Project description:The modification of the The modification of the tolerance of xylose-fermenting yeast is an urgent issue for improving ethanol production. In this study, multiple genes involving in superoxide dismutase, glutathione biosynthesis, NADPH regeneration and acetic acid degradation were overexpressed using stress-induced promoters, which is selected from the transcriptome data. Stress-induced promoters were used to realize the feedback control of the tolerant genes, which can ultimately improve the tolerance and ethanol production. We reported the stress-induced promoters for overexpressing tolerant genes and increasing yeast tolerance in a feedback manner
Project description:Increasing yeast robustness against biomass-derived inhibitors and insoluble solids is essential for the realization of a bio-based economy. The xylose-fermenting Saccharomyces cereivisiae F12 strain was subjected to an adaptive laboratory evolution experiment in the presence of these stressors. The resulting evolved strain exhibit better fermentation performance in terms of xylose consumption and ethanol yields than the parental strain. The overexpression of genes related to cell wall integrity and the stress response, together with the downregulation of protein synthesis and iron transport and homeostasis were highlighted as the major biological processes responsible for the improved phenotype.
Project description:Creating Saccharomyces yeasts capable of efficient fermentation of pentoses such as xylose remains a key challenge in the production of ethanol from lignocellulosic biomass. Metabolic engineering of industrial Saccharomyces cerevisiae strains has yielded xylose-fermenting strains, but these strains have not yet achieved industrial viability due largely to xylose fermentation being prohibitively slower than that of glucose. Recently, it has been shown that naturally occurring xylose-utilizing Saccharomyces species exist. Uncovering the genetic architecture of such strains will shed further light on xylose metabolism, suggesting additional engineering approaches or possibly even the development of xylose-fermenting yeasts that are not genetically modified. We previously identified a hybrid yeast strain, the genome of which is largely Saccharomyces uvarum, which has the ability to grow on xylose as the sole carbon source. Despite the sterility of this hybrid strain, we were able to develop novel methods to genetically characterize its xylose utilization phenotype, using bulk segregant analysis in conjunction with high-throughput sequencing. We found that its growth in xylose is governed by at least two genetic loci: one of the loci maps to a known xylose-pathway gene, a novel allele of the aldo-keto reductase gene GRE3, while a second locus maps to an allele of APJ1, a chaperonin gene not previously connected to xylose metabolism. Our work demonstrates that the power of sequencing combined with bulk segregant analysis can also be applied to a non-genetically-tractable hybrid strain that contains a complex, polygenic trait, and it identifies new avenues for metabolic engineering as well as for construction of non-genetically modified xylose-fermenting strains.
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.
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. Experiment Overall Design: Three aerobic batch fermentations were carried out both on 50 g l-1 glucose and on 50 g l-1 xylose to compare the yeast transcriptome and proteome of cells growing on xylose with that of glucose repressed and glucose derepressed cells. Samples of the xylose-grown cells were harvested at 72 h from the start of the xylose cultures with 32 g l-1 of residual xylose present. Samples of the glucose repressed cells were harvested at 5 h from the start of the glucose cultures with 37 g l-1 of residual glucose present. Samples of the glucose derepressed cells were harvested at 24 h from the start of the glucose cultures containing no glucose but 13 g l-1 of accumulated ethanol.
Project description:The main objective is to improve xylose fermentation by deletion of PHO80 gene in recombinant xylose-fermenting yeast strains. Microarray analysis was performed to investigate effects of PHO80 deletion on the gene expression profile of xylose-fermenting strains.
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:We previously reported that a recombinant Candida utilis strain expressing a Candida shehatae xylose reductase K275R/N277D, a C. shehatae xylitol dehydrogenase, and xylulokinase from Pichia stipitis produced ethanol from xylose. However, its productivity was low. In this study, metabolomic (CE-TOF MS) and transcriptomic (microarray) analyses were performed to characterize xylose metabolism by the engineered C. utilis and to identify key genetic changes contributing to efficient xylose utilization. Metabolomic analysis revealed that the xylose-fermenting strain accumulated more pentose phosphate pathway intermediates, more NADH, and more glycolytic intermediates upstream of glyceraldehyde 3-phosphate than wild-type. Transcriptomic analysis of the strain grown on xylose indicated a significant increase in expression of genes encoding tricarboxylic acid cycle enzymes, respiratory enzymes, and enzymes involved in ethanol oxidation. To decrease the NADH/NAD+ ratio and increase ethanol yield from the fermentation of xylose, ADH1 encoding NADH-dependent alcohol dehydrogenase was overexpressed. The resultant strain exhibited a 17% increase in ethanol production and a 22% decrease in xylitol accumulation relative to the control.
Project description:To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation.