WO2007035693A9 - Isonitrile biosynthetic genes and uses thereof - Google Patents

Abstract

Novel isonitrile biosynthetic enzymes isnA and isnB which together possess a catalytic activity of converting an amine-containing compound to an isonitrile-containing compound are described. The genes that encode such enzymes, recombinant vectors containing one or both of such genes, a host cell transformed with said vector, methods for the production and isolation of the enzymes of the invention are also described. Methods for the identification of bioactive, isonitrile-containing natural products derived from the biosynthetic activity of the enzymes of the invention, and methods for synthetically producing isonitrile-containing compounds from the isolated enzymes of the invention are also described.

Description

ISONITRILE BIOSYNTHETIC GENES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Serial No. 60/718,357, filed September 19, 2005, the contents of which is herein incorporated by reference in their entirety.

GOVERNMENT SUPPORT

[002] The invention was supported, in whole, or in part, by NIH grant number CA24487.

The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[003] For more than 50 years, cultured soil microbes have played a significant role in drug discovery, ever since Selman Waksman isolated the small molecule antibiotics streptomycin and actinomycin, using cultured soil bacteria obtained in a New Jersey swamp (S. F. Brady, C. J.

Chao, J. Clardy, J. Am. Chem. Soc. 2002).

[004] Cultured soil bacteria have been an important source of biologically active, naturally occurring small molecules. Uncultured soil bacteria, which outnumber their cultured counterparts by at least two orders of magnitude, are likely to be an equally important source of such molecules (Brady et al., supra). It is estimated that existing culture techniques reveal only a minuscule fraction of soil microbes and due to isolation issues, a potentially untapped source of biologically active small molecules are unidentifiable and unaccessible. In an attempt to access the biosynthetic potential of uncultured bacteria, the inventor has explored an approach that involves the cloning and heterologous expression of DNA extracted directly from environmental samples (environmental DNA, eDNA) into easily cultured bacteria.

[005] In earlier reports, the inventor has described the identification and characterization of small molecule antibiotics from antibacterially active eDNA clones found by using a high- tliroughput phenotypic screen (Brady et al., supra). This approach directly couples the biosynthesis of each natural product that is found to a relatively small piece of cloned eDNA and therefore permits the characterization of both new natural products and their biosynthetic gene clusters simultaneously.

[006] Naturally occurring isonitrile-containing compounds have been shown to possess antibacterial properties. In addition, natural and synthetic isonitrile-containing compounds have been studied for their potential use inter alia in cancer therapy and in multi-drug resistance. Naturally occurring isonitriles have been known for fifty years, but their biosynthesis, even the source of the constituent atoms, remains obscure. Elucidation of the genes involved in the biosynthesis of isonitrile-containing compounds would be useful in the production and screening of potential isonitrile-containing therapeutics.

SUMMARY OF THE INVENTION

[007] The present invention provides isolated and/or recombinant materials and associated methods for expressing and isolating isonitrile biosynthetic enzymes capable of converting amine-containing compounds to isonitrile containing compounds, such as tryptophan to its corresponding 3-(isonitrileethylene) indole. Characterization of an antibacterially active eDNA clone has led the inventors to the identification of several isonitrile-containing natural-product antibiotics and in turn, the first isonitrile synthase, isnA and its associated isonitrile biosynthetic gene cluster.

[008] Thus, in one embodiment, the invention provides an isolated and/or recombinant polynucleotide sequences that is substantially homologous to SEQ ID NO: 1 or any fragment thereof encoding a protein having isonitrile synthase activity such as isnA (SEQ ID NO: 2). The invention also provides other isolated and/or recombinant materials useful for the biosynthesis of an isonitrile-containing compound from the isonitrile biosynthetic gene cluster. These additional materials include isolated and/or recombinant DNA compounds that encode an oxidative decarboxylase enzyme, isnB (SEQ ID NO: 4) also involved in the biosynthesis of isonitrile- containing compounds, and the recombinant protein enzymes that can be produced from these nucleic acids in the recombinant host cells of the invention.

[009] In one embodiment, the invention provides a recombinant expression vector that comprises a control sequence positioned to drive expression of isnA or isnB. [0010] In another embodiment, the invention provides a recombinant expression vector that comprises isnA and isnB and at least one control element positioned to drive expression of one or both genes. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the isnA and isnB gene products.

