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Intermediates and enzymes of the non-mevalonate isoprenoid pathway

Intermediates and enzymes of the non-mevalonate isoprenoid pathway description/claims

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.

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 producing them. Further, the invention relates to their use for the formation or production of intermediates or products or enzymes of said biosynthetic pathway as well as to enzymes and intermediates. Further, the invention relates to the screening for inhibitors or enzymes for said biosynthetic pathway.

The system of biosynthetic pathways in any organism is highly streamlined, whereby a few central trunk pathways branch into a great number of peripheral pathways. The central trunk pathways involve starting materials which are highly integrated. Therefore, central or trunk pathways are highly regulated. At the same time they are crucial for any attempts to interfere with the metabolism of any organism either by an inhibitor or by metabolic engineering.

The isoprenoid pathways are a prime example for this metabolic organisation. They are very long and highly branched, leading to some 30,000 isoprenoid or terpenoid compounds. They all seem to derive from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). They are produced by two alternative trunk pathways (reviewed in Eisenreich et al., 2001).

By the classical research of Bloch, Cornforth, Lynen and co-workers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) have become established as key intermediates in the biosynthesis of isoprenoids via mevalonate. However, many bacteria, plastids of all plants, and the protozoon Plasmodium falciparum synthesize IPP and DMAPP by an alternative pathway via 1-deoxy-D-xylulose 5-phosphate. The discovery of the pathway was mainly based on the incorporation of isotope-labelled 1-deoxy-D-xylulose into the isoprenoid side chain of menaquinones from Escherichia coli (Arigoni and Schwarz, 1999).

This mevalonate-independent pathway has so far only been partially explored (FIG. 1). For a better understanding of these aspects of the invention, the pathway shall be briefly explained. It can be divided into three segments:

In a first pathway segment shown in FIG. 1, pyruvate (1) is condensed with glyceraldehyde 3-phosphate (2) to 1-deoxy-D-xylulose 5-phosphate (DXP) (3). Subsequently, DXP is converted into 2C-methyl-D-erythritol 4-phosphate (MEP) (4) by a two-step reaction comprising a rearrangement and a reduction. This establishes the 5-carbon isoprenoid skeleton.

In the subsequent segment of the mevalonate-independent pathway (FIG. 1), MEP (4) is first condensed with CTP to 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) (5) by 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (PCT/EP00/07548). CDP-ME (5) is subsequently ATP-dependent phosphorylated by 4-diphosphocytidyl-2C-methyl-D-erythritol kinase yielding 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDP-MEP) (6). The intermediate is subsequently converted into 2C-methyl-D-erythritol 2,4-cyclodiphosphate (cMEPP) (7) by 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (PCT/EP00/07548). These three enzymatic steps form a biosynthetic unit which activates the isoprenoid C5-skeleton for the third pathway segment (Rohdich et al., 1999; Lüttgen et al., 2000; Herz et al., 2000).

Bioinformatic studies (German Patent Application 10027821.3), as well as studies with mutants of Synechocystis sp. (Cunningham et al., 2000) and Escherichia coli (Campos et al., 2001; Altincicek et al., 2001) demonstrate the involvement of lytB and gcpE genes in the isoprenoid pathway. However, the function and the reaction catalyzed by the corresponding gene products are still unknown.

Recently, a kinase (XylB) has been described that catalyzes the conversion of 1-deoxy-D-xylulose into 1-deoxy-D-xylulose 5-phosphate at high rates (Wungsintaweekul et al., 2000).

Genes and enzymes participating in further downstream reactions have been described. However, the gene functions, the intermediates, and the mechanisms leading to the products are still unknown.

For numerous pathogenic eubacteria as well as for the malaria parasite P. falciparum, the enzymes involved in the non-mevalonate pathway are essential. The intermediates of the mevalonate-independent pathway cannot be assimilated from the environment by pathogenic eubacteria and P. falciparum. The enzymes of the alternative isoprenoid pathway do not occur in mammalia which synthesize their isoprenoids and terpenoids exclusively via the mevalonate pathway. Moreover, the idiosyncratic nature of the reactions in this pathway reduces the risk of cross-inhibitions with other, notably mammalian enzymes.

Therefore, enzymes of the alternative isoprenoid pathway seem to be specially suited as targets for novel agents against pathogenic microorganisms and herbicides. The elucidation of unknown steps and the identification of these targets, e.g. genes and cognate enzymes of these pathways is obligatory for this purpose. A further source of interest in the non-mevalonate pathway derives from the fact certain pathogens like Mycobacteria, Plasmodia, Escherichia etc. use this pathway to activate γδ T cells (Fournié and Bonneville, 1996). Therefore, γδ T cells likely act as a first line of defense against infections by such pathogens. Intermediates of the non-mevalonate pathway have been suggested to be responsible for γδ T cell activation (Jomaa et al., 1999). Recently, it was show that E. coli strains lost the ability to stimulate γδ T cells when the dxr or the gcpE gene was knocked out (Altincicek et al., 2001).

Moreover, there is a great biotechnological interest in these pathways, since they lead to valuable vitamins and isoprenoid or terpenoid products.

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.

It is an object of the invention to provide enzymes and nucleic acids coding for said enzymes as well as intermediates for the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to isopentenyl diphosphate and/or dimethylallyl diphosphate.

It has surprisingly been found that the intermediate in the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to isopentenyl diphosphate and/or dimethylallyl diphosphate is 1-hydroxy-2-methyl-2-butenyl 4-diphosphate. This intermediate is formed by an enzyme encoded by gcpE as designated in the E. coli genome. It has further been found that this enzyme prefers as reductant NADH or NADPH. Further, it has been found that it is promoted by Co2+.

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