EP1520025A2 - Nucleic acid molecules involved in the synthesis of melithiazols - Google Patents

Abstract

The present invention relates to the isolation and characterization of novel polyketide synthase and nonribosomal peptide synthetase genes from the myxobacterium Melittangium lichenicola Me I 46 that are necessary for the biosynthesis of melithiazols. Further, the present invention relates to methods of improving the pharmacological properties of melithiazols by biocombinatorial methods. The invention also relates to such improved melithiazols as well as to pharmaceutical compositions comprising such improved melithiazols.

Description

Nucleic acid molecules involved in the synthesis of melithiazols

The present invention relates to the isolation and characterization of novel polyketide synthase and nonribosomal peptide synthetase genes from the myxobacterium Melittangium lichenicola Me I 46 that are necessary for the biosynthesis of melithiazols. Further, the present invention relates to methods of improving the pharmacological properties of melithiazols by biocombinatorial methods. The invention also relates to such improved melithiazols as well as to pharmaceutical compositions comprising such improved melithiazols.

A number of documents is cited in this specification. The disclosure content of these documents including manufactures' manuals is herewith incorporated by reference.

Polyketides are compounds synthesized from activated short chain carboxylic acids, whereas non-ribosomally biosynthesised polypeptides are build up from amino acids. Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) are complex modular biosynthetic systems that catalyse the stepwise condensation of these building blocks, as well as their modification (e.g. keto reduction, dehydration, enoylreduction, N-methylation, heterocyclization, oxidation; epimerisation) (Cane, D.E. Chemical Reviews 97 (1997), 2463-2706). Bot types of enzymatic systems are responsible for the production of an immense variety of natural products in microorganisms. The melithiazols are a unique group of ^β-methoxy-acrylate fungicides produced by the myxobacterium Melittangium lichenicola (DE 94 4410 449; WO 95/26414). They are highly active against a broad range of fungi and yeasts with low mammalian toxicity. Practical applicability is presently limited to low production of the wild strain and by complicated chemical synthesis (see e.g. EP 00 116 43; US 98 069 35). The technical problem underlying the present invention was therefore to provide means and methods for the easy and convenient production of melithiazols or precursors or building blocks thereof. This technical problem is solved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a nucleic acid molecule encoding a (poly)peptide involved in the synthesis of a melithiazol, said nucleid acid molecule being selected from the group of (a) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1 (Mel B); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 2 (Mel C); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 3 (Mel D); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 4 (Mel E); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 5 (Mel F); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 6 (Mel G); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 7 (Mel H); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 8 (ORF 1); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 9 (ORF 2); a nucleic acid molecule encoding the polypept de of SEQ ID NO : 10 (ORF 3); a nucleic acid molecule encoding the polypept de of SEQ ID NO : 11 (ORF 4); a nucleic acid molecule encoding the polypept de of SEQ ID NO : 12 (ORF 5); a nucleic acid molecule encoding the polypept de of SEQ ID NO : 13 (ORF 6); a nucleic acid molecule encoding the polypept de of SEQ ID NO: 14 (ORF 7); (b) a nucleic acid molecule having the DNA sequence of SEQ ID NO. 15 (mel B); SEQ ID NO: 16 (mel C); SEQ ID NO: 17 (mel D); SEQ ID NO: 18 (mel E); SEQ ID NO: 19 (mel F); SEQ ID NO: 20 (mel G); SEQ ID NO: 21 (mel H); SEQ ID NO: 22 (ORF 1); SEQ ID NO: 23 (ORF 2); SEQ ID NO: 24 (ORF 3); SEQ ID NO: 25 (ORF 4); SEQ ID NO: 26 (ORF 5); SEQ ID NO: 27 (ORF 6); or SEQ ID NO: 28 (ORF 7); (c) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol and having an amino acid sequence that is at least 80%, preferably at least 90% and most preferred at least 99% identical to the amino acid sequence of (a) or (b); (d) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol and having the amino acid sequence of (a) or (b) with at least one conservative amino acid substitution; (e) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol and having an amino acid sequence that is an isoform of the amino acid sequence of any of (a) to (d); and (f) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol the complementary strand of which hybridizes under stringent conditions to the DNA molecule encoding the amino acid sequence of any of (a) to (e).

In accordance with the invention, the ORFs 5 and 6 are, throughout the specification, also referred to as nucleic acid molecules encoding Mel J and Mel K, respectively.

Preferably, the nucleic acid molecules are expressible from appropriate regulatory sequences such as promoters which include the natural regulatory sequences. Nucleic acid molecules as referred to above also comprising regulatory sequences are a preferred embodiment of the invention.

The term "(poly)peptide" refers to peptides as well as polypeptides. In accordance with conventional nomenclature, the term peptide denotes a molecule having up to 30 amino acids. Consequently, a polypeptide is defined as having more than 30 amino acids.

The term "(poly)peptide involved in the synthesis of a melithiazol" refers to peptides or polypeptides that catalyze one or more steps in the synthesis of this type of compound. These peptides or polypeptides, recombinantly produced in accordance with the present invention, may be identical with their naturally occurring counterparts. Alternatively, they may constitute a modified version of said naturally molecule wherein the modification may have been effected, for example, by biocombinatorial methods such as are explained in more detail herein below.

The term "stringent hybridization conditions" (i.e. conditions of high stringency) refers to conditions which comprise, e.g. an overnight incubation of the probe with the nucleic acid to be probed affixed to a filter (e.g. by performing a Southern blot) at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1-0.5 x SSC at about 65°C. Alternatively, high stringency conditions refer to hybridization in, e.g. 0.2-0.5x SSC, 0.1 % SDS at 65°C followed by washing as indicated above. The hybridisation solution would advantageously also comprise the other compounds as mentioned above. Also contemplated are nucleic acid molecules that hybridize to the nucleic acid molecules of the invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaH₂P0₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50°C with 1 X SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Preferred in accordance with the present inventions are polynucleotides which are capable of hybridizing to the complementary strand of the nucleic acid molecules of the invention or parts thereof, under stringent hybridization conditions (under conditions of high stringency), i.e. which do not cross hybridize to unrelated polynucleotides.

The nucleic acid molecules that are homologous to the above-described molecules (i.e. that display the mentioned degree of similarity or identity) and that represent derivatives of these molecules usually are variations of these molecules that represent modifications having the same biological function. They can be naturally occurring variations, for example sequences from other organisms, or mutations that can either occur naturally or that have been introduced by specific mutagenesis. Furthermore, the variations can be synthetically produced sequences. Percentage identity may be defined, for example, by using one of the following publicity available computer programs: BLAST (available from NCBI), Clustal W (available from EMBL).

Generally, by means of conventional molecular biological processes it is possible (see, e.g., Sambrook et al., "Molecular Cloning, A Laboratory Manual" 2^nd edition 1989, CSH Press, Cold Spring Harbor) to introduce different mutations into the nucleic acid molecules of the invention. One possibility is the production of deletion mutants in which nucleic acid molecules are produced by continuous deletions from the 5'- or 3'-terminus of the coding DNA sequence and that lead to the synthesis of proteins that are shortened accordingly. Another possibility is the introduction of single-point mutation at positions where a modification of the amino acid sequence influences, e.g., the enzyme activity or the regulation of the enzyme. By this method muteins can be produced, for example, that possess a modified K_m-value or that are no longer subject to the regulation mechanisms that normally exist in the cell, e.g. with regard to allosteric regulation or covalent modification. Such muteins may be identified, e.g. by methods of the present invention (see below), to be valuable in the production of e.g. therapeutically useful melithiazols displaying, for example, an enhanced fungicidal activity.

The amino acid sequences and nucleic acid sequences referred to above by a sequence identifier can also be obtained by the information provided in Figure 2 in combination with Table 1.

Nucleic acid molecules that hybridize to the molecules of the invention can be isolated, e.g., from genomic DNA of bacteria of the genus Melittangium. It should be noted that, in accordance with the present invention, it is preferred that the nucleic acid molecules are identical to or are derived from coding sequences from this genus and particularly from the species Melittangium lichenicola. In order to identify and isolate such nucleic acid molecules the molecules of the invention or parts of these molecules or the reverse complements of these molecules can be used, for example by means of hybridization according to conventional methods (see, e.g., Sambrook et al., 1989). As a hybridization probe nucleic acid molecules can be used, for example, that have exactly or basically the nucleotide sequence referred to in Table 1 or parts of these sequences or sequences complementary thereto. The fragments used as hybridization probe can be synthetic fragments that were produced by means of conventional synthesis methods and the sequence of which basically corresponds to the sequence of a nucleic acid molecule of the invention.

The term "isoform" means, in connection with the term "gene" or "nucleic acid", a form of a gene or nucleic acid including a derivative of a gene or nucleic acid resulting from, for example, alternative promoter usage or RNA editing. Isoforms can be detected by

(a) in silico analysis (e.g. by clustering analysis of any types of expressed sequences or the corresponding proteins, by alignment of expressed sequences with chromosomal DNA, by interspecies comparisons or by analysis of the coding as well as non-coding sequences like promoters).

(b) any type of hybridisation techniques (e.g. Northern blots, nuclease protection assays, microarrays) starting from RNA (as described in Higgins, S.J., Hames, D., "Nucleic acid hybridisation, a practical approach" IRL Press, Oxford, 1985.; Sambrook, loc. cit.).