[0011] In another embodiment, the invention provides a method for producing isonitrile- containing compound in recombinant host cells, which method comprises expressing isnA and isnB or any active portions thereof in a host cell and maintaining the host cell under conditions that allow the production of isonitrile-containing compounds via the biosynthetic activity of the isnA and isnB. [0012] In another embodiment, the invention provides recombinant materials for the production of libraries of isonitrile-containing compounds wherein the isonitrile-containing members of the library are synthesized by the isonitrile biosynthetic enzymes of the invention. The resulting isonitrile-containing compounds can be further modified to convert to other useful compounds, such as antibiotics or compounds that are useful intermediates in the preparation of antibiotics.

[0013] In another related embodiment, the invention provides a method to prepare a nucleic acid that encodes a modified isonitrile biosynthetic enzyme, which method comprises using the isnA and/or isnB encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, insertion, or replacement. The thus modified isnA and/or isnB encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce enzymes having desired characteristics.

[0014] The invention also provides novel isonitrile-containing compounds and antibiotics or other useful compounds derived therefrom and methods of screening for such compounds. The compounds of the invention can be used to identify new pharmacophores for therapeutic use. The compounds of the invention may also be used as an intermediate in the manufacture of another compound, such as a pharmaceutical compound.

[0015] These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. The natural product antibiotic 1 was isolated from the culture broth of a 5.5 kB antibacterially active subclone of the eDNA cosmid, pCSLGlδ. isnA and isnB are necessary and sufficient to confer the production of 1 to E. coli.

[0017] Figure 2. M/Z values observed for 1 obtained from cultures of E. coli transformed with isnA/B and grown in either ¹⁴N-tryptophan or ¹⁵N-tryptophan. To prevent the loss of the labeled amino group a transaminase deficient (aspC, UvE and tyrB) E. coli strain was used in these feeding experiments.

[0018] Figure 3. Feeding experiments in E. coli indicate isnA uses tryptophan in the biosynthesis of 1. The proposed intermediate 2 is suggested by feeding experiments using the methylester 3.

[0019] Figure 4. Gene clusters present in sequenced bacterial genomes that contain homologs to isnA, the isonitrile synthase involved in the biosynthesis of 1. None of the bacteria from which these gene clusters are derived has been reported to produce isonitrile containing natural products. Compounds 3-8 were isolated from the E. coli cultures expressing these gene clusters.

[0020] Figure 5. Compound 1 is derived from tryptophan and the C2 carbon of ribulose-5-P.

Predicted m/z values for isotopically labeled 1 are shown.

[0021] Figure 6. The systematic differential labeling of 1 produced by E. coli strains deficient in specific primary metabolic enzymes (=) indicates that the isonitrile carbon is derived from the C2 carbon (C) of a pentose phosphate pathway intermediate. Color is used to designate the region of metabolism that was ruled out with feeding experiments in different E. coli mutants

(a. a. = amino acids).

[0022] Figure 7. M/Z values observed for 1 obtained from cultures of E. coli transformed with isnA/B and grown in uniformly, Cl and C2 labeled 13C-glucose.

[0023] Figure 8. The tautomerization of aldo and keto sugars may explain the use of different regio- and stereochemical sugar isomers in the enzymatic synthesis of 1.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The invention provides an isolated isonitrile biosynthetic enzyme gene cluster as shown in Fig. 1, comprising enzymes isnA and isnB of bacterial origin which together possess a catalytic activity of converting an amine-containing compound to an isonitrile-containing compound, the genes that encode such enzymes, recombinant vectors containing one or both of such genes, a host cell transformed with said vectors and methods for the production and isolation of the enzymes of the invention and methods for the production, identification and characterization of their biosynthetic products.

[0025] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0026] As used interchangeably herein, the terms "polynucleotide" and "nucleic acid" refer to the single- or double-stranded DNA or RNA of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5' (upstream) end to the 3' (downstream) end. As used interchangeably herein, the terms "polynucleotide sequence" and "nucleic acid sequences" refer to the sequence of a polynucleotide or nucleic acid. [0027] Bacteria and eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes. Genes can be clustered in structures referred to as "gene clusters" on a single chromosome and can be made up of one or more operons. Each gene cluster or operon can include up to 20 or more genes, usually from 2 to 6 genes and may additionally include one or more promoters and/or other regulatory sequences capable of initiating transcription of one or more of the genes.

