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The use of fossil fuels to obtain energy is responsible for a large part of greenhouse gas emissions into the atmosphere. Second generation bioethanol (2G ethanol) appears as a great option because it is a cleaner and renewable source and does not compromise food security. However, there are still several bottlenecks that limit the efficiency of 2G ethanol production. Through the use of bioinformatics tools, such as differential expression analysis, co-expression and gene interaction networks of previously generated RNA-Seq and microarray transcriptomic data, we compared the metabolism of two xylose fermenting yeasts with the insertion of the XR/XDH and XI pathways under contrasting conditions of 2G ethanol fermentation (glucose vs xylose uptake). The coexpression analysis for the XR/XDH pathway was performed using three fermentation conditions: (i) only glucose medium, (ii) mixed sugar medium (including the glucose and the xylose fermentation phase), and (iii) only xylose medium. However, the coexpression analysis of the XI pathway has four collection points extracted throughout the fermentation in order to encompass all phases: (1) Predominant consumption of glucose, (2) transition between consumption of glucose and xylose, (3) predominant consumption of xylose and (4) end of fermentation. Beyond the interactomes, we found the hub genes from network topology metrics (degree, betweenness and closeness centrality) performing the union of the pairwise intersection of top 20 genes for each metric. The hub genes found in the interactome metrics analysis were then validated in the co-expression results, and it was considered a key genes linked to 2G fermentation if they had |GS|>0.2, |MM|>0.5, and were present in a module with module-sample relationship R>0.8 and p-value<=0.05. As a result, from the two possible pathways to consume xylose (XR/XDH and XI pathways), we generated both interactomes, which has a total of 479 and 571 nodes, respectively. Moreover, co-expression analysis provided us key modules related to each 2G ethanol fermentation phase of these two metabolic pathways of study (XR/XDH and XI). The enrichment of the key modules found showed processes related to response to stress, response to stimulus and nitrogen compound biosynthetic process for the XI pathway, and generation of precursor metabolites and energy, energy derivation by oxidation of organic compounds, aerobic respiration, ATP biosynthetic process, fungal-type cell wall organization, proteolysis, ribosome biogenesis, and gene expression for the XR/XDH pathway. In our analysis, for XR/XDH pathway, we found 8 key genes related to gluconeogenesis, reactive oxygen species metabolic process, protein polyubiquitination, DNA replication, recombinational repair, beta-alanine biosynthetic process, fatty acid beta-oxidation and endoplasmic reticulum to Golgi vesicle-mediated transport. Moreover, for the XI pathway, we found 10 key genes related to cellular response to oxidative stress, glutamate catabolic process, protein ubiquitination, lactate metabolic process, glucose 6-phosphate metabolic process, glycogen biosynthetic process, mitochondrial electron transport, ATP synthesis, glycolytic process, pyruvate metabolic process and proteolysis. These results can be used to understand the role of each group of genes in each 2G ethanol fermentation step through XI and XR/XDH pathways.

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1	Alexandra Cardelli(a230577@dac.unicamp.br)
2	Gonçalo Pereira(goncalo@unicamp.br)
3	Lucas Carvalho(lucasmigueel@gmail.com)

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