Dataset Information

Simple and Practical Multigram Synthesis of d-Xylonate Using a Recombinant Xylose Dehydrogenase.

ABSTRACT: An efficient multienzyme system for the preparative synthesis of d-xylonate, a chemical with versatile industrial applications, is described. The multienzyme system is based on d-xylose oxidation catalyzed by the xylose dehydrogenase from Calulobacter crescentus and the use of catalytic amounts of NAD+. The cofactor is regenerated in situ by coupling the reduction of acetaldehyde into ethanol catalyzed by alcohol dehydrogenase from Clostridium kluyveri. Excellent conversions (>95%) were obtained in a process that allows easy product isolation by simple evaporation of the volatile buffer and byproducts.

SUBMITTER: Sanchez-Moreno I

PROVIDER: S-EPMC6648848 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Simple and Practical Multigram Synthesis of d-Xylonate Using a Recombinant Xylose Dehydrogenase.

Sánchez-Moreno Israel I Benito-Arenas Raúl R Montero-Calle Pilar P Hermida Carmen C García-Junceda Eduardo E Fernández-Mayoralas Alfonso A

ACS omega 20190618 6

An efficient multienzyme system for the preparative synthesis of d-xylonate, a chemical with versatile industrial applications, is described. The multienzyme system is based on d-xylose oxidation catalyzed by the xylose dehydrogenase from Calulobacter crescentus and the use of catalytic amounts of NAD+. The cofactor is regenerated in situ by coupling the reduction of acetaldehyde into ethanol catalyzed by alcohol dehydrogenase from Clostridium kluyveri. Excellent convers ...[more]

PMID: 31460157

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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.

Dataset Information

Simple and Practical Multigram Synthesis of d-Xylonate Using a Recombinant Xylose Dehydrogenase.

Publications

Simple and Practical Multigram Synthesis of d-Xylonate Using a Recombinant Xylose Dehydrogenase.

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