[0028] A "control element" as used herein includes a promoter and optionally includes operator sequences and other elements such as ribosome binding sites, depending on the nature of the host. Regulatory sequences that allow for regulation of expression of a heterologous gene relative to the growth of the host cell may also be included. Examples of such regulatory sequences known to those of skill in the art are those that drive expression of a gene to be turned off in response to a chemical or physical stimulus.

[0029] As used herein, the term "promoter" refers to a polynucleotide molecule that, in its native state, is located upstream or 5' to a translational start codon of an open reading frame (or protein-coding region) and that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. When operably linked or positioned to drive expression of a polynucleotide molecule, a promoter typically causes the polynucleotide molecule to be transcribed in a manner that is similar to that of which the promoter is normally associated with.

[0030] As used herein, the term "substantially homologous" refers to polynucleotide molecules that demonstrate a substantial percent sequence identity with the promoters provided herein, wherein the polynucleotide molecules function (e.g., in bacteria) to direct transcription and have at least about 40%, such as at least about 50%, or at least about 60%, e.g. at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as 98% or 99% sequence identity with the polynucleotide sequences of the promoters described herein. In one preferred embodiment, polynucleotide molecules of the invention have at least about 85% sequence identity with the polynucleotide sequences of SEQ ID NOS: 1, 3 or 5.

[0031] As used herein, the term "percent sequence identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference polynucleotide molecule (or its complementary strand) as compared to a test polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA₅ and TFASTA available as part of the GCG^®. Wisconsin Package^® (Accelrys Inc., San Diego, Calif). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction times 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.

[0032] As used herein, the term "homology" refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide positional identity, i.e., sequence similarity or identity. As used herein, the term homology also refers to the concept of similar functional properties among different polynucleotide molecules. Polynucleotide molecules are homologous when under certain conditions they specifically hybridize to form a duplex molecule. Under these conditions, referred to as stringency conditions, one polynucleotide molecule can be used as a probe or primer to identify other polynucleotide molecules that share homology. Thus, the use of the polynucleotides of the invention, including those which do not encode active protein, are envisioned and can be used as probes for these genes or related genes.

[0033] The term "stringent conditions" is functionally defined with regard to the hybridization of a nucleic-acid probe to a target nucleic acid (i.e., to a particular nucleic-acid sequence of interest) by the specific hybridization procedure discussed in Molecular Cloning: A Laboratory Manual, 3^rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000 (referred to herein as Sambrook, et al.). Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of polynucleotide molecule fragments. Depending on the application envisioned one would desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. [0034] For applications requiring high selectivity, one will typically desire to employ relatively high stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 7O⁰C. A high stringent condition, for example, is to wash the hybridization filter at least twice with high-stringency wash buffer (0.2x SSC, 0.1% SDS, 65⁰C). Appropriate moderate stringency conditions that promote DNA hybridization, for example, 6.Ox sodium chloride/sodium citrate (SSC) at about 45⁰C, followed by a wash of 2.Ox SSC at 5O⁰C, are known to those skilled in the art. Additionally, the salt concentration in the wash step can be selected from a low stringency of about 2.Ox SSC at 5O⁰C to a high stringency of about 0.2x SSC at 5O⁰C. Additionally, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22⁰C, to high stringency conditions at about 65⁰C Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed. Such selective conditions tolerate little mismatch between the probe and the template or target strand. Detection of polynucleotide molecules via hybridization is well known to those of skill in the art. Homology can also be determined by computer programs that align polynucleotide sequences and estimate the ability of polynucleotide molecules to form duplex molecules under certain stringency conditions. Polynucleotide molecules from different sources that share a high degree of homology are referred to as "homologues".

[0035] A "variant" is a polynucleotide containing changes in which one or more nucleotides of the invention having isnA or isnB catalytic activity is deleted, added, and/or substituted, preferably while substantially maintaining a catalytic function of isnA or isnB. For example, one or more base pairs may be deleted from the 5' or 3' end of a promoter to produce a "truncated" promoter. One or more base pairs can also be inserted, deleted, or substituted internally to a promoter. Those of skill in the art are familiar with the standard resource materials that describe specific conditions and procedures for the construction, manipulation, and isolation of macromolecules (e.g., polynucleotide molecules, plasmids, etc.), as well as the generation of recombinant organisms and the screening and isolation of polynucleotide molecules.