(c) PCR-applications as well as hybridisation techniques starting from single strand or double strand cDNA obtained by reverse transcription, as described for example in Stoss, O., Stoilov, P., Hartmann, A.M., Nayler, O., Stamm, S. The in vivo minigene approach to analyse tissue-specific splicing. Brain Res. Protoc. (1999), 3:383-394.

Isoforms of (poly)peptides are preferably encoded by said isoforms of genes/nucleic acids.

The nucleic acid molecules described herein coding for the (poly)peptides of the invention and isoforms (isogenes) and mutant forms of said proteins are contributing to the synthesis of melithiazols. The identification of said genes was finally achieved by an approach that relied on the assumption that similarities might be present in the genes encoding PKS and NRPS involved in the synthesis of other secondary metabolites and the genes encoding corresponding polypeptides involved in the synthesis of melithiazol. One particular problem associated with this approach was the fact that the genome of myxobacteria contains a large variety of gene clusters that give rise to different secondary metabolites such as polyketides (e. g. Silakowski et al., Gene 275 (2001), 233- 240). Many building blocks of the polyketides are rather similar or even identical. Therefore, one might assume that the enzymes involved in the synthesis of such metabolites are not readily distinguishable from enzymes involved in the synthesis of structurally related compounds. Specifically, the correlation of certain (poly)peptides (also referred to as proteins) or of nucleic acid molecules encoding said (poly)peptides with the synthesis of certain metabolites such as melithiazol cannot be ascertained without further ado due to the number of different gene clusters expected in the genome of such organisms. This situation is further complicated by the fact that most of these gene clusters have so far not been identified. On the other hand, one might have assumed that similar enzymatic steps are performed by enzymes that are analogous, but not homologous in different genera of myxobacteria. Thus, it might very well have been that enzymes performing the same or a similar function do not concur in their primary amino acid sequences and are therefore quite different with respect to the DNA sequence that encodes these (poly)peptides.

An additional problem the present inventors were faced with was to identify genes/nucleic acid sequences that were indeed involved in the synthesis of melithiazol. In other terms, the identification of the responsible gene cluster was prima facie not sufficient to allow a valid conclusion with regard to the actual involvement of the ORFs (open reading frames) identified in the synthesis of melithiazols.

It could be experimentally supported, as also shown in example 9, that the MelJ gene and MelK gene are involved in the biosynthesis of melithiazol. The heterologous expression of the putative methyl transferase MelK and the hydrolase MelJ from the melithiazol gene cluster in a myxothiazol A producer resulted in the formation of myxothiazol Z. This clearly established that MelK and MelJ are responsible for the transformation of the β-methoxyacrylate-amide into the β-methoxyacrylate-ester.

Example 3 and Figure 3 show one strategy for the chromosomal integration of melJK from M. lichenicola into S. aurantiaca. Figure 4 shows that the resulting product is myxothiazol Z.

In order to support the expectation that the (dehydro-isobutyrate) starter of the melithiazol biosynthesis is derived from the amino acid valine, deuterated valine was fed to culture broth and incorporation rates of of about 7 % were detected. Example 5 shows an experimental setup which was used by the inventors. It could also be shown by the inventors that the three methoxy groups are derived from S-Adenosyl-Methionine. The transfer of the methyl groups is achieved by the melK gene, which is therefore a SAM dependent methyl transferase. A feeding experiment with [¹³CH₃]-Methionin is shown in example 4.

Feeding experiments showed that the amid-N of myxothiaziol is derived from glycine. Because of the specificity code of adenylation domains it can be assumed that MelG also activates glycine. Example 6 shows an experimental setup used by the inventors for feeding ¹⁵N-glycine and Table 3 refers to the specificity conferring amino acids in the amino acid binding pockets of the adenylation domains.

The inventors could furthermore show that MelJ is a protein with all characteristics of the nitrilase superfamily of proteins, which includes the amidases. The active site cysteine can be found in position 145 and the catalytic triad glutamate₄₄-lysinen₂-cysteineι₄5 is responsible for hydrolysis (Pace and Brenner, 2001). Figure 5 shows an alignment of MelJ with five proteins of the nitrilase superfamily.

It was also possible to show by homology searches that MelK is a SAM dependent methyltransferase with a poorly conserved SAM binding site. Example 10 describes more in detail the analysis of the database amino acid sequence alignment of MelK-related sequences shown in Figure 6.

The present inventors generated a variety of probes from fragments of the genes mtaEFG of the myxothiazole biosynthetic gene cluster from S. aurantica DVV4/3- 1 , described in Silakoski et al., J. Biol. Chem. 274 (1999), 37391-37399. Analysis of the nucleotide sequence determined from two overlapping cosmids surprisingly revealed co-linearity of the myxothiazole biosynthetic genes with the melithiazol biosynthetic genes within a region that turned out to be very similar (melC-melG) as judged by the structural similarities of the two molecules.

The nucleic acid molecules of the invention, alone or in a combination representing part or the complete gene cluster involved in the synthesis of melithiazol will now allow the convenient recombinant production of melithiazols or building blocks or precursors thereof in suitable host cells. It is, in accordance with the invention, important to note that whereas the recombinant production of melithiazol employing the complete gene cluster is particularly advantageous, certain polypeptides encoded by single genes of said gene cluster may also advantageously be used, for example, in biocombinatorial experiments carried out, for example, in bacteria of the genera Melittangium or Stigmatella or in the synthesis of building blocks or precursors.

In one embodiment, the following genes are used:

a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1 (Mel B); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 (Mel C); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 3 (Mel D); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 4 (Mel E); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 5 (Mel F); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 6 (Mel G); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 7 (Mel H); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 12 (ORF 5/melJ); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 13 (ORF 6/melK) and a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 9 (ORF 2). In a further embodiment, a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1 (Mel B); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 (Mel C); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 3 (Mel D); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 4 (Mel E); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 5 (Mel F); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 6 (Mel G); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 7 (Mel H); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 12 (ORF 5/melJ); and a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 13 (ORF 6/melK) were used.

The genes involved in the synthesis of melithiazols are termed, in accordance with the invention "mel" genes. The corresponding (poly)peptides are termed "Mel" (poly)peptides/proteins. The proteins/polypeptides have been assigned the following functions:

Mel B comprises the following domains: KS, AT and ACP. The AT domain is responsible for loading the acyl carrier protein (ACP) with isobutyryl-CoA. The ACP is responsible for the forwarding of the intermediate.

Mel C comprises the following domains: HC, A, PCP (peptidyl carrier protein) and Ox. The HC domain is necessary for the coupling of cysteine with the starting unit and the catalysis of the ring closing reaction. The A domain is necessary for the activation of cysteine and the loading of the PCP. The PCT domain is necessary for the forwarding of the intermediate.

Mel D comprises the following domains: HC, A, Ox, A, PCP, KS, AT, DH, KR and ACP. The HC domain is necessary for the coupling of cysteine with the above intermediate. The A domain is required for the activation of cysteine and the loading of the PCP. PCP is necessary for the forwarding of the intermediate. The KS domain is required for coupling the extender unit with the above-mentioned intermediate. The AT domain is required for loading of ACP with malonyl CoA. The DH domain is necessary for the catalysis of the hydratization of the β- hydroxy intermediate. The KR domain is required for the reduction of the β- ketoacyl intermediate to the secondary alcohol. The ACP domain is required for forwarding the intermediate.

Mel E comprises the following domains: KS, AT, OMT, KR and ACP. The KS domain is required for coupling the extender unit with the above-mentioned intermediate. The AT domain is required for loading the ACP with methyl malonyl CoA. The OMT domain is required for methylating the β-OH-intermediate. The KR domain is required for the reduction of the β-ketoacyl intermediate to the secondary alcohol.

Mel F comprises the following domains: KS, AT, OMT and ACP. The KS domain is required for coupling the extender unit with the above-mentioned intermediate. The AT domain is required for loading the ACP with malonyl CoA. The OMT domain is required for methylating the β-OH-intermediate. The ACP domain is required for forwarding the intermediate.

Mel G comprises the following domains: C, A, MonoOx, A, PCP and TE. The C domain is required for coupling the above-mentioned intermediate with the amino acid glycine. The A domain is required for activating glycine and loading the PCP. The MonoOx is exptected to be required for hydroxilating the α-carbon of the above-mentioned amino acid and for splitting the instable intermediate thereby producing melithiazol. The TE unit is required for liberating the α-keto acid.

The invention also relates to nucleic acid molecules encoding the above recited domains as well as to (poly)peptides consisting of said domains.

Mel H shows homologies to heptadienisomorases. ORF 5 encodes a hydrolase whereas ORF 6 is expected to encode a methyltransferase. Corresponding proteins are expected to contribute to the derivatisation of the amid into the methylester.

ORF 7 is involved in the ABC transport and confers to the organism producing the fungicide the necessary resistance.

ORF 2 is expected to contribute to the establishment of a double bond in the starting unit.

Deduced functions of ORFs found in the melthiazol bisynthetic gene cluster are also shown in table 2.

In a preferred embodiment of the nucleic acid molecule of the present invention the nucleic acid molecule is DNA. The DNA may be genomic DNA or cDNA. Alternatively, it may be of semisynthetic or synthetic origin.

In another preferred embodiment of the nucleic acid molecule of the present invention the nucleic acid molecule is RNA. More preferably, said RNA is mRNA.

The invention also relates to a gene cluster comprising at least two nucleic acid molecules of the invention.

The gene cluster of the invention may correspond to the naturally occuring gene cluster but may also be an artificially generated gene cluster. In such an artificially generated gene cluster the order and/or orientation of genes may be changed. As is apparent from e.g. Table 1 , some of the genes have overlapping coding sequences. These coding sequences, for example, may be rearranged to have the same orientation and to be spatially separated. It is important to note that the term "gene cluster comprising at least two nucleic acid molecules" also refers to the naturally occuring situation where two coding sequences overlap and are orientated in opposition direction. Thus, each coding sequence, irrespective of whether it overlaps with another coding sequence or not and irrespective of its orientation is considered, in accordance with the invention, as a nucleic acid molecule of the invention. Two overlapping coding sequences are therefore and for the purposes of the invention, also viewed as two different nucleic acid molecules.

In a preferred embodiment of the present invention the gene cluster comprises (a) at least two, preferably three, more preferred up to 5, even more preferred at least 6 and most preferred all of a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1 (Mel B); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 (Mel C); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 3 (Mel D); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 4 (Mel E); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 5 (Mel F); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 6 (Mel G); and a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 7 (Mel H); or (b) at least two, preferably three, more preferred up to 5, even more preferred at least 6 and most preferred all of a nucleic acid molecule comprising SEQ ID NO: 15 (melB); SEQ ID NO: 16(melC); SEQ ID NO: 17(melD); SEQ ID NO: 18(melE); SEQ ID NO: 19(melF); SEQ ID NO: 20(melG) and SEQ ID NO: 21 (melH); or (c) a nucleic acid molecule encoding a variety of (poly)peptides being involved in the synthesis of a melithiazol and having an amino acid sequence that is at least 80%, preferably at least 90% and most preferred at least 99% identical to the amino acid sequences of (a) or (b); (d) a nucleic acid molecule encoding a variety of (poly)peptides being involved in the synthesis of a melithiazol and having the amino acid sequences of (a) or (b) with at least one conservative amino acid substitution; (e) a nucleic acid molecule encoding a variety of (poly)peptides being involved in the synthesis of a melithiazol and having amino acid sequences that are isoforms of the amino acid sequences of any of (a) to (d); and (f) a nucleic acid molecule encoding a variety of (poly)peptides being involved in the synthesis of a melithiazol the complementary stand of which hybridizes under stringent conditions to the DNA molecule encoding the amino acid sequence of any of (a) to (e).

It is understood in accordance with the present invention that the variety of (poly)peptides referred to in items (c) to (f) of this embodiment preferably exactly represents the functions of the (poly)peptides mentioned in items (a) and (b) and are thus functional variants thereof. In other terms, the variety of (poly)peptides necessarily or advantageously performs all functions performed by Mel B, Mel C, Mel D, Mel E, Mel F, Mel G and Mel H. Advantageously, the gene cluster further comprises at least one of the ORFs identified herein above as ORFs 1 to 7 or variants thereof as disclose above and preferably all of ORFs 1 to 7 or variants thereof.

This embodiment is particularly advantageous since it allows the complete or essentially the complete synthesis of melithiazols.

In addition, the invention relates to a vector comprising a nucleic acid molecule of the invention.

The vectors may particularly be plasmids, cosmids, viruses or bacteriophages used conventionally in genetic engineering that comprise the nucleic acid molecule of the invention. Preferably, said vector is an expression vector. Expression vectors e.g. derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus or plasmid, may be used for delivery of the nucleic acid molecules (also referred to as polynucleotides) of the invention into targeted cell populations. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (, 2001). The vectors containing the polynucleotides of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas, e.g., calcium phosphate or DEAE-Dextran mediated transfection or electroporation may be used for other cellular hosts; see Sambrook, supra.

Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions. Preferably, the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic cells. Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDMδ, pRc/CMV, pcDNAI , pcDNA3, the Echo™ Cloning System (Invitrogen), pSPORTI (GIBCO BRL) or pRevTet- On/pRevTet-Off or pCI (Promega).

Further, the invention relates to a vector comprising a plurality of different nucleic acid molecules of the invention. In this embodiment, the same vector carries a variety of the aforementioned nucleic acid molecules of the invention.

Additionally, the invention relates to a vector comprising the gene cluster of the present invention.

The invention also relates to a host cell transformed or transfected with the vector of the present invention wherein said vector carries a nucleic acid molecule of the invention, a plurality of different nucleic acid molecules of the invention or the gene cluster of the invention. The vector according to the first variation of this embodiment may, for example, be used as propagating material for a number of selected genes that may be useful in biocombinatorial experiments. Additionally, these vectors may be the starting point of biocombinatorial experiments in so far as further vectors containing additional genes necessary for the production of secondary metabolites may be introduced into said host cells. The second variation may be used much in the same way as the first variation but may further be used in the production of at least a portion or precursor of a melithiazol. The host cell according to the third variation, i.e. the host cell transformed or transfected with the complete cluster of genes, may in addition to the functions described for the previously described variations, be used for the production or essentially the production of melithiazols. The above does not exclude further applications of the host cells of the invention.

Advantageously, the nucleic acid molecules are expressed after homologous recombination in and stable transformation of the host cell. This holds also true for the following embodiments of host cells.

Namely, the invention also relates to a host cell transformed or transfected with a plurality of vectors of the invention.

In variation to a host cell transformed with a vector comprising different nucleic acid molecules of the invention or the gene cluster of the invention, the host may be transformed or transfected with a plurality of vectors wherein each vector advantageously contains a different gene from the cluster.

In a preferred embodiment of the invention, the host cell is a bacterium.

In more preferred embodiment of the invention, the host cell is a myxobacterium, an actinomycete, a pseudomonad or Escherichia coli.

In a particularly preferred embodiment the host cell of the invention further comprises a nucleic acid molecule encoding a phosphopantetheinyl transferase in expressible form.

This transferase is necessary for the post-translational activation of proteins encoded by the nucleic acid molecules of the invention. The ACP and PCP domains present in these proteins affected by the transferase have been discussed above. Such transferases are present in host cells such as Melittangium or Stigmatella host cells and are available in the art. Acyl carrier proteins (ACP) and peptidyl carrier proteins (PCP) have to be converted from the inactive apo- into the active holo-form. This activation is achieved by the attachment of the 4-phosphopantetheine cofactor to an highly conserved serine residue (see R.H. Lambalot et al., Chem. Biol. 3 (1996) 923-936).

For further guidance, see, for example, Sanchez et al., Chem. Biol. 8 (2001), 725-728.

The invention also relates to a method of producing a melithiazol or a precursor of melithiazol comprising culturing the host of the present invention under suitable conditions and isolating the melithiazol produced.

Culturing conditions rely on the particular host cell that is used for the production of the melithiazols. For example, if Stigmatella or Melittangium is used as a host culture conditions may be employed as described in this specification. If E.coli is employed as a host, conventional conditions such as described in Sambrook, loc. cit., may be used. The product may be obtained after breaking up the bacteria according to conventional procedures; see Sambrook et al., loc. cit. For example, it may be obtained by extraction of the bacterial cell mass, culture supernatant or whole broth with organic solvents or adsorber resins (see Bόhlendorf B., et al., (1999), Eur. J. Org. Chem. 2601 - 2608). Optionally, the product is exported from the cells.

The invention furthermore relates to a method of producing a (poly)peptide involved in the synthesis of a melithiazol comprising culturing the host of the present invention under suitable conditions and isolating the (poly)peptide produced.

As regards the culturing conditions employed, it is referred to the previous embodiment. The proteins produced can either be isolated from the cells or exported into the culture medium. In both cases, purification can be achieved by conventional methods involving, for example, ion exchange, affinity and size chromatographic methods.

The invention, in an additional embodiment, relates to a (poly)peptide encoded by the nucleic acid molecule of the invention or produced by the aforementioned method of the invention.

In addition, the invention relates to a method of improving a pharmacological property of a melithiazol comprising the steps of (a) replacing, in a host cell transformed as transfected with the vector of the invention that comprises either the different nucleic acid molecules of the invention or the gene cluster of the invention, or in the host cell of the invention that further comprises a nucleic acid molecule encoding the aforementioned transferase in expressible form, at least one nucleic acid molecule of the invention by a corresponding nucleic acid molecule from a heterologous source; and (b) testing for the desired improvement of said pharmacological property. The desired improvement in this and the following embodiments is tested, advantageously, vis-a-vis the corresponding pharmacological property of naturally occuring melithiazol that may be obtained as described herein above. The properties may, also in the other embodiments of the method of the invention, be tested in an appropriate mouse model.

This embodiment of the invention represents one advantageous option of performing biocombinatorial experiments for the improvement of the pharmacological properties of melithiazols. In general and for example such biocombinatorial experiments have been described for the improvement of other metabolites in Hutchinson and McDaniel, Curr. Opin. Investig. Drugs, Dec; 2 (12) (2001), 1681-1690; Yoon et al., Chem. Biol. Feb; 9 (2), (2002) 203-214; Floss, J. Ind. Microbiol. Biotechnol., Sep; 27 (3), (2001), 183-194; Rodriguez and McDaniel, Curr. Opin. Microbiol. Oct; 4 (5), (2001), 526-534. The principles of exerting biocombinatorics described in these documents can be mutatis mutandis applied to the improvement of melithiazols as well. For example, if the host cell is a bacterium of the genus Melittangium, a nucleic acid molecule from the genus Stigmatella may be used to replace in the host cell a nucleic acid molecule known or suspected to confer the same or a similar function in the synthesis of a structurally similar or dissimilar metabolite. Of course, more than one nucleic acid sequence may be replaced. Usually, after each replacement step the host cell will be monitored for the properties of the new product. Similarly, Stigmatella may represent the host that receives nucleic acid molecules from another organism such as Melittangium lichenicola .

Corresponding nucleic acid molecules from heterologous sources include nucleic molecules that encode homologous proteins/(poly)peptides, i.e. proteins/(poly)peptides having the same or a corresponding function in a different organism. In an particularly preferred embodiment of the invention, at least one cysteine in melithiazol is replaced by a serine. As a consequence, one of the thiazole rings is replaced by an oxazole ring. This brings about improved characteristics such as improved activity. The test for the desired pharmacological activity depends on the specific activity tested and can be designed by the skilled artisan without further ado. Testing includes methods adapted to high throughput analysis.

The invention further relates to a method for improving a pharmacological property of a melithiazol comprising the steps of (a) subjecting, in a host cell transformed or transfected with the vector of the invention that comprises either the different nucleic acid molecules of the invention or the gene cluster of the invention, or in the host cell of the invention that further comprises a nucleic acid molecule encoding the aforementioned transferase in expressible form, at least one nucleic acid molecule of the invention to mutagenesis; and (b) testing for the desired improvement of said pharmacological property.

This embodiment of the invention is a variation of the previously described embodiment that relies on the modification of genes by mutagenesis, rather than on the exchange of genes by different genes, and the subsequent testing whether the mutation/any of the mutations has brought about the desired improvement of the pharmaological activity. Methods of mutagenizing genes are well known in the art and described, for example, in Sambrook et al., loc. cit., and Wu & Kaiser, J. Bacteriol. 178 (1996), 5817-5821.

The invention also relates to a method for improving a pharmacological property of a melithiazol comprising the steps of (a) subjecting, in a host cell transformed or transfected with the vector of the invention that comprises either an or the different nucleic acid molecules of the invention, a nucleic acid molecule of the invention to mutagenesis; (b) testing for an improved activity of the (poly)peptide encoded by said nucleic acid molecule; (c) combining said nucleic acid molecule in expressible form in a host cell with the remainder of the nucleic acid molecules present in the gene cluster of the invention or with corresponding nucleic acid molecules from a heterologous source in expressible form; (d) allowing production of the melithiazol; and (e) testing for the desired improvement of said pharmacological property. The host cell used for the production of the metabolite should also comprise a nucleic acid encoding a phosphopantetheinyl transferase in expressible form.

The invention further relates to a method of improving a pharmacological property of a melithiazol comprising the steps of (aa) deleting from or inactivating in a host cell transformed or transfected with the vector comprising the gene cluster of the invention, one or more nucleic acid molecules according to the invention, or (ab) providing a host cell transformed or transfected with the vector of the invention comprising a plurality of different nucleic acid molecules of the invention or with a plurality of vectors comprising each different nucleic acid molecules according to the invention, wherein said plurality of different nucleic acid molecules is less than the genes contained in the gene cluster of the invention and wherein in the host cells further comprise a nucleic acid molecule encoding a phosphopantetheinyl transferase in expressible from; b) allowing for expression of the nucleic acid molecules; c) assessing whether a compound with fungicidal activity is produced; and/or d) assessing whether the compound produced has an improved pharmacological property.

In accordance with this embodiment of the method of the invention, the host cell contains a variety of genes necessary for the production of melithiazol but not all genes. In the absence of at least one of said genes, it can be tested, whether the secondary metabolite produced (if at all produced) confers improved biological properties as compared to the naturally occuring melithiazol. The assessment step referred to in items (c) and (d) can essentially be done in two ways: Either, only one of these steps can be carried out. Thus, only a functional property that demonstrates an improved pharmacological property needs to be tested for without first testing for the presence of the molecule. Of course, the functional activity may also immediately and unambiguously give rise to conclusions with regard to the presence to said molecule. Alternatively, the test for the presence of the fungicidal activity allows the conclusion that a melithiazol derivative has been obtained. Naturally, the experimentator would take care that the host cell cannot produce any other fungicidal activity. The presence of the fungicidal activity in combination with the necessity to use a smaller gene cluster is perse advantageous since a reduction of the number of genes confers metabolic advantages for the microorganism which may thus produce larger amounts of the metabolite.

In a preferred embodiment of the nucleic acid molecule of the present invention the deletion or absence of said nucleic acid molecule results in the absence of the first or second thiazole ring of melithiazol. The remaining thiazole ring may additionally be changed to an oxazole ring as described above. Again, these changes are expected to confer improved characteristics such as improved activity. In addition, it is of advantage to have a smaller compound available.

The invention also relates to a method of improving a desired property of or conferring a desired property to a secondary metabolite different from melithiazol comprising the steps of (a) providing a host cell comprising a gene cluster or a part of a gene cluster wherein the gene products of said gene cluster are involved and sufficient for the synthesis of said secondary metabolite; (b) transforming or transfecting said host cell with at least one vector of the invention comprising a plurality of different nucleic acid molecules of the invention or with a plurality of vectors comprising each different nucleic acid molecules according to the invention, wherein said plurality of different nucleic acid molecules is less than the genes contained in the gene cluster of the invention; (c) allowing for expression of the nucleic acid molecules; (d) assessing whether a secondary metabolite is produced; and/or (e) assessing whether the secondary metabolite produced displays the desired property or the improved desired property.

This embodiment is another option of carrying out a biocombinatorial synthesis of secondary metabolites. In host cells, for example, of the genus Stigmatella, existing gene clusters may be targeted or combined with nucleic acids found in accordance with the present invention. In so far, the secondary metabolites produced by the host cell may be improved. Alternatively, the host cells may comprise a genetically engineered cluster of genes where at least one of the naturally occuring genes has been deleted or replaced by a homologous gene from another organism. By introducing at least one nucleic acid molecule of the invention in expressible form into said host cells, the genes existing in said host cells may be replaced or supplemented by the newly incoming genes. This approach can also be used to select for metabolites with improved properties.

The desired property may be any desired property including a pharmacological property. Depending on the property that is desired, the test assay will be designed by the person skilled in the art. Generally, such test assays can be taken from the literature and require no undue burden even if they need to be adapted for the purposes of the present invention.

In a preferred embodiment of the method of the invention, said pharmacological property is selected from an improved range of fungicidal activity, and lower toxitity for the human or animal patient.

In a further preferred embodiment the method of the invention further comprises modifying the melithiazol as a lead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophylic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines or combinations thereof.

The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch-Analysis and Related Approaches", VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Deutsche Apotheker Zeitung 140(8), 813-823, 2000).

The invention further relates to a melithiazol obtainable by the method of the present invention.

The melithiazol obtained or obtainable by the method of the invention is an improved version of the natural compound. Since the modification relies in the production by recombinant means which were not available prior to the present invention, these improved melithiazols are novel and could also not be provided by the prior art without inventive skills. The improved melithiazols will find wide applications as fungicides.

In addition, the invention relates to a composition comprising the melithiazol of the invention.

The composition may further comprise packaging material such as a vial, appropriate storage buffers and a manual of instruction. The composition may be formulated, for example, as a spray and be useful in extinguishing contaminating fungi in laboratories, house holds etc.

Preferably, the composition is a pharmaceutical composition. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and/or diluent. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg, however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The invention also relates to a method of producing a pharmaceutical composition comprising the steps of the method of the present invention of producing and/or improving melithiazol or a secondary metabolite different from melithiazol and further the step of formulating the melithiazol or secondary metabolite thus obtained with a pharmaceutically acceptable carrier or diluent.

The formulation step may be effected to methods well known in the art.

The figures show:

Figure 1 : Chemical formula of melithiazol.

Figure 2: DNA sequence comprising the melithiazol biosynthetic gene cluster.

Figure 3:

3A: Schematic diagram showing the chromosomal integration of melJK from M. lichenicola Me I46 into S. aurantiaca DW4/3-1

Genes are shown as arrows, the homologous region from mtaH used for integration is hatched. P indicates the presumed bi-directional promotor regulating the transcription of melJK. Fsp\ shows the position of the recognition sequence of the respective restriction enzyme.

3B: Geneotype of the merodiploid strain ESW602 resulting from integration of pESW243 into the chromosome of S. aurantiaca DW4/3-1

3C: Southern blot analysis of ESW602 and S. aurantiaca DW4/3-1 after restriction with Fsp\

A DNA fragment containing the promotor region of melJK, melJK and melH was used as probe (3.2 kb). The size of the hybridizing fragments was estimated using DIG labelled DNA molecular weight marker III (Roche Diagnostics). Hybridizing fragments and their sizes are indicated in 6A and 6B. S. aurantiaca DW4/3-1 shows a signal, because mtaH and melH are very similar on the DNA level (78,6 % identity over 680 bp).

Figure 4: HPLC analysis of the extracts from S. aurantiaca DW4/3-1 , mutant ESW602 and the myxothiazol A and Z producer M. fulvus Mx f333/8

Figure 5: Catalytic triade described for the nitrilase superfamily (Pace and Brenner, 2001) .

Five proteins of the nitrilase superfamily are aligned with MelJ. Amino acids shown in bold are conserved in all proteins, whereas shaded amino acids are found in at least four of them. Amino acids forming the catalytic triade are marked with an asterisk. The corresponding regions of nitrilases from Arabidopsis thaliana (NIT3), Fusarium lateritium (CHY1), Gloeocercospora sorghi (CHT), Klebsiella pneumoniae (BXN) and Streptococcus pneumoniae are shown.

