| Congresso Brasileiro de Microbiologia 2023 | Resumo: 362-1 | ||||
Resumo:Second-generation ethanol is one of the most promising biofuels to replace fossil fuels. Therefore, optimizing its production is the main target of study in the area. The fermentation step is still a limiting point for the profitable production of 2G ethanol, and the co-fermentation of glucose and xylose is the main objective. The yeast used in this process (Saccharomyces cerevisiae) does not ferment pentoses, such as xylose, nor does it have an efficient transport system for this type of sugar. Thus, studies with transporters have been carried out to increase the efficiency of the bioconversion of lignocellulosic biomass to ethanol, by improving the transport of xylose expressing sugars transporters from organisms capable of naturally fermenting pentoses. Therefore, the objective of this work was to optimize the consumption of xylose, through the construction of a genetically improved industrial strain of S. cerevisiae. For this purpose, they were initially analyzed using RT-qPCR which MFS (Major Facilitator Superfamily) transporter genes were differentially expressed in a Trichoderma reesei culture in Mendels-Andreotti medium with 2% xylose. Three genes were selected to continue the research. Based on the gene expression, a phylogenetic analysis was performed to enable the comparison of selected transporters (MFS1, MFS2, and MFS3) with other xylose transporters already characterized. Molecular docking analysis was also performed to investigate the affinity of these transporters for xylose and to select the best genes for heterologous expression. MFS1 and MFS2 were chosen for heterologous expression. Then, the redox pathway of xylose metabolism was inserted into a strain of S. cerevisiae (URAΔ352, TRP1Δ63). After confirming the construction of S. cerevisiae able of metabolizing xylose, MFS1, and MFS2 were inserted into the yeast. Yeasts transformed with MFS1 and MFS2 were inoculated under three conditions: a) glucose; b) xylose; c) glucose + xylose. It was observed that expression of MFS1 significantly improved xylose transport when compared to yeast without heterologous transporters. The expression of MFS3 in this system still requires further analysis to conclude the ability of this protein to transport xylose. Despite this improvement in xylose consumption, due to MFS1 expression, xylose transporters naturally have a higher affinity for glucose than for xylose. And, as observed in our trial of sugar consumption and ethanol production, glucose is always consumed first in mixtures of glucose and xylose, causing a diauxic growth of the strains and reducing the efficiency of the process. Therefore, we also made the in silico construction of MFS1 with mutations that mimetizing phosphorylations and mutations for insertion of motifs involved in the recognition of sugars, inserting amino acids that would guarantee selectivity to xylose. Next, molecular docking was performed between the mutants of MFS1 and xylose and glucose. Mutations that increased affinity for xylose while decreasing affinity for glucose were selected for heterologous expression in S. cerevisiae. With this, we hope to develop even more efficient yeasts for the transport of xylose with the engineering of MFS1. Palavras-chave: Trichoderma reesei, MFS, Xylose, Fermentation, Saccharomyces cerevisiae Agência de fomento:CAPES, CNPq, FAPESP | |||||