[0036] As used herein, the term "construct" refers to any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a polynucleotide molecule where one or more polynucleotide molecule has been linked in a functionally operative manner. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art; see for example, Sambrook, et al.

[0037] As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, connected with a second expressible polynucleotide molecule, such as a gene of the invention, where the polynucleotide molecules are so arranged that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a single contiguous polynucleotide molecule and more preferably are adjacent. For example, a promoter is operably linked to a gene if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0038] As used herein, the term "transformed" refers to a cell, tissue, organ, or organism into which has been introduced a foreign polynucleotide molecule, such as a construct. Preferably, the introduced polynucleotide molecule is integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced polynucleotide molecule is inherited by subsequent progeny. A "transgenic" or "transformed" cell or organism also includes progeny of the cell or organism.

[0039] As used herein the term "isonitrile biosynthetic enzyme activity" means a protein or polypeptide capable of catalyzing one or more steps in the conversion of an amine (e.g. tryptophan) to an isonitrile (e.g., 3-(isonitrileethylene)indole).

Nucleic Acids, Constructs and Vectors

[0040] The present invention relates to isolated and/or recombinant (including, e.g., essentially pure) nucleic acids of bacterial origin having sequences which encode enzymes responsible for the biosynthesis of isonitriles. In one embodiment, the nucleic acid or portion thereof encodes a protein or polypeptide having at least one functional characteristic of an isonitrile synthase, such as a catalytic activity (e.g., catalysis of isonitrile formation from an amine). The present invention also relates more specifically to isolated and/or recombinant nucleic acids or a portion thereof having sequences substantially homologous to the sequence of isnA as shown in SEQ ID NO 1. [0041] In another embodiment, the invention provides the nucleic acid or portion thereof encoding a protein or polypeptide having at least one functional characteristic of an isonitrile oxidative decarboxylase, such as a catalytic activity (e.g., catalysis of the oxidative removal of a carboxyl moiety from its substrate. The present invention also relates more specifically to isolated and/or recombinant nucleic acids or a portion thereof having sequences substantially homologous to the sequence of isnB as shown in SEQ ID NO 3.

[0042] The invention also provides a recombinant expression vector comprising the isnA (SEQ ID NO: 1) and the isnB (SEQ ID NO: 3) genes or any functional portions thereof having catalytic activity, and or more control sequences positioned to express such genes or catalytic portions thereof. In one preferred embodiment, the vector comprising all or a portion of isnA and isnB comprises the nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence substantially homologous to the nucleic acid sequence of SEQ ID NO: 5.

[0043] The invention further relates to isolated and/or recombinant nucleic acids that are characterized by (1) their ability to hybridize to (a) a nucleic acid of the invention as described herein, (b) the complement of (a), or (c) to portions of either of the preceding, (2) by their ability to encode one or more polypeptides having the amino acid sequence of an isonitrile synthase and/or an isonitrile oxidative decarboxylase, such as the amino acid sequences of SEQ ID NOS: 2 or 4 or functional equivalents thereof (e.g., a polypeptide that converts an amine moiety to an isonitrile) or (3) by both characteristics.

[0044] Isolated and/or recombinant nucleic acids meeting these criteria comprise nucleic acids having sequences identical to sequences of naturally occurring isnA and isnB and portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues are modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

[0045] Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions, for example. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 1, Suppl. 26, 1991), the teachings of which are hereby incorporated by reference. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Thus, high or moderate stringency conditions can be determined empirically, depending in part upon the characteristics of the known DNA to which other unknown nucleic acids are being compared for sequence similarity.

[0046] Isolated and/or recombinant nucleic acids that are characterized by their ability to hybridize to a nucleic acid of the invention or to the complement of such nucleic acids (e.g. under high or moderate stringency conditions), may further encode a protein or polypeptide having at least one functional characteristic of isnA or an isnB such as a catalytic activity (e.g., converting an amine-containing compound to an isonitrile-containing compound). The catalytic or binding function of a protein or polypeptide encoded by hybridizing nucleic acid may be detected by standard enzymatic assays for activity or binding (e.g., assays which monitor isonitrile formation as disclosed in the Examples). Enzymatic assays, complementation tests, or other suitable methods can also be used in procedures for the identification and/or isolation of nucleic acids which encode a polypeptide that is, for example, substantially homologous to the amino acid sequences of SEQ ID NOS: 2 and 4 or the functional equivalents of these polypeptides.

[0047] Nucleic acids of the present invention can be used in the production of proteins or polypeptides. For example, a nucleic acid sequence of the invention can be incorporated into various constructs and vectors created for further manipulation of sequences or for production of the encoded polypeptide in suitable host cells.

[0048] Nucleic acids referred to herein as "isolated" are nucleic acids separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. "Isolated" nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids which are isolated. Nucleic acids referred to herein as "recombinant" are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial recombination, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. "Recombinant" nucleic acids are also these that result from recombination events that occur through the natural mechanisms of cells, but are selected for after the introduction to the cells of nucleic acids designed to allow and make probable a desired recombination event. Compositions containing such nucleic acids are often characterized by the presence of heterologous nucleic acids or the autologous nucleic acids.

[0049] In another embodiment, the invention provides an antisense nucleic acid, which is complementary, in whole or in part, to a target molecule comprising a sense strand, and can hybridize with the target molecule. The target can be a polynucleotide of the invention, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart). When introduced into a cell, antisense nucleic acid can inhibit the expression of the gene encoded by the sense strand. Antisense nucleic acids can be produced by standard techniques.

Proteins

[0050] The invention also relates to proteins or polypeptides encoded by nucleic acids of the present invention. The proteins and polypeptides of the present invention can be isolated and/or recombinant. Proteins or polypeptides referred to herein as "isolated" are proteins or polypeptides purified to a state beyond that in which they exist in cells. "Isolated" proteins or polypeptides include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, including essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis, or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. Proteins or polypeptides referred to herein as "recombinant" are proteins or polypeptides produced by the expression of recombinant nucleic acids. Compositions containing such proteins are often characterized by the presence heterologous proteins and contaminants or the absence of autologous proteins or contaminants.

[0051] In accordance with the invention, transformation of a host is carried out using a vector comprising a polynucleotide or construct of the invention and then culturing of the thus obtained transformant is carried out under generally used conditions, thereby allowing the strain to produce a polypeptide having the isonitrile biosynthetic enzyme activity. Examples of the host to be used include microorganisms, animal cells and plant cells. Examples of the microorganisms include Escherichia coli, microorganisms belonging to the genus Pseudomonas, Bacillus, Streptomyces, Lactococcus, Erwinia etc., yeasts belonging to the genus Saccharomyces, Pichia, Kluyveromyces, etc., and filamentous fungi belonging to the genus, Aspergillus, Penicillium, Trichoderma, etc. Examples of animal cells include animal cells utilizing the baculovirus expression system. [0052] Confirmation of the expression and expressed product can be made easily by the use of an antibody specific for the isnA and/or isnB enzymes, or functional portions thereof, and the expression can also be confirmed by measuring the enzyme activity of isnA and/or isnB or functional portions thereof. Purification of isnA or isnB or functional portions thereof from the transformant culture medium can be carried out by optional combination of centrifugation, UF concentration, salting out and various types of chromatography such as of ion exchange resins. Purification may also be accomplished using antibodies as described below.

[0053] In a preferred embodiment, the protein or portion thereof has at least one functional characteristic of an isnA or isnB enzyme such as isonitrile biosynthetic catalytic activity (e.g. catalysis of an amine moiety to an isonitrile moiety). As such, these include, for example, naturally occurring enzymes, variants (e.g. mutants) of those proteins and/or portions thereof. Such variants include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides in which one or more residues are modified, and mutants comprising one or more modified residues.

[0054] Since the primary structure and gene structure of the isnA and isnB enzymes are described by the present invention, it is possible to obtain a gene coding for the amino acid sequence of a natural enzyme or enzymes capable of enzymatically catalyzing the formation of an isonitrile-containing compound from amine-containing compound, in which one or more amino acid residues of the amino acid sequence are modified by at least one of deletion, addition, insertion and substitution, by introducing random mutation or site-specific mutation using the gene of the present invention. This method renders possible preparation of a gene coding for an isonitrile synthase and/or any associated isonitrile oxidative decarboxylases which have the same or similar activity but possess other desired properties such as optimum temperature, temperature stability, optimum pH and pH stability, substrate specificity, etc. are slightly changed, and it also renders possible production of such isonitrile synthase and isonitrile oxidative decarboxylase enzymes by means of gene engineering techniques.

[0055] Site-specific mutation can be introduced easily by the use, for example, of commercially available kits. Examples of such kits include Mutan™-G (manufactured by Takara Shuzo) in which the gapped duplex method is used; Mutan™-K (manufactured by Takara Shuzo) in which the Kunkel method is used; Mutan™-Express Km (manufactured by Takara Shuzo) in which the ODA method is used and QuickChange® Site-Directed Mutagenesis Kit (manufactured by STRAT AGENE) in which primers for mutation use and Pyrococcus furiosus DNA polymerase are used, as well as TaKaRa LA PCR in vitro Mutagenesis Kit (manufactured by Takara Shuzo) and Mutant™-Super Express Km (manufactured by Takara Shuzo) as kits in which PCR is used.

[0056] Thus, the primary structure and gene structure of the isnA and isnB enzymes provided by the present invention render possible production of an inexpensive and high purity polypeptide having the isonitrile biosynthetic enzyme activity.

[0057] The invention further relates to fusion proteins, comprising isnA, isnB or functional portions thereof (as described above) as a first moiety, linked to second moiety not occurring in the enzyme as found in nature. Thus, the second moiety can be an amino acid or polypeptide. The first moiety can be in an N-terminal location, C-terminal location or internal to the fusion protein. In one embodiment, the fusion protein comprises isnA, isnB or functional portions thereof as the first moiety, and a second moiety comprising a linker sequence, and affinity ligand.

[0058] Fusion proteins can be produced by a variety of methods. For example, a fusion protein can be produced by the insertion of an aaRS gene or portion thereof into a suitable expression vector, such as Bluescript SK+/- (Stratagene), pGEX-4T-2 (Pharmacia) and pET-15b (Novagen). The resulting construct is then introduced into a suitable host cell for expression. Upon expression, fusion protein can be purified from a cell lysate by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).

[0059] The invention also relates to isolated and/or recombinant portions of isnA and/or isnB. For example, a portion of isnA or isnB can also refer to one of two or more distinct subunits of each of these enzymes. Portions of the respective enzymes can be made which have full or partial function (or no function) on their own, or which when mixed together spontaneously assemble with one or more other polypeptides to reconstitute a functional protein having at least one function characteristic of isnA and/or isnB.

[0060] The invention further relates to antibodies that bind to an isolated and/or recombinant isnA and/or isnB enzymes or functional portion(s) thereof, including portions of antibodies (e.g., peptides), which can specifically recognize and bind to isnA and/or isnB or portions thereof. These antibodies can be used in methods to purify the enzymes or portions thereof by various methods of immunoaffϊnity chromatography, or to selectively inactivate one of the enzyme's active sites, or to study other aspects of the enzyme's structure, for example.

Uses of the Invention

[0061] The isonitrile biosynthetic enzymes of the invention and polynucleotides encoding such enzymes and portions thereof are useful in the biosynthetic preparation and identification of novel, bioactive, isonitrile-containing natural products. Heterologously expressed isonitrile- containing natural products such as those of Formulas 1-8, from the polynucleotides and host cells of the invention may be structurally characterized and assayed for bioactivity using a wide variety of human disease related assays to identify new isonitrile-containing small molecules that can serve either as lead structures for new therapeutics or tools to study disease biology. These isonitrile-containing compounds or compounds comprising functional groups related to isonitriles may serve as intermediates in the formation of other unique chemical moieties such as thiazalines and diazalones. Thus, one method of the invention comprises producing isonitrile- containing natural products comprising the steps of: a) providing a cell culture comprising recombinant host cells capable of expressing isnA and/or isnB or functional portions thereof; and b) maintaining said culture under conditions in which isonitrile-containing compounds are biosynthetically produced. The invention further provides methods of identifying bioactive isonitrile-containing natural compounds comprising the steps of: a) providing a cell culture comprising recombinant host cells capable of expressing isnA and/or isnB or functional portions thereof; and b) maintaining said culture under conditions in which isonitrile-containing compounds are biosynthetically produced; and c) assaying the culture for the presence of bioactive isonitrile-containing compounds.

[0062] Assays for bioactivity include but are not limited to antibacterial and antifungal cytotoxicity assays to identify novel natural products with antimicrobial activity. Other high throughput screens relevant to human disease biology are also contemplated by the present invention. Bioactive, isonitrile-containing compounds produced by the isonitrile biosynthetic enzymes of the invention are arrayed in 96-well plates and incorporated into the large-scale screening programs.

[0063] The isonitrile biosynthetic enzymes of the invention also have applicability in the chemical industry for use in biocatalysis of isonitriles and functional groups related to isonitrile groups such as N-formyl groups. These isonitrile-containing compounds or compounds comprising functional groups related to isonitriles may serve as intermediates in the formation of other unique chemical moieties such as thiazalines and diazalones. In accordance with the invention, the invention provides a method of synthesizing an isonitrile -containing compound comprising the step of contacting an enzyme of the invention with an amine-containing substrate under conditions in which the isonitrile-containing compound or a compound comprising a functional group related to an isonitrile group, is produced.

[0064] There is a critical need in the chemical industry for efficient catalysts for the practical synthesis of optically pure materials; enzymes, such as isonitrile biosynthesis enzymes can provide the optimal solution. The synthesis of polymers, pharmaceuticals, natural products and agrochemicals is often hampered by expensive processes which produce harmful byproducts and which suffer from low enantioselectivity. Enzymes have a number of remarkable advantages which can overcome these problems in catalysis: they act on single functional groups, they distinguish between similar functional groups on a single molecule, and they distinguish between enantiomers. Moreover, they are biodegradable and function at very low mole fractions in reaction mixtures. Because of their chemo-, regio- and stereospecificity, enzymes present a unique opportunity to optimally achieve desired selective transformations. These are often extremely difficult to duplicate chemically, especially in single-step reactions. The elimination of the need for protection groups, selectivity, the ability to carry out multi-step transformations in a single reaction vessel, along with the concomitant reduction in environmental burden, has led to the increased demand for enzymes in chemical and pharmaceutical industries.

[0065] Enzyme-based processes have been gradually replacing many conventional chemical- based methods. A current limitation to more widespread industrial use is primarily due to the relatively small number of commercially available enzymes. Furthermore, virtually all of the enzymes known so far have come from cultured organisms, mostly bacteria. Traditional enzyme discovery programs rely solely on cultured microorganisms for their screening programs and are thus only accessing a small fraction of natural diversity. Thus the isonitrile biosynthetic enzymes of the invention of uncultured soil bacterial origin represent a unique class of enzymes suitable for use in industries including but not limited to biocatalysis in the chemical, pharmaceutical, textile, and agrochemical industries wherein biosynthetic conversion of an amine- to an isonitrile-containing compound is desirable.

Compounds

[0066] The present invention further comprises isonitrile-containing compounds having antibacterial activity produced from the isonitrile biosynthetic gene cluster of the invention and identified in accordance with the methods of the invention. Preferably, the compounds have the following formula:

Ar- CH=CH-NC or Ar-CH-CH (COOH)-NC

[0067] Wherein the Ar is a substituted or unsubstituted aromatic or heteroaromatic group. The terms "aryl" or "aromatic" as used herein, refer to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. In yet another embodiment, the process can convert and amino acid to the corresponding isonitrile, e.g., an isonitrile having the formula:

R=CH-NC or

R-CH (COOH)-NC

[0068] Wherein R is a natural or synthetic amino acid side chain (e.g., a substituted or unsubstituted aliphatic or aromatic group).

[0069] The terms "substituted aryl" or "substituted aromatic," as used herein, refer to an aryl or aromatic group substituted by one, two, three or more aromatic substituents. [0070] The terms "heteroaryl" or "heteroaromatic," as used herein, refer to a mono-, bi- or tri-cyclic aromatic radical or ring having from five to ten ring atoms of which at least one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may be optionally oxidized. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like. The heteroaromatic ring may be bonded to the chemical structure through a carbon or hetero atom.

