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Supplementation of intracellular XylR leads to coutilization of hemicellulose sugars.

ABSTRACT: Escherichia coli has the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in using E. coli for the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR augmentation must be finely tuned for robust coutilization of these two hemicellulosic sugars. Using a novel technique for scarless gene insertion, an additional copy of xylR was inserted into the araBAD operon. The resulting strain was superior at cometabolizing mixtures of arabinose and xylose and was able to produce at least 36% more ethanol than wild-type strains. This strain is a useful starting point for the development of an E. coli biocatalyst that can simultaneously convert all biomass sugars.

SUBMITTER: Groff D

PROVIDER: S-EPMC3302627 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Supplementation of intracellular XylR leads to coutilization of hemicellulose sugars.

Groff Dan D Benke Peter I PI Batth Tanveer S TS Bokinsky Gregory G Petzold Christopher J CJ Adams Paul D PD Keasling Jay D JD

Applied and environmental microbiology 20120127 7

Escherichia coli has the potential to be a powerful biocatalyst for the conversion of lignocellulosic biomass into useful materials such as biofuels and polymers. One important challenge in using E. coli for the transformation of biomass sugars is diauxie, or sequential utilization of different types of sugars. We demonstrate that, by increasing the intracellular levels of the transcription factor XylR, the preferential consumption of arabinose before xylose can be eliminated. In addition, XylR ...[more]

PMID: 22286982

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Project description:We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.

Dataset Information

Supplementation of intracellular XylR leads to coutilization of hemicellulose sugars.

Publications

Supplementation of intracellular XylR leads to coutilization of hemicellulose sugars.

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