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Intermediates and enzymes of the non-mevalonate isoprenoid pathway

a technology of intermediates and enzymes, which is applied in the direction of lyases, transferases, protozoa, etc., can solve the problems of hampered previous attempts to approach these goals and low biosynthesis rate along these pathways

Inactive Publication Date: 2008-12-25
BACHER ADELBERT +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new system for the efficient biosynthesis of isoprenoid products, which are important chemicals used in many industries. The system involves genetically modified strains of E. coli that are fed with specific substrates and promoters to produce desired intermediates and products. The system can be used for the structure elucidation of unknown intermediates and the assignment of biological functions of putative genes or cognate enzymes in the non-mevalonate biosynthetic pathway. The system can also be used for screening chemical libraries for potential herbicides, antimalarials, and antimicrobial substances. Overall, the patent provides a novel approach for the production of isoprenoids and related compounds.

Problems solved by technology

Previous attempts to approach these goals have been hampered by the low rate of biosynthesis along these pathways in wild-type cells studied so far.

Method used

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  • Intermediates and enzymes of the non-mevalonate isoprenoid pathway
  • Intermediates and enzymes of the non-mevalonate isoprenoid pathway
  • Intermediates and enzymes of the non-mevalonate isoprenoid pathway

Examples

Experimental program
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Effect test

example 1

Construction of a Vector Carrying the xylB Gene of Escherichia coli Capable for Transcription and Expression of D-xylulokinase

[0147]Chromosomal DNA from Escherichia coli strain XL1-Blue (Bullock et al. 1987; commercial source: Stratagene, LaJolla, Calif., USA) is isolated according to a method described by Meade et al. 1982.

[0148]The E. coli ORF xylB (accession no. gb AE000433) from base pair (bp) position 8596 to 10144 is amplified by PCR using chromosomal E. coli DNA as template. The reaction mixture contains 10 pmol of the primer 5′-CCGTCGGAATTCGAGGAGAAATTAACCATGTATATCGGGATAGATCTTGG-3′ (SEQ ID NO:1), 10 pmol of the primer 5′-GCAGTGAAGCTTTTACGCCATTAATGGCAGAAGTTGC-3′ (SEQ ID NO:2), 20 ng of chromosomal DNA, 2 U of Taq DNA polymerase (Eurogentec, Seraing, Belgium) and 20 nmol of dNTPs in a total volume of 100 μl containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-hydrochloride, pH 8.8 and 0.1% (w / w) Triton X-100.

[0149]The mixture is denaturated for 3 min at 94° C. Then 30 PCR cycles for ...

example 2

Construction of a Vector Carrying the XylB and dxr Genes of Escherichia coli Capable for Transcription and Expression of D-xylulokinase and DXP reductoisomerase

[0154]The E. coli ORF dxr (accession no. gb AE000126) from base pair (bp) position 9887 to 11083 is amplified by PCR using chromosomal E. coli DNA as template. The reaction mixture contains 10 pmol of the primer 5′-CTAGCCAAGCTTGAGGAGAAATTAACCATGAAGCAACTCACCATTCTGG-3′ (SEQ ID NO:3), 10 pmol of the primer 5′-GGAGATGTCGACTCAGCTTGCGAGACGC-3′ (SEQ ID NO:4), 20 ng of chromosomal DNA, 2 U of Taq DNA polymerase (Eurogentec) and 20 nmol of dNTPs in a total volume of 100 μl containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-hydrochloride, pH 8.8 and 0.1% (w / w) Triton X-100.

[0155]The mixture is denaturated for 3 min at 94° C. Then 30 PCR cycles for 60 sec at 94° C., 60 sec at 50° C. and 75 sec at 72° C. followed. After further incubation for 10 min at 72° C., the mixture is cooled to 4° C. An aliquot of 2 μl is subjected to agarose gel elec...

example 3

Construction of a Vector Carrying the XylB, dxr and ispD Genes of Escherichia coli Capable for Transcription and Expression of D-xylulokinase, DXP Reductoisomerase and CDP-ME Synthase

[0161]The E. coli ORF ispD (accession no. gb AE000358) from base pair (bp) position 6754 to 7464 is amplified by PCR using chromosomal E. coli DNA as template. The reaction mixture contains 10 pmol of the primer 5′-CCGGGAGTCGACGAGGAGAAATTAACCATGGCAACCACTCATTTGGATG-3′ (SEQ ID NO:5), 10 pmol of the primer 5′-GTCCAACTCGAGTTATGTATTCTCCTTGATGG-3′ (SEQ ID NO:6), 20 ng of chromosomal DNA, 2 U of Taq DNA polymerase (Eurogentec) and 20 nmol of dNTPs in a total volume of 100 μl containing 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-hydrochloride, pH 8.8 and 0.1% (w / w) Triton X-100.

[0162]The mixture is denaturated for 3 min at 94° C. Then 30 PCR cycles for 30 sec at 94° C., 30 sec at 50° C. and 45 sec at 72° C. followed. After further incubation for 10 min at 72° C., the mixture is cooled to 4° C. An aliquot of 2 μl is su...

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Abstract

The invention provides a protein in a form that is functional for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate notably in its (E)-form of the non-mevalonate biosynthetic pathway to isoprenoids. The invention also provides a protein in a form that is functional for the enzymatic conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, notably in its (E)-form, to isopentenyl diphosphate and / or dimethylallyl diphosphate. Further, screening methods for inhibitors of these proteins are provided. Further, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate is provided and chemical and enzymatic methods of its preparation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional application of U.S. application Ser. No. 10 / 474,536, having a filing date of Apr. 7, 2004, which is a 35 U.S.C. § 371 national phase application of International PCT Application Serial No. PCT / EP02 / 04005 filed Apr. 10, 2002, which claims priority to German Patent Application No. 101 18 166.3, filed Apr. 11, 2001, German Patent Application No. 101 30 236.3, filed Jun. 22, 2001, German Patent Application No. 101 55 084.7, filed Nov. 9, 2001, and German Patent Application No. 102 01 458.2, filed Jan. 16, 2002. The contents of these applications are hereby incorporated by reference as if recited in full herein.FIELD OF THE INVENTION[0002]The present invention relates to cells, cell cultures or organisms or parts thereof for the efficient formation of a biosynthetic product or intermediate or enzyme of a 1-deoxy-D-xylulose 5-phosphate-dependent biosynthetic pathway. Further, the invention relates to vectors for...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/10C12N15/54C40B30/08C40B30/06C12N1/11C12N1/15C12N1/21C12N5/10A01K67/027A01H5/00C07F9/02A61K31/66C07K16/00C12P21/02G01N33/53C12Q1/68C12P9/00C12N15/11G01N33/50A61K31/663A61P31/04A61P37/02A61P37/04A61P43/00C07B61/00C07C33/025C07C33/035C07C47/263C07F9/09C07F9/113C07H19/10C07K16/44C12N1/19C12N9/02C12N9/16C12N9/88C12N9/99C12N15/09C12N15/52C12N15/55C12Q1/02G01N33/15G01N33/569
CPCC07B2200/05C07C33/025C07F9/098C07F9/113C07H19/10C12N15/52C12P9/00C40B40/00G01N2500/04A61P31/04A61P37/02A61P37/04A61P43/00
Inventor BACHER, ADELBERTROHDICH, FELIXADAM, PETRAAMSLINGER, SABINEEISENREICH, WOLFGANGHECHT, STEFAN
Owner BACHER ADELBERT
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