Project description:Xylose induced effects on metabolism and gene expression during anaerobic growth of an engineered Saccharomyces cerevisiae on mixed glucose-xylose medium were quantified. Gene expression of S. cerevisiae harbouring an XR-XDH pathway for xylose utilisation was analysed from early cultivation when mainly glucose was metabolised, to times when xylose was co-consumed in the presence of low glucose concentrations, and finally, to glucose depletion and solely xylose being consumed. Cultivations on glucose as a sole carbon source were used as a control. Genome-scale dynamic flux balance analysis models were developed and simulated to analyse the metabolic dynamics of S. cerevisiae in the cultivations. Model simulations quantitatively estimated xylose dependent dynamics of fluxes and challenges to the metabolic network utilisation. Increased relative xylose utilisation was predicted to induce two-directionality of glycolytic flux and a redox challenge already at low glucose concentrations. Xylose effects on gene expression were observed also when glucose was still abundant. Remarkably, xylose was observed to specifically delay the glucose-dependent repression of particular genes in mixed glucose-xylose cultures compared to glucose cultures. The delay occurred during similar metabolic flux activities in the both cultures. Xylose is abundantly present together with glucose in lignocellulosic streams that would be available for the valorisation to biochemicals or biofuels. Yeast S. cerevisiae has superior characteristics for a host of the bioconversion except that it strongly prefers glucose and the co-consumption of xylose is yet a challenge. Further, since xylose is not a natural substrate of S. cerevisiae, the regulatory response it induces in an engineered yeast strain cannot be expected to have evolved for its utilisation. Dynamic cultivation experiments on mixed glucose-xylose medium having glucose cultures as control integrated with mathematical modelling allowed to resolve specific effects of xylose on the gene expression and metabolism of engineered S. cerevisiae in the presence of varying amounts of glucose.

Project description:Resistance of Saccharomyces cerevisiae to high furfural concentration is based on NADPH-dependent reduction by at least two oxireductases. Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural resistant strain and its parent in micro-aerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower furfural concentrations, NADH-dependent oxireductases are the main defence mechanism. At concentrations above 15 mM, however, [1-13C]-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through pentose-phosphate pathway increases and that NADPH-dependent oxireductases became the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is up-regulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterised ORF YKL071W and 4 further, likely NADPH-dependent oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inverse engineered significantly increased furfural resistance in the parent strain, thereby demonstrating these two enzymes to be key elements of the resistance phenotype. Experiment Overall Design: RNA levels were measured in glucose limited, micro-aerobic chemostat cultures with different concentrations of the growth inhibitor furfural. Two strains were compared: TMB3400-FT30-3 is a strain that has been evolutionary adapted to withstand high furfural concentrations. TMB3400 is its less resistant parent. Number of biological replicates: 2-3.

Dataset Information

Saccharomyces cerevisiae

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure