Project description:Saccharomyces cerevisiae cannot metabolize non-glucose sugars including cellobiose, xylose, xylodextrins in nature, which are prevalent in plant cell wall. Here, one engineered S. cerevisiae strain, which expresses a cellodextrin transporter gene (cdt-1) and an intracellular β-glucosidase gene (codon-optimized gh1-1) from Neurospora crassa; XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene), and XKS1 (xylulose kinase gene) from Scheffersomyces stipitis, as well as cdt-2 (coding for cellodextrin transporter 2), gh43-2 (coding for β-xylosidase) and gh43-7 (coding for a xylosyl-xylitol-specific β-xylosidase) from N. crassa, can utilize the above non-glucose sugars. We sequenced mRNA from exponential cultures of the engineered S. cerevisiae grown on glucose, cellobiose, xylose or xylodextrins as a single carbon source in both aerobic and anaerobic conditions in biological triplicate. Differences in gene expression between non-glucose sugar and glucose metabolism revealed by RNA deep sequencing indicated that non-glucose sugar metabolism induced mitochondrial activation and reduced amino acid and protein biosynthesis under fermentation conditions.
Project description:Agricultural wastes and other non-food sources can be used to produce biofuels. Despite multiple attempts using engineered yeast strains expressing exogenous genes, the native Saccharomyces cerevisiae produces low amount of second generations of biofuels. Here, we focused on Znf1, a non-fermentable carbon transcription factor and the suppressor protein Bud21 to overcome this challenge. Several mutants of engineered S. cerevisiae strains were engineered to enhance production of biofuels and xylose-derived compounds such as xylitol. This study demonstrates Znf1's novel transcriptional regulatory control of xylose and offer an initial step toward a more sustainable production of advanced biofuels from xylose.
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:Saccharomyces cerevisiae cannot metabolize xylodextrins in nature. One engineered S. cerevisiae strain, which expresses XYL1 (xylose reductase gene), XYL2 (xylitol dehydrogenase gene), and XKS1 (xylulose kinase gene) from Scheffersomyces stipitis, and cdt-2 (coding for cellodextrin transporter 2), gh43-2 (coding for β-xylosidase) and gh43-7 (coding for a xylosyl-xylitol-specific β-xylosidase) from N. crassa, can utilize xylodextrins in aerobic condtions but not anaerobic conditions. We sequenced mRNA from anaerobically fed-batch cultures of the engineered S. cerevisiae grown on xylodextrins with or without the continuous feeding of xylose in biological duplicate. Dynamic changes of gene expression during xylose feeding experiment revealed by RNA deep sequencing indicated that xylose helps anaerobically xylodextrin-grown cells to recover mithondrial function thereby resuming xylodextrin consumption. Furthermore, different portions of genes involved in ribosome biogenesis showed either decreased or increased transcriptions. The underlying mechanism remains to be elucidated.
Project description:The ascomycetes Saccharomyces cerevisiae, Candida albicans and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode xylose reductase and xylitol dehydrogenase required to convert xylose to xylulose, on which all three fungi grow. We have created C. albicans strains deleted for either or both the xylose reductase gene GRE3, and the xylitol dehydrogenase gene XYL2. As expected, all the mutant strains cannot grow on xylose, while the gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are complemented efficiently by the XYL1 and XYL2 from S. stipitis respectively. Intriguingly, the S. cerevisiae GRE3 and SOR1 genes can complement the gre3 and xyl2 mutants respectively, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work establishes that C. albicans strains engineered to lack essential steps for xylose metabolism provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source. Transcription profile of cells in xylose compared to glucose. Two sets: Candida albicans, 1 condition ; Saccharomyces cerevisiae 2 conditions / in xylose (SX) or no sugar (S) (replicates with dye-swap)
Project description:The aim of present study is to understand the impact of xylose utilization on the Saccharomyces cerevisiae physiology after initial genetic engineering and in a strain with an improved xylose utilization phenotype.
Project description:HMF and furfural were pulse added to xylose-utilizing Saccharomyces cerevisiae during either the glucose consumption phase or the xylose consumption phase. Transcriptome samples were collected before and one hour after pulsing of inhibitors.