Figure 6: Alignment of the MelK-subfamily of SAM dependent methyl transferases

Kagan and Clarke motifs are indicated by red (motif I), orange (post I), yellow (motif II), and blue (motif III) triangles. Black triangles indicate residues binding the SAM-cofactor in 1JG4 via side-chain atoms, while magenta boxes indicate residues forming less sequence specific backbone or hydrophobic contacts to the cofactor in 1JG4. The absolutely conserved Gly in position 110 in 1JG4 is not in direct contact to the cofactor. Amino acid sequences used for the alignment were derived from the following organisms (database entries in brackets): Mycobacterium leprae (Q9X7D5), Homo sapiens LCMT (see text), Caenorhabditis elegans (YKG4_CAEEL), Streptomyces glaucescens (TCMP_STRGA), Pseudomonas aeruginosa (Q9I2N5), Rhizobium meliloti (Q92Y12), Mycobacterium tuberculosis (YX99_MYCTU), Streptomyces cqelicolor (Q9ZBH0), Actinosynnema pretiosum ASM 10 (see text), Streptomyces coelicolor (Q9FBX1), Anabaena sp. (Q8YKG6), Methanosarcina acetivorans (Q8TLW9), Methanosarcina mazei (Q8PWV7), Mycobacterium tuberculosis (033293), Oceanobacillus iheyensis (BAC13195), Bacillus subtilis (Q45500), Arabidopsis thaliana (Q93YR1), Oryza sativa (AAN65019), Rhizobium loti (Q98LR1), Pseudomonas aeruginosa (Q9HX18), Archaeoglobus fulgibus (PIM1_ARCFU), Methanococcus jannaschii (PIMT_METJA), Pyrococcus horikoshii (PIMT_PYRHO), Aeropyrum pernix (PIMT_AERPE), Pyrococcus furiosus (1 JG4).

The examples illustrate the invention.

Examplel: Cloning of ORFs involved in melithiazol synthesis

The chemical structures of myxothiazol and melithiazol show certain similarities. A biosynthetic comparison of the two fungicides was initiated, because of the following reasons. Myxothiazol biosynthesis starts with a leucine derived 3- methylbutyryl-CoA starter unit, whereas at the beginning of melithiazol biosynthesis an oxidated isobutyrate of unknown origin is used (see below). In the central part of the molecule, myxothiazol contains a bisthiazole moiety that is formed via the incorporation of two cysteins into the polyketide backbone. Although two putative oxidation domains have been identified, it remains unclear how the oxidation of the assumed thiazoline intermediate is performed. Melthiazol shows a thiazoline-thiazole moiety. The comparison of the corresponding genes of the melithiazol biosynthetic gene cluster should give insight into ring-oxidation. The next part of the molecules are identical, so screening probes were derived from the corresponding DNA-regions (mtaE and mtaF, see below). Myxothiazol terminates in an amide, while Melithiazol features a methyl-ester. Melittangium lichenicola Me I 46, the organism which produces melithiazol, was cultivated in M7 medium (0,5 % Probion ME 069, 0,1 % CaCl₂ * H₂0, 0,1 , Mg₂S0₄ * 7H₂0 , 0,1 % yeast extract, 0,5 % soluble starch, 1 % HEPES, pH adjusted to 7,4 with 20 % KOH, supplemented with 0,2 % glucose and 0,1 mg/ml cyanocobalmin after sterilisation). This strain is only exemplarily employed. The skilled person may also use other organisms, e.g., of the same species, which advantageous would be tested for the production of melithiazol beforehand. 100- ml batch cultures in 250 ml Erlenmeyer flasks were incubated at 30°C on a gyratory shaker at 160 rpm for about 3 days. After harvesting the cells chromosomal DNA of M. lichenicola Me I 46 was isolated as described (Neuman, B., Pospiech A. & Schairer H. U. (1992) Trends in Genetics 8, 332-333) and used for the generation of a cosmid library as described (Silakowski, B., Schairer, H. U., Ehret, H., Kunze, B., Weinig, S., Nordsiek, G., Brandt, P., Blδcker, H., Hofle, G., Beyer, S. & Mϋller, R. (1999) J. Biol. Chem. 274, 37391-37399). The resulting cosmid library consisted of 1920 clones, which were screened for the presence of melithiazol biosynthetic genes using fragments of genes mtaEFG of the myxothiazol biosynthetic gene cluster from S. aurantiaca DW4/3-1. The probes were derived from the regions coding for the putative O-methyltransferases of MtaE and MtaF, respectively the adenylation domain of MtaG. For the amplification of the probes the following primers were used: Primer METE1 5^'- cag age teg agg tea tgt tgc agt cgc-3', Primer METE4 5^'-gct eta gat gag ccc gaa gcg ctt gga c-3' (primer pair 1), Primer FSW1 5'-agg tgg ggc cga age cga cgt tg , Primer FSW4 5'-gga tgc cgt gca ggt get tct-3^'(pimer pair 2), Primer GMOX1 5^'- gga tga acg egg aaa ccg tct-3^', Primer GMOX4 5^'-agg cgc age gcg gat teg at-3^' (primer pair 3).

A standard PCR was carried out using Taq polymerase (Life Technologies, Inc) according to the manufacturers protocol.

Cosmids M1 and M2 gave signals with the mtaE and mtaF derived probes, whereas cosmid M2 additionally hybridizied with the mtaG derived probe. Analysis of the nucleotide sequence determined from the two cosmids revealed an overlap of 5372 bp, starting at position 34039. Comparison with the myxothiazol gene cluster showed colinearity of the melithiazol biosynthetic genes within the melC-melG region as judged by the structural similarities between the two molecules.

Comparison on the Protein level Identity (%) MtaB / MelB

KS₂-AT₃ / KS-AT 66%

ACP₃ / ACP 69%

MtaC / MelC 63%

MtaD / MelD 60%

MtaE / MelE 66%

MtaF / MelF 69%

MtaG / MelG 70%

MtaH / MelH 74%

Example 2: Functional analysis of ORFs involved in melithiazol synthesis.

Analysis of the open reading frames (ORFs) found is presented in table 1. The domain structure of each gene is given. ORF2 is most likely involved in the oxidation of the isobutyryl starter molecule that is used for melithiazol biosynthesis resulting in the exo-double bond in the molecule. Isobutyryl-CoA was identified as starter molecule in feeding experiments with deuterated valine, which resulted in approximately 7% incorporation into melithiazol after degradation of valine into isobutyryl-CoA. MelB loads isobutyryl-CoA onto the PKS. MelC-MelG produce the backbone of the molecule in analogy to the myxothiazol structure. The adenylation domain of MelC activates Cysteine and attaches it on the PCP domain. Under the catalysis of the heterocyclisation domain the thiazoline-ring is formed. The oxidation domain within melC is inactive. In the next round of chain elongation performed by MelD another cysteine is incorporated. In case of the second heterocycle the assumed thiazoline-intermediate is oxidated to an thiazole-ring. MelD also contains a PKS- part, which extends the backbone with an acetate unit. MelE incorporates an propionate unit and mythylates the β-hydroxy-intermediate. The last building block of the molecule is attached by MelF. MelG probably catalysis the formation of the amide. The methyl-ester in melithiazol is subsequently produced via the action of ORF5 and ORF6, which methylate the imine-form of the amide and subsequently hydrolyse this intermediate. The origin of the methyl group in the ester was established using labelling experiments employing ¹³C-methionine and subsequent NMR analysis of the isolated melithiazol. The formation of the methyl ester has been analysed in a myxothiazol Z producing strain, which generated the methyl ester from the myxothiazol A amide (Steinmetz, H., Forsche, E., Reichenbach, H. & Hofle, G. (2000) Tetrahedron 56, 1681-1684). ORF7 is assumed to be an ABC transporter involved in the export of the compound out of the cell.

Example 3: Heterologous expression of melJ and melK

Hydrolysis of cosmid M1 with Stu\ results in four DNA-fragments with sizes between 3.1 and 3.2 kb. The target fragment harbours at its 5'-end a 309 bp deletion of melH and the complete genes melJ und melK. Additional 670 bp in front of melK presumably harbour the promotor region regulating the transcription of melJK. For heterologous expression of melJ and melK in S. aurantiaca DW4/3-1, the mixture of 4 fragments was subcloned into pCR^®-XL-TOPO (Invitrogen) after restriction of cosmid M1 and extension of the fragments using Taςr-DNA-polymerase. Resulting clones were analyzed by restriction and sequencing resulting in the identification of plasmid pMSW12 harbouring the 3264 bp target fragment. To allow homologous recombination in S. aurantiaca DW4/3-1 via mtaH, the insert of pESW26 was isolated after digestion with NotUSpel and cloned into Not\IXba\ predigested pMSW12, which resulted in pESW243. The construction of the above mentioned plasmids is shown in table 4.

Example 4: Feeding experiment employing labelled methionine: Methionin

1000 ml of M7-medium in 21 Erlenmeyer flasks was inoculated with 50 ml of a culture of M. lichenicola Me I46 (3 days old) and grown at 30°C (170 rpm in a gyratory shaker). 10 mg of [¹³CH₃]-methionine (Campro Scientific, Veenendaal, The Netherlands) were dissolved in 10 ml water and sterilized using a membrane filter. The compound was pulse-fed as follows: 6 mg after 24h, 2 mg each after 48 and 72h. 1 % (v/v) XAD-adsorber resin (Rohm & Haas, Germany) was added after 72h. After incubating for 96h, cells and resin were harvested by centrifugation and the pellet was extracted and analyzed as described previously by ¹³C-NMR (Bόhlendorf et al., 1999). Incorporation rates were calculated by comparison to an internal standard.

