Project description:Background: Bacterial fermentation of carbohydrates from sustainable lignocellulosic biomass into biofuels by the anaerobic bacterium Clostridium acetobutylicum is a promising alternative energy source to fossil fuels. Understanding the complex metabolic pathways it employs and how they are regulated will contribute to improved biofuel production. Recently, it has been demonstrated that xylose is not appreciably fermented in the presence of arabinose, suggesting a hierarchy of pentose utilization in this organism. Our goal is to uncover if transcriptional regulation contributes to this hierarchy. Results: Growth and sugar consumption rates showed that arabinose, like glucose, actively represses xylose utilization in cultures fermenting xylose. RNA-Seq revealed there was a large overlap in differentially regulated genes after addition of arabinose or glucose, suggesting a common mechanism of regulation. A putative ORF was identified that may be important for transcriptional regulation in response to the nutritional state of the cells. Conclusions: Decreased xylose consumption, increased acetate production, and transcription of the phosphoketolase gene (CA_C1343) revealed a transition of pentose catabolism from the pentose phosphate pathway to the phosphoketolase pathway after addition of arabinose. Together, these results substantiate the claim that arabinose is utilized preferentially over xylose in C. acetobutylicum. Furthermore, they demonstrate that this phenomenon is modulated in part at the transcriptional level, and they provide valuable insight into potential mechanisms for altering pentose utilization to modulate fermentation products for biofuel production.
Project description:In this study, we reported the updated chromosome and plasmid sequence of ZM4, and its two engineered xylose-utilizing strains derivatives (strains 2032 and 8b). The majority of plasmid genes have either homologs from other organisms or some conserved domains. Our bioinformatics analysis suggested that several plasmid genes may be essential genes of ZM4. To further validate the function of these plasmid genes, an RNA-Seq pipeline was developed for Z. mobilis and used to compare the gene expression under various conditions, including anaerobic and aerobic conditions, and in different biomass hydrolysates. Overall, these plasmids genes are more responsive to different concentrated hydrolysates than the different oxygen concentrations tested (such as anaerobic vs. aerobic conditions). Moreover, our results also indicate that the plasmid copy number is affected by growth conditions and foreign gene insertion while the chromosomal gene expression is relatively stable across different conditions in different strain backgrounds.
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 this study, the recombinant Trichoderma reesei strain HJ48 was employed to investigate the differences between anaerobic fermentation of xylose and glucose, through genome-wide transcription analysis. Analysis of the genes induced under fermentation condition has revealed novel features in T. reesei. Our results how that many genes related to ribosome were expressed more highly with xylose than with glucose in HJ48.
Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, Gram-positive anaerobe, which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2 and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to co-utilize glucose and xylose, make this bacterium an attractive candidate for microbial bioenergy production. Glycolytic pathways and an ABC-type sugar transporter were significantly up-regulated during growth on glucose and xylose, indicating that C. saccharolyticus co-ferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks represents a highly desirable feature of a lignocellulose-utilizing, biofuel-producing bacterium. Keywords: substrate response
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.