Washington State University

Mark R Wildung

Rodney Bruce Croteau

Anne Schoendorf

Kevin D Walker

John E Crock

Charles Cullen Burke

Bernd M Lange

Jonathan Gershenzon

David G McCaskill

Transacylase enzymes and the use of such enzymes to produce paclitaxel, related taxoids, as well as intermediates in the paclitaxel biosynthetic pathway are disclosed. Also disclosed are nucleic acid sequences encoding the transacylase enzymes.

Transacylases of the paclitaxel biosynthetic pathway

Transacylase enzymes of and the use of such enzymes to produce Taxol&trade;, related taxoids, as well as intermediates in the Taxol&trade; biosynthetic pathway are disclosed. Examples of specific enzymes described herein include taxadienol 5-O-acetyl transacylase (TAX1) and 10-deacetylbaccatin III-10-O-acetyl transferase (TAX6). Also disclosed are nucleic acid sequences encoding the transacylase enzymes.

Nucleic acid molecules encoding 10-deacetylbaccatin III-O-acetyl transferase and related products

The taxadiene synthase gene of Pacific yew has been cloned and its nucleic acid and polypeptide sequence is presented. Truncation or removal of the transit peptide increases expression of the cloned taxadiene synthase gene expression in cells.

Compositions and methods for Taxol biosynthesis

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Geranyl diphosphate synthase large subunit, and methods of use

Transacylase enzymes and the use of such enzymes to produce Taxol&trade;, related taxoids, as well as intermediates in the Taxol&trade; biosynthetic pathway are disclosed. Also disclosed are nucleic acid sequences encoding the transacylase enzymes.

A cDNA encoding (E)-&bgr;-farnesene synthase from peppermint (,) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO: 1) is provided which codes for the expression of (E)-&bgr;-farnesene synthase (SEQ ID NO:2), from peppermint (,). In other aspects replicable recombinant cloning vehicles are provided which code for (E)-&bgr;-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-&bgr;-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-&bgr;-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-&bgr;-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-&bgr;-farnesene synthase may be used to obtain expression or enhanced expression of (E)-&bgr;-farnesene synthase in plants in order to enhance the production of (E)-&bgr;-farnesene, or may be otherwise employed for the regulation or expression of (E)-&bgr;-farnesene synthase, or the production of its product.

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (mentha x piperita, L.) that produces the aphid alarm pheromone (E)-&bgr;-farnesene

cDNAs encoding 1-deoxyxylulose-5-phosphate synthase from peppermint (,) have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) are provided which code for the expression of 1-deoxyxylulose-5-phosphate synthase from plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, replicable recombinant cloning vehicles are provided which code for plant 1-deoxyxylulose-5-phosphate synthases, or for a base sequence sufficiently complementary to at least a portion of 1-deoxyxylulose-5-phosphate synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a plant 1-deoxyxylulose-5-phosphate synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant 1-deoxyxylulose-5-phosphate synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant 1-deoxyxylulose-5-phosphate synthase may be used to obtain expression or enhanced expression of 1-deoxyxylulose-5-phosphate synthase in plants in order to enhance the production of 1-deoxyxylulose-5-phosphate, or its derivatives such as isopentenyl diphosphate (BP), or may be otherwise employed for the regulation or expression of 1-deoxyxylulose-5-phosphate synthase, or the production of its products.

Nucleic and amino acid sequences relating to a novel transketolase, and methods for the expression thereof

The taxadiene synthase gene of Pacific yew has been cloned and its nucleic acid and polypeptide sequence is presented. Truncation or removal of the transit peptide increases expression of the cloned taxadiene synthase gene expression in E. coli cells.

Compositions and methods for taxol biosynthesis

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

Isolation and bacterial expression of a sesquiterpene synthase CDNA

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

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Colfax, WA, United States of America

Mark R Wildung