Example 5: Feeding experiment employing labelled valin: d8-DL-Valin

50 ml M7-medium in 250 ml Erlenmeyer flasks were inoculated with 2 ml of a culture of M. lichenicola Me I46 (3 days old) and grown at 30°C (170 rpm in a gyratory shaker). 15 mg of deuterated d8-DL-Valin (Cambridge Isotope Laboratories, Andover, USA) were added in equal portions after 24h, 48 and 72 h. After 72 h, XAD-adsorber resin was added (1 % (v/v) and cells were grown for another 24 h. Subsequently, cells and resin were harvested by centrifugation and extracted twice (first using 30 ml methanol and then with 20 ml acetone). Combined extracts were dried under vacuo and redissolved in 500 μl methanol. The analysis was performed after injection of 5 μl of the extract into a HPLC-MS.

Example 6: Feeding experiment employing ¹⁵N-Glycine, ¹⁵N-Glutamate and ¹⁵N-Ammonium Chloride

S. aurantiaca DW 4/3-1 was inoculated in 20 ml tryptone-starch medium (Mahmud et al., 2002) and cultivated at 28 °C (200 rpm in a gyratory shaker) for two days. 10 ml of the culture was then transferred to a 500 ml Erlenmeyer flask containing 200 ml tryptone-starch medium and the flask was shaken at 30 °C (160 rpm). 30 mg of each of the labelled precursors (¹⁵N-Glycine, ¹⁵N-glutamate and ⁵N-ammonium chloride) was dissolved in 2 ml water and sterilized through a 0.22 μm diameter pore size ultrafilter (Millipore, Millex-GV4). The labelled precursors were pulse-fed in two equal portions 24 and 48 h after inoculation. The cells were harvested after four days of cultivation and the products were extracted with acetone. Myxothiazol was purified by silica gel column chromatography (n-hex:EtOAc 5:1 ~ n-hex-EtOAc 1 :1 , EtOAc) and subsequently by HPLC (YMC-Pack ODS-AQ, 250x10 mm, methanol-water 82:18). The incorporation rate of ¹⁵N into myxothiazol was determined using selected ion monitoring (SIM) in a electrospray mass spectrometer. Example 7: Model for the melithiazol biosynthetic pathway

The biosynthesis switches from PKS type biochemistry (MelB) to NRPS (MelCD), back to PKS (MelDEF) and finally back to NRPS (MelG). MelD belongs to the few known proteins in which a NRPS module is covalently linked to a PKS module, which makes it an ideal target to study PKS/NRPS interaction. The modular structure of type I PKSs usually starts with an AT or a CoA-ligase domain responsible for the recognition (and, in the case of CoA-ligases, for activation) of the starter molecule followed by transfer of the activated substrate to the first ACP domain (compare the biosynthetic gene clusters of erythromycin (Donadio et al., 1991), rapamycin (Molnar et al., 1996; Schwecke et al., 1995) and rifamycin (August et al., 1998). Alternative starters can be used to initiate the biosynthesis of the polyketides but most frequently acetyl-CoA, malonyl-CoA, propionyl-CoA or methylmalonyl-CoA are employed. If activated dicarboxylic acids are used, modified KS domains can be found at the beginning of the first module. These have lost their condensation activity but effectively decarboxylate the ACP-bound dicarboxylic acid giving rise to the starter moiety. Because the active site cysteine of these KS domains is mutated to glutamine, they have been designated KSo-domains (Bisang et al., 1999). In the case of melB, the modular organisation looks similar: the protein starts with a KS domain that is followed by an AT and an ACP domain.

Several myxobacterial PKSs show an atypical arrangement starter modules with ACP-KS-AT-AT-KR-ACP (Gaitatzis et al., 2002; Ligon et al., 2002; Silakowski et al., 2001b; Silakowski et al., 1999). It has been shown that in these systems the first AT is responsible for loading of the starter molecule, whereas the second AT loads the first extender unit ((Wilkinson et al., 2001) and PF Leadlay, CJ Wilkinson, SW and RM unpublished). Different melithiazols have been described but M. lichenicola Me I46 only produces melithiazol A (and melithiazol C, which is presumably a degradation product), which is characterized by a dehydro-isobutyrate starter molecule (Bδhlendorf et al., 1999). Myxobacteria frequently employ activated short branched chain carboxylic acids as starter molecules for PKS, e.g. isobutyrate and 2-methyl-butyrate in myxalamid and isovalerate in myxothiazol biosynthesis (Silakowski et al., 2001b; Silakowski et al., 1999; Trowitzsch-Kienast et al., 1986). Although these starter molecules are generally thought to be derived from branched chain amino acids by amino acid degradation, it has recently become clear that in myxobacteria a novel branch of the mevalonate pathway is used as an alternative source of at least isovalerate (Mahmud et al., 2002). It is possible that the dehydro- isobutyrate starter of melithiazol is derived from valine degradation and subsequent oxidation. In order to verify this hypothesis, deuterated valine was fed to the culture broth and incorporation rates of about 7% were detected using LC-MS analysis (data not shown). In addition, a mass increase of +4 was detected, which indicates that valine is also degraded to isobutyrate and then oxidized to methyl-malonate in M. lichenicola Me I46, which resembles the metabolic situation in streptomycetes (Reynolds et al., 1988; Vrijbloed et al., 1999). As melithiazol is assembled using one methyl-malonate unit (activated by the AT of MelF, see below), this pathway reasonably explains the presence of the +4 peak, because the incorporated extender unit would be labelled with four deuterii (one located at carbon 2 and three at the methyl branch of the propionate extender). The active site cysteine is clearly present in position 189 of the KS of MelB, which indicates that it does not represent a KSQ domain. Since it might be possible that the AT domain loads isobutyryl-CoA onto the ACP of MelB and because there is no indication from the primary sequence that the KS is inactive, the function of the MelB-KS domain cannot unambiguously be characterized. It is not clear either on which stage of the biosynthesis the oxidation of the isopropyl-moiety occurs; ORF2 is the protein most likely involved, because it resembles amino acid oxidases. The oxidation might be performed during each step from the amino acid to the ACP- bound isobutyrate.

Next, MelC activates cysteine (the A-domain perfectly matches the nonribosomal code (Challis et al., 2000; Stachelhaus et al., 1999)), performs the first heterocyclization and transfers the thiazoline-intermediate to MelD. Interestingly, MelC contains an Ox-domain located behind the PCP, which is similar to the structure of MtaC. Nevertheless, M. lichenicola Me I46 is only known to produce thiazoline-thiazole type melithiazols, which raises the question of the function of this Ox-domain in both biosynthetic systems. Further biochemical experiments are described below. MelD is similar to MtaD and harbours two modules; one NRPS module with another Ox-domain that is inserted in between the core motifs of A- domains A8 and A9 instead of the localization behind the PCP in MelC. The thiazoline-thiazole intermediate is then transferred to the PKS module of MelD, which extends with malonate and reduces the β-keto-thioester to its enoyl-form. Large spacer regions (S) are located between the DH and the KR domains of the PKS modules of MelD and between the O-MT and KR domains of MelE. These have been described for almost all PKS and fatty acid biosynthetic systems (Cane, 1997; Smith, 1994) but their function is not clear. S regions can also be found in all myxobacterial PKS systems sequenced so far and show an identity of up to 35% on the amino acid level.

MelE is another enzyme; in between the AT domain and the spacer region, an O- MT domain can be found, which has only been reported in two other biosynthetic systems, both from myxobacteria (Gaitatzis et al., 2002; Silakowski et al., 1999). This domain is assumed to methylate the intermediate after ketoreduction giving rise to the methyl-ether. The O-MT domain is similar to those of MelF, MtaE, MtaF, StiD and StiE and shows the typical primary amino acid sequences involved in SAM binding (Kagan and Clarke, 1994). Feeding experiments employing ¹³C-labelled methionine were performed, the results of which prove that both O-MT domains employ SAM as cofactor (incorporation rates for the three SAM derived methyl groups in melithiazol were between 16 and 21 %). The methylmalonate extended intermediate is transferred to MelF, which activates malonate. Again similar biochemistry is performed in comparison to the unique myxothiazol system: An O-MT domain gives rise to the methylation of the enol-form of the β-keto intermediate bound to the ACP. Interestingly, the O-MT sequences of MelF and MtaF show more identities than those of MelE and MelF, which indicates that they are specialized for different biochemical reactions.

MelG resembling MtaG was found at the end of the PKS/NRPS part of the gene cluster. MtaG is believed to be involved in the formation of the terminal amide of myxothiazol. Thus, genes responsible for methyl-ester formation might start from the thioesterified myxothiazol-acid bound to the ACP of MelF. It was therefore concluded that melithiazol is formed via the amide form of the melithiazol-acid. Example 8: Establishing the biosynthetic precursors of the terminal methyl- ester and amide functionalities by feeding experiments

The terminal gene of the myxothiazol cluster (mtaG) resembles an unusual NRPS with an integrated monooxygenase (Monoθx)-domain. MtaG might therefore be involved in the formation of the terminal amide via the addition of an extra amino acid to the MtaF-ACP bound myxothiazol acid. Subsequently, the carbon backbone of this amino acid is removed by the action of the monooxygenase domain within mtaG giving rise to the terminal amide structure (Silakowski et al., 1999).

