Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae.

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5'-end of the gene, site-directed mutagenesis, and codon optimization.

A vector set for systematic metabolic engineering in Saccharomyces cerevisiae.

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 µ-containing plasmids.

Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface.

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al.

DNA deformation energy as an indirect recognition mechanism in protein-DNA interactions.

Proteins that bind to specific locations in genomic DNA control many basic cellular functions. Proteins detect their binding sites using both direct and indirect recognition mechanisms. Deformation energy, which models the energy required to bend DNA from its native shape to its shape when bound to a protein, has been shown to be an indirect recognition mechanism for one particular protein, Integration Host Factor (IHF).

Indirect recognition in sequence-specific DNA binding by Escherichia coli integration host factor: the role of DNA deformation energy.

Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif.

Activation of transcription initiation from a stable RNA promoter by a Fis protein-mediated DNA structural transmission mechanism.

The leuV operon of Escherichia coli encodes three of the four genes for the tRNA1Leu isoacceptors. Transcription from this and other stable RNA promoters is known to be affected by a cis-acting UP element and by Fis protein interactions with the carboxyl-terminal domain of the alpha-subunits of RNA polymerase. In this report, we suggest that transcription from the leuV promoter also is activated by a Fis-mediated, DNA supercoiling-dependent mechanism similar to the IHF-mediated mechanism described previously for the ilvP(G) promoter (S.