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:To determine the genomic location of a gene that permits xylose utilization we conducted bulk segregant analysis (BSA) using Affymetrix yeast tiling arrays. BSA works by taking advantage of DNA sequence polymorphisms between different strains and the fact that it is relatively easy to pool large numbers of meiotic spore products (segregants) in yeast. Pooling segregants based on their phenotype allows the region of the genome responsible for the phenotype to be detected. This is because DNA polymorphisms in regions unlinked to the locus causing the phenotype will segregate randomly and be “evened” out, while around the genomic region of interest, sequences or polymorphisms responsible for the trait will be present in all positive segregants, and absent in all negative segregants. In our case, a Simi White wine strain (S. cerevisiae) carrying the locus responsible for xylose utilization was crossed to a laboratory strain of Saccharomyces cerevisiae; this strain was estimated to carry DNA polymorphisms relative to the laboratory strain at a level of approximately .5%. Spores from the Simi White / S288c diploid were screened for the xylose utilization phenotype and 39 positive spores were combined into one pool and 39 negative spores into another pool, and genomic DNA (gDNA) was isolated from each pool. We then hybridized the positive and negative gDNA pools to tiling microarrays that were based on the S288c reference genome with the expectation that regions of the genome derived from Simi White will hybridize less robustly to the array because of the DNA polymorphisms between Simi White and S288c. Log2 ratios of probe intensities were calculated (negative/positive), and a peak appeared in the chromosome XV right subtelomeric region that corresponds to less robust hybridization to the microarray of the positive pool gDNA coming from this region of the genome
Project description:In the present study transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at genome-wide level how signalling and carbon catabolite repression differed in cells grown on either glucose or xylose. The more detailed knowledge about is xylose sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is it rather recognised as a non-fermentable carbon source is important in achieving understanding for further engineering this yeast for more efficient anaerobic fermentation of xylose.
Project description:In response to carbon source switching from glucose to non-glucose, such as ethanol and galactose, yeast cells can directionally preprogram cellular metabolism to efficiently utilize the nutrients. However, the understanding of cellular responsive network to utilize a non-natural carbon source, such as xylose, is limited due to the incomplete knowledge on the xylose response mechanisms. Here, through optimization of the xylose assimilation pathway together with combinational evaluation of reported targets, we generated a series of mutants with varied growth ability. However, understanding how cells respond to xylose and remodel cellular metabolic network is far insufficient based on current information. Therefore, genome-scale transcriptional analysis was performed to unravel the cellular reprograming mechanisms underlying the improved growth phenotype.