Dataset Information

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway.

ABSTRACT:

Background

Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation.

Results

A new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized.

Conclusion

Pentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.

SUBMITTER: Bettiga M

PROVIDER: S-EPMC2720912 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Publications

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway.

Bettiga Maurizio M Bengtsson Oskar O Hahn-Hägerdal Bärbel B Gorwa-Grauslund Marie F MF

Microbial cell factories 20090724

<h4>Background</h4>Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation.<h4>Results</h4>A new recombinant S. cerevisiae strain expressing an improved ...[more]

PMID: 19630951

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Project description:BackgroundOver the last decades, many strategies to procure and improve xylose consumption in Saccharomyces cerevisiae have been reported. This includes the introduction of efficient xylose-assimilating enzymes, the improvement of xylose transport, or the alteration of the sugar signaling response. However, different strain backgrounds are often used, making it difficult to determine if the findings are transferrable both to other xylose-consuming strains and to other xylose-assimilation pathways. For example, the influence of anomerization rates between α- and β-xylopyranose in pathway optimization and sugar sensing is relatively unexplored.ResultsIn this study, we tested the effect of expressing a xylose epimerase in S. cerevisiae strains carrying different xylose-consuming routes. First, XIs originating from three different species in isogenic S. cerevisiae strains were tested and the XI from Lachnoclostridium phytofermentans was found to give the best performance. The benefit of increasing the anomerization rate of xylose by adding a xylose epimerase to the XI strains was confirmed, as higher biomass formation and faster xylose consumption were obtained. However, the impact of xylose epimerase was XI-dependent, indicating that anomer preference may differ from enzyme to enzyme. The addition of the xylose epimerase in xylose reductase/xylitol dehydrogenase (XR/XDH)-carrying strains gave no improvement in xylose assimilation, suggesting that the XR from Spathaspora passalidarum had no anomer preference, in contrast to other reported XRs. The reduction in accumulated xylitol that was observed when the xylose epimerase was added may be associated with the upregulation of genes encoding endogenous aldose reductases which could be affected by the anomerization rate. Finally, xylose epimerase addition did not affect the sugar signaling, whereas the type of xylose pathway (XI vs. XR/XDH) did.ConclusionsAlthough xylose anomer specificity is often overlooked, the addition of xylose epimerase should be considered as a key engineering step, especially when using the best-performing XI enzyme from L. phytofermentans. Additional research into the binding mechanism of xylose is needed to elucidate the enzyme-specific effect and decrease in xylitol accumulation. Finally, the differences in sugar signaling responses between XI and XR/XDH strains indicate that either the redox balance or the growth rate impacts the SNF1/Mig1p sensing pathway.

Project description:BackgroundConsiderable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.ResultsGenes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells.ConclusionThe results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.

Project description:BACKGROUND:Xylose-based ethanol production by recombinant S. cerevisiae is of great interest to basic and applied bioenergy research. By expressing three different fungal pathways in two S. cerevisiae hosts respectively, we found that the xylose utilization efficiency by recombinant S. cerevisiae depends not only on the choice of xylose pathway but also on the choice of host, exhibiting an obvious host or context dependence. To investigate molecular mechanisms of this context dependence, we applied RNA-seq analysis in this study for a systematic characterization of the xylose utilization via different pathways in different S. cerevisiae hosts. RESULTS:Based on the RNA-seq analysis, the transcripts that were regulated during xylose utilization have been identified. Three transcription factors involved in regulation of amino acid metabolism, responses to oxidative stresses, and degradation of aggregated proteins, respectively, were found to participate in xylose metabolism regulation regardless of which pathway was expressed and which host the xylose pathway was expressed in. Nine transcription factors, involved in homeostasis, regulation of amino acid metabolism, and stress responses, were identified as the key modules responsible for the host-specific responses to the same xylose pathway. In addition, the transcriptional regulations of xylose utilization in different yeast hosts were compared to two reference regulation patterns, which indicated that diverse regulation strategies were adopted by different hosts for improved xylose utilization. CONCLUSIONS:This study provides the first transcriptomic study of the host dependence of xylose utilization in S. cerevisiae. Both the conserved regulatory modules for xylose metabolism and the key modules responsible for host dependence were identified. As indicated by the functions of the conserved transcription factors involved in xylose metabolism regulation, the xylose utilization in recombinant S. cerevisiae may be affected by both carbohydrate metabolism regulation and stress responses. Based on the comparison of transcriptional regulation patterns, the metabolic optimizations of xylose utilization in different hosts went toward different directions, which may explain the host dependence observed in this study. The knowledge revealed by this study could provide valuable insights towards the improvement of metabolic engineering strategies for cellulosic ethanol production.

Project description:Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is critically important for robust and efficient renewable biofuel production from lignocellulosic biomass.

Dataset Information

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway.

Background

Results

Conclusion

Publications

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway.

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