Terminal amides are found in many bioactive substances from mammals and insects, including many peptide hormones (Kulathila et al., 1999; Schoof et al., 1997). Generally, they arise from the oxidative cleavage of C-terminal glycine- extended precursors by a bifunctional copper-zinc dependent enzyme, peptidylglycine α-amidating monooxygenase (PAM) (Kulathila et al., 1999; Schoof et al., 1997). The process requires two reaction steps, the first involves ascorbate- and copper-dependent aerobic hydroxylation of the glycine α-carbon by peptidylglycine α-hydroxylating monooxygenase (PHM) and the second step, the cleavage of the resulting carbinolamide, is catalyzed by a zinc-dependent enzyme, peptidylamidoglycolate lyase (PAL). We assumed that the monooxygenase in MtaG would perform a similar reaction and hydroxylate the amino acid extended myxothiazol in α-position, which could result in an intermediate that can fall apart spontaneously (or catalyzed by MtaG), giving rise to myxothiazol and glyoxylic acid thioesterified to the PCP of MtaG. The thioesterase at the C- terminus of MtaG would thus be needed to release the glyoxylate from the PCP to deblock the enzyme.

To strengthen this hypothesis, we tried to identify the nitrogen source of the terminal amide of myxothiazol in a series of incorporation experiments. The putative donor amino acids were chosen after analysis of the MtaG-region responsible for the specificity of A-domains for their putative binding pocket (Challis et al., 2000; Stachelhaus et al., 1999), which is rather difficult in MtaG (and MelG as well) due to the insertion of the monooxygenase domain next to the binding pocket. Nevertheless, both amino acid regions were indicative of glycine as the amino acid used. The feeding of ¹⁵N-labelled glycine might therefore result in incorporation of the label into myxothiazol if the above mentioned theory is correct. Indeed, using mass spectrometry of isolated myxothiazol, the result of the feeding experiment showed 40.6 % incorporation of ¹⁵N-glycine into the compound, whereas ¹⁵N- labeled glutamate and ammonium chloride were not incorporated at all. This suggests that the terminal amide of myxothiazol is derived directly from an extra glycine that is intermediary attached to the molecule. Interestingly, serine has been shown to be the nitrogen source of the terminal amide of nosiheptide (Mocek et al., 1993), which indicates a similar mechanism for amide formation in the producer Streptomyces actuosus and possibly for other natural products with terminal amide moieties as well (e.g thiostreptone).

Currently, two more examples of NRPS with inserted MonoOx-domains can be found in the databases (hypothetical proteins from Nostoc punctiforme and Ralstonia solanacearum, which show the domain structure A-MonoOx-PCP-KS- AT-ACP-Aminotransferase-C-A-PCP— and — KS-AT-ACP-Aminotransferase- MonoOx-C-A-PCP— , respectively). None of these proteins seems to be involved in terminal amide formation, because the MonoOx-domains are not inserted into C-terminal modules. Neither are they located within an A-domain. Nevertheless, both proteins belong to the class of PKS/NRPS hybrids. In contrast, the unusual arrangement of domains in MtaG can be found in MelG as well. It is thus reasoned that melithiazol in close biosynthetic analogy is also formed via the amide intermediate. This would represent a unique mechanism to generate a methyl-ester, because the glycine extender has to be removed completely in order to generate the ester. It seemed likely that melithiazol is biosynthesized by a methyl transferase (which would presumably employ SAM as cofactor) and a hydrolase. The gene products of melK and melJ, which are located directly downstream of melG, indeed show some similarities to such enzymes. To verify the dependence of the methyl-ester formation on a SAM dependent enzyme, a feeding experiment employing [¹³CH₃]-methionine was performed and showed incorporation rates of 16-21% into the three methoxy-goups of melithiazol A, which was detected using ¹³C-NMR spectroscopy. Similar results have been obtained in feeding experiments with Myxococcus fulvus Mx f333/8, the producer of the methyl-ester of myxothiazol A (designated myxothiazol Z) (Steinmetz et al., 2000). Example 9: Heterologous expression of the putative methyl transferase MelK and the hydrolase MelJ from the melithiazol gene cluster in the myxothiazol A producer

As outlined in Example 8, MelK and MelJ seemed to be responsible for the formation of the methyl-ester. In addition, it had been speculated in earlier studies employing M. fulvus Mx f333/8 that myxothiazol Z is formed from myxothiazol A via the iminoester-intermediate (Steinmetz et al., 2000). Melithiazol A might also be derived from a melithiazol-amide analogously, which would employ the imino-ester of the amide as an intermediate. Alternatively, one could speculate that the methyl- ester is formed similar to the methylation of bacterial chemotaxis transmembrane receptors, e.g. by the protein methyl transferase CheR and the methylesterase/amidase CheB from Salmonella typhimurium (Djordjevic and Stock, 1997; Koshland Jr., 1988). The alternative route would thus involve the free myxothiazol acid produced by MelJ which is subsequently methylated. MelJ is a protein with all the hallmarks of the nitrilase superfamily of proteins, which includes the amidases. The active site cysteine can be found in position 145 and the catalytic triad glutamate ₄-lysinen2-cysteineι₄5 is responsible for hydrolysis (Pace and Brenner, 2001).

In order to verify the involvement of MelK and MelJ in methyl-ester formation, the operon harbouring both genes and their presumed promotor region was transferred into the myxothiazol A producer S. aurantiaca DW4/3-1. For homologous recombination, the DNA region of MtaH was used, because this gene was known not to be necessary for myxothiazol formation (see below). After transfer of both genes into the myxothiazol producer, the genotype of the mutant strain was analysed by Southern hybridizations verifying the correctness of the integration in mutant ESW602 (see figure 3). ESW602 was analyzed for the production of secondary metabolites which resulted in the detection of myxothiazol A and myxothiazol Z in the culture broth (see figure 4, LC-MS analysis: myxothiazol A: m + H⁺ = 503, myxothiazol Z: m + H⁺ = 488), which clearly establishes that MelK and MelJ are responsible for the transformation of the β-methoxyacrylate-amide into the β-methoxyacrylate-ester.

Example 10: MelK is a SAM dependent methyl transferase with poorly conserved SAM binding site

Homology searches with MelK in Swall using Fasta3 (Pearson and Lipman, 1988) give 41 related sequences, mostly from Mycobacteria, Streptomyces and Methanosarcina with unknown function, together with the methyltransferase Asm10 from the Actinosynnema pretiosum ansamitocin biosynthetic gene cluster. Asm 10 is the only biochemically characterized member of this group of proteins. The recombinant enzyme catalyzes the final step in ansamitocin biosynthesis, the SAM-dependent N-methylation of the amid linkage (Yu et al., manuscript in preparation). No significant similarities to structurally known SAM dependent methyl transferases specific either for small molecule, protein, DNA, or RNA could be detected. For these, it has been demonstrated that they share only little sequence identity but incorporate a highly conserved structural fold and that the cofactor-binding residues are poorly conserved (Martin and McMillan, 2002). In contrast to the O-MT domains of MelE and MelF, which match the formerly proposed core sequences perfectly (Kagan and Clarke, 1994)), these signature sequences are difficult to identify in MelK and the related sequences. For a brain protein phosphatase 2A leucine carboxyl methyltransferase (LCMT) it was shown recently that LCMT uses SAM as cofactor and harbours four regions, which coincide only fairly well with the Kagan and Clarke binding motifs I, II and III and the additional post I motif (De Baere et al., 1999). A profile fittig alignment of the MelK-related sequences with the protein-L-isoaspartate O-methyltransferase from Pyrococcus furiosus (PDB-id 1JG4) and other sequences from the Pimt- branch of SAM-dependent methyltransferases [EC 2.1.1.77] shows reasonable similarity of the motifs identified by Kagan and Clarke in the MelK-related sequences as well, making it plausible to include these enzymes in the large family of SAM-dependent methyltransferases. In the alignment (figure 6) showing MelK-related sequences from diverse organisms together with some typical Pimt methyltransferases the Kagan and Clarke motifs are indicated by red (motif I), orange (post I), yellow (motif II), and blue (motif III) triangles. Black triangles indicate residues binding the SAM-cofactor in 1JG4 via side-chain atoms, which should therefore be sequence-specific, while magenta boxes indicate residues forming less sequence specific backbone or hydrophobic contacts to the cofactor in 1 JG4. Two of the four residues forming side-chain contacts to the cofactor in 1JG4 are only conservatively replaced (Gly/Ala 108 and Glu/Asp 128), all other residues show high diversity, which has already been observed by Martin and McMillan as common to the methyltransferases. The absolutely conserved Gly in position 110 in 1 JG4 is not in direct contact to the cofactor, but on one side of the binding pocket in a position where any side chain would interfere with the cofactor sulfur atom. Thus, it can be expected that MelK, as Asm10 and LCMT, binds the SAM-cofactor similarly to the other methyltransferases and contains the typical SAM-binding fold.

