Thermogen Inc.

Other

Biotechnology Research Development Corporation

David C Demirjian

Malcolm J Casadaban

Veronika Vonstein

J Mark Weber

Nikos C Pagratis

Michael Fonstein

John Aikens

Larry Allen

Veronica Vonstein

Mikhail Peredultchuk

Badal C Saha

Paul Taylor

Ian G Fotheringham

Francis Michael Racine

Nathan Wymer

George L Gaines

Yoshikiyo Sakaibara

The invention provides biosynthetic routes to xylitol production that do not require pure-xylose for synthesis and that can utilize inexpensive substrates such as hemicellulose hydrolysates.

Methods for production of xylitol in microorganisms

We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including ., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan ). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu Km transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

Recombinant DNA and methods for producing thermostable enzymes

The present invention relates to a system for identifying, isolating and utilizing promoter elements useful for expression of nucleotide sequences and the proteins encoded thereby in a thermophile. In one embodiment, a recombinant DNA molecule is provided, and comprises a reporter sequence, a putative thermophile promoter, a selectable marker sequence, and a 3&prime; and a 5&prime; DNA targeting sequence that are together capable of causing integration of at least a portion of said DNA molecule into the genome of a thermophile. Further, within the recombinant DNA, the reporter sequence is under the transcriptional control of a promoter which functions in a thermophile to form a promoter/reporter cassette, the promote/reporter cassette is flanked by said 3&prime; and said 5&prime; DNA targeting sequences, and the promoter/reporter cassette is positioned in the opposite orientation of the DNA targeting sequences.

Methods for isolation of thermophile promoters

Thermus promoters for gene expression

The instant invention encompasses isolated stable esterase enzymes characterized by the ability to remain stable at certain temperatures, substrate specificities, and activity profile.

Stable biocatalysts for ester hydrolysis

The instant disclosure teaches transposons and methods of the instant invention for making Protein Fusions by rapid, random shuffling of protein domains to produce novel protein fusions. This system is generally applicable to production of multifunctional chimeric proteins in vivo and in vitro. The methods and constructs of the instant invention can be used to randomly create both carboxy- and amino-terminal protein fusions in vivo. The methods and contructs of the instant invention are useful in the development of a protein domain library, in the construction of multifunctional enzymes, and in the accelerated evolution of new enzymatic activities.

Protein fusion method and constructs

We have developed a new gene transfer system for extreme thermophiles of the genus Thermus, including Thermus flavus., using a chromosomal gene, and a thermostable derivative of the kanamycin-resistance gene (kan.sup.tr2). A plasmid mediated gene-replacement process is used to insert it into the chromosome resulting in the production of Leu.sup.- Km.sup.r transformants. This system not only allows stable, single-copy gene insertion into the chromosome of an extreme thermophile, but can be used in the thermo-genetic process described here to generate thermo-stabilized enzymes and proteins for industrial processes. This host-vector environment makes it possible to generate further thermo-stabilizing mutations in the kan gene beyond those levels previously reported.

Thermophile gene transfer

Growing community of inventors

Chicago, IL, United States of America

David C Demirjian