expression of these enzymes is mainly regulated at the transcription level, and A. niger and T. reesei also showed differences in TFs content and in their expression. Several sugar transporters that were induced in both fungal strains could be new players on biomass degradation besides their role in sugar uptake. Interestingly, our findings revealed that in both strains several genes that code for proteins of unknown function and pro-oxidant, antioxidant, and detoxification enzymes were induced during growth in SEB as carbon source, but their specific roles on lignocellulose degradation remain to be elucidated. Conclusions This is the first report of a time-course experiment monitoring the degradation of pretreated bagasse by two important fungi using the RNA-seq technology. It was possible to identify a set of genes that might be applied in several biotechnology fields. The data suggest that these two microorganisms employ different strategies for biomass breakdown. This knowledge can be exploited for the rational design of enzymatic cocktails and 2G ethanol production improvement