Table 1:

KS: Ketoacylsynthase FMN: Oxidation domain including a flavin binding site

AT: Acyltransferase MonoOx: Monooxygenase domain including a flavin binding site

DH: Dehydratase

ER: Enoylreduktase

KR: Ketoreduktase TE: Thioesterase

ACP: Acyl Carrier Protein

S: Spacer Bp: base pairs

OMT- O-Methyltransferase A: Adenylation domain

HC: Heterocyclisation domain C: Condensation domain

Table 2: Deduced functions of ORFs found in the melithiazol biosynthetic gene cluster

Table 3: Specificity conferring amino acids in the amino acid binding pockets of the A-domains in the mta and the mel biosynthetic gene clusters (Challis et al., 2000; Stachelhaus et al., 1999)

Table 4: Plasmids generated and used in this work

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Claims

Claims

1. A nucleic acid molecule encoding a (poly)peptide involved in the synthesis of a melithiazol, said nucleid acid molecule being selected from the group of

(a) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1 (Mel B); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 (Mel C); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 3 (Mel D); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 4 (Mel E); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 5 (Mel F); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 6 (Mel G); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 7 (Mel H); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 8 (ORF 1); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 9 (ORF 2); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 10 (ORF 3); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 11 (ORF 4); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 12 (ORF 5); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 13 (ORF 6); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 14 (ORF 7);

(b) a nucleic acid molecule having the DNA sequence of SEQ ID NO: 15 (mel B); SEQ ID NO: 16 (mel C); SEQ ID NO: 17 (mel D); SEQ ID NO: 18 (mel E); SEQ ID NO: 19 (mel F); SEQ ID NO: 20 (mel G); SEQ ID NO: 21 (mel H); SEQ ID NO: 22 (ORF 1); SEQ ID NO: 23 (ORF 2); SEQ ID NO: 24 (ORF 3); SEQ ID NO: 25 (ORF 4); SEQ ID NO: 26 (ORF 5); SEQ ID NO: 27(ORF 6); or SEQ ID NO: 28 (ORF 7);

(c) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol and having an amino acid sequence that is at least 80%, preferably at least 90% and most preferred at least 99% identical to the amino acid sequence of (a) or (b);

(d) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol and having the amino acid sequence of (a) or (b) with at least one conservative amino acid substitution;

(e) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol and having an amino acid sequence that is an isoform of the amino acid sequence of any of (a) to (d); and

(f) a nucleic acid molecule encoding a (poly)peptide being involved in the synthesis of a melithiazol the complementary stand of which hybridizes under stringent conditions to the DNA molecule encoding the amino acid sequence of any of (a) to (e).

2. The nucleic acid molecule of claim 1 which is DNA.

3. The nucleic acid molecule of claim 1 which is RNA.

4. A gene cluster comprising at least two nucleic acid molecules of claim 1 or 2.

5. The gene cluster of claim 4 which comprises

(a) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1 (Mel B); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 2 (Mel C); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 3 (Mel D); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 4 (Mel E); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 5 (Mel F); a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 6 (Mel G); and a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 7 (Mel H); or

(b) a nucleic acid molecule having the DNA sequence of SEQ ID NO: 15 (mel B); SEQ ID NO: 16 (mel C); SEQ ID NO: 17 (mel D); SEQ ID NO: 18 (mel E); SEQ ID NO: 19 (mel F); SEQ ID NO: 20 (mel G); and SEQ ID NO: 21 (mel H);

(c) a nucleic acid molecule encoding a variety of (poly) peptides being involved in the synthesis of a melithiazol and having an amino acid sequence that is at least 80%, preferably at least 90% and most preferred at least 99% identical to the amino acid sequences of (a) or (b);

(d) a nucleic acid molecule encoding a variety of (poly)peptides being involved in the synthesis of a melithiazol and having the amino acid sequences of (a) or (b) with at least one conservative amino acid substitution;

(e) a nucleic acid molecule encoding a variety of (poly)peptides being involved in the synthesis of a melithiazol and having an amino acid sequence that are isoforms of the amino acid sequences of any of (a) to (d); or

(f) a nucleic acid molecule encoding a variety of (poly) peptides being involved in the synthesis of a melithiazol the complementary stand of which hybridizes under stringent conditions to the DNA molecule encoding the amino acid sequence of any of (a) to (e).

6. A vector comprising a nucleic acid molecule of claim 1 or 2.

7. A vector comprising a plurality of different nucleic acid molecules of claim 1 or 2.

8. A vector comprising the gene cluster of claim 4 or 5.

9. A host cell transformed or transfected with the vector of any one of claim 6 to 8.

10. A host cell transformed with a plurality of vectors of claim 6.

11. The host cell of claim 9 or 10 which is a bacterium.

12. The host cell of claim 11 which is a myxobacterium, an actinomycete, a pseudomonad or Escherichia coli.

13. The host cell of any one of claim 9 to 12, further comprising a nucleic acid molecule encoding a phosphopantetheinyl transferase in expressible form.

14. A method of producing a melithiazol comprising culturing the host cell of any one of claims 9 to 13 under suitable conditions and isolating the melithiazol produced.

15. A method of producing a (poly)peptide involved in the synthesis of a melithiazol comprising culturing the host cell of any one of claims 9 to 13 under suitable conditions and isolating the (poly)peptide produced.

16. A (poly)peptide encoded by the nucleic acid molecule of any one of claims 1 to 3 or produced according to the method of claim 15.

17. A method of improving a pharmacological property of a melithiazol comprising the steps of

(a) replacing, in a host cell transformed or transfected with the vector of claim 7 or 8, or in the host cell of claim 13, at least one nucleic acid molecule of claim 1 or 2 by a corresponding nucleic acid molecule from a heterologous source; and

(b) testing for the desired improvement of said pharmacological property.

18. A method for improving a pharmacological property of a melithiazol comprising the steps of

(a) subjecting, in a host cell transformed or transfected with the vector of claim 7 or 8 or in the host cell of claim 13, at least one nucleic acid molecule of claim 1 or 2 to mutagenesis; and

(b) testing for the desired improvement of said pharmacological property.

19. A method for improving a pharmacological property of a melithiazol comprising the steps of (a) subjecting, in a host cell transformed or transfected with the vector of claim 6 or 7, a nucleic acid molecule of claim 1 or 2 to mutagenesis;

(b) testing for an improved activity of the (poly)peptide encoded by said nucleic acid molecule;

(c) combining said nucleic acid molecule in expressible form in a host cell with the remainder of the nucleic acid molecules present in the gene cluster of claim 3 or 4 or with corresponding nucleic acid molecules from a heterologous source in expressible form;

(d) allowing production of the melithiazol; and

(e) testing for the desired improvement of said pharmacological property.

20. A method of improving a pharmacological property of a melithiazol comprising the steps of

(aa) deleting from or inactivating in a host cell transformed or transfected with the vector of claim 8 one or more nucleic acid molecules according to claim 1 or 2, or

(ab) providing a host cell transformed or transfected with the vector of claim 7 or with a plurality of vectors comprising each different nucleic acid molecules according to claim 1 or 2, wherein said plurality of different nucleic acid molecules is less than the genes contained in the gene cluster of claim 4 and wherein the host cell further comprises a nucleic acid molecule encoding a phosphopantetheinyl transferase in expressible form;

b) allowing for expression of the nucleic acid molecules;

c) assessing whether a compound with fungicidal activity is produced; and/or

d) assessing whether the compound produced has an improved pharmacological property.

21. The method of claim 20 wherein the deletion or absence of said nucleic acid molecule results in the absence of the first thiazol ring of melithiazol.

22. A method of improving a desired property of or conferring a desired property to a secondary metabolite different from melithiazol comprising the steps of

(a) providing a host cell comprising a gene cluster or a part of a gene cluster wherein the gene products of said gene cluster are involved and sufficient for the synthesis of said secondary metabolite;

(b) transforming or transfecting said host cell with at least one vector of claim 6 or 7 or with a plurality of vectors comprising each different nucleic acid molecules according to claim 1 or 2, wherein said plurality of different nucleic acid molecules is less than the genes contained in the gene cluster of claim 4;

(c) allowing for expression of the nucleic acid molecules;

(d) assessing whether a secondary metabolite is produced; and/or

(e) assessing whether the secondary metabolite produced displays the desired property or the improved desired property.

23. The method of any one of claims 17 to 22 wherein said pharmacological property is selected from an improved range of fungicidal activity, and lower toxicity for the human or animal patient

24. The method of anyone of claims 14 and 17 to 23 further comprising modifying the melithiazol as a lead compound to achieve

(i) modified site of action, spectrum of activity, organ specificity, and/or

(ii) improved potency, and/or

(iii) decreased toxicity (improved therapeutic index), and/or

(iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or

(vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or

(vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or

(viii) improved general specificity, organ/tissue specificity, and/or

(ix) optimized application form and route

by

(i) esterification of carboxyl groups, or

(ii) esterification of hydroxyl groups with carbon acids, or

(iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or

(iv) formation of pharmaceutically acceptable salts, or

(v) formation of pharmaceutically acceptable complexes, or

(vi) synthesis of pharmacologically active polymers, or

(vii) introduction of hydrophylic moieties, or

(viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or

(ix) modification by introduction of isosteric or bioisosteric moieties, or

(x) synthesis of homologous compounds, or

(xi) introduction of branched side chains, or

(xii) conversion of alkyl substituents to cyclic analogues, or

(xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or

(xv) synthesis of Mannich bases, imines, or

(xvi) transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidines

or combinations thereof.

25. A melithiazol obtainable by the method of any one of claim 17 to 24.

26. A composition comprising the melithiazol of claim 25.

27. The composition of claim 26 which is a pharmaceutical composition.

28. A method of producing a pharmaceutical composition comprising the steps of the method of any one of claims 17 to 24 and further the step of formulating the melithiazol or the secondary metabolite thus obtained with a pharmaceutically acceptable carrier or diluent.