[0071] The terms "substituted heteroaryl" or "substituted heteroaromatic," as used herein, refer to a heteroaryl or heteroaromatic group, substituted by one, two, three, or more aromatic substituents.

[0072] Suitable substituents include, but are not limited to, F, Cl, Br, I, OH, protected hydroxy, aliphatic ethers, aromatic ethers, oxo, NO2, CN, Cl-C12-alkyl optionally substituted with halogen (such as perhaloalkyls), C2-C12-alkenyl optionally substituted with halogen, C2- C12-alkynyl optionally substituted with halogen^" NH2, protected amino, NH -Cl-C12-alkyl~NH -C2-C12-alkenyl,^~NH -C2-C12-alkenyl^~NH -C3-C12-cycloalkyl^~NH -aryl,~NH -heteroaryl~NH -heterocycloalkyl, dialkylamino, diarylamino, diheteroarylamino, O-Cl-C12-alkyl, O-C2-C12- alkenyC O-C2-C 12-alkynyl^~ O-C3 -C 12-cycloalkyl^~ O-aryl^~ O-heteroaryl^~ O-heterocycloalkyl, ^"C(O)- Cl-C12-alkyl~C(O)- C2-C12-alkenyl~C(O)- C2-C12-alkynyl^~C(O)-C3-C12-cycloalkyl, ^~ C(O)-aryl^~ C(O)-heteroaryl^~ C(O)-heterocycloalkyl^~ CONH2^~ CONH- C 1 -C 12-alkyl^~ CONH- C2-C 12-alkenyC CONH- C2-C 12-alkynyC C0NH-C3 -C 12-cycloalkyl,^" CONH-aryl,^" CONH- heteroaryl^~CONH-heterocycloalkyl^~CO2- Cl-C12-alkyl^~CO2- C2-C12-alkenyl,^"CO2- C2-C12- alkynyl^" CO2-C3 -C 12-cycloalkyl,^~ C02-aryl^~ CO2-heteroaryC CO2-heterocycloalkyl^~ D C02- C 1 - C 12-alkyl,^~ 0C02- C2-C 12-alkenyl,^" 0C02- C2-C 12-alkynyl,^~ OCO2-C3-C 12-cycloalkyl^~ 0C02- aryi OCO2-heteroaryl^~ OCO2-heterocycloalkyl,^~ 0C0NH2,^" OCONH- Cl-Cl 2-alkyl^~ OCONH- C2-C12-alkenyC OCONH- C2-C12-alkynyl,^~ OCONH- C3-C12-cycloalkyl,^~ OCONH- aryl, ^~0C0NH- heteroaryl,^" OCONH- heterocycloalkyCNHC(O)- Cl-C12-alkyl,^"NHC(O)-C2-C12- alkenyl^" NHC(O)-C2-C12-alkynyl^~NHC(O)-C3-C12-cycloalkyl,^"NHC(O)-aryl,^~ NHC(O)- heteroaryl^~NHC(0)-heterocycloalkyl,^"NHC02- Cl-C12-alkyl^~NHC02- C2-C12-alkenyl, ^"NHC02- C2-C12-alkynyl,^~NHCO2- C3-C12-cycloalkyl^~NHCO2- aryl^~NHC02- heteroaryl, ^"NHC02- heterocycloalkyl,^~NHC(O)NH2, NHC(O)NH- Cl-C12-alkyl,^~NHC(O)NH-C2-C12- alkenyi;NHC(O)NH-C2-C12-alkynyi;NHC(O)NH-C3-C12-cycloalkyl,^~NHC(O)NH-aryl₃ ^"NHC(0)NH-heteroaryl^~NHC(0)NH-heterocycloalkyl, NHC(S)NH2, NHC(S)NH- C1-C12- alkyl,^~NHC(S)NH-C2-C12-alkenyl^~NHC(S)NH-C2-C12-alkynyl,^~NHC(S)NH-C3-C12- cycloalkyl^~NHC(S)NH-aryl,^~NHC(S)NH-heteroaryl,^"NHC(S)NH-heterocycloalkyl, ^~NHC(NH)NH2, NHC(NH)NH- Cl-Cl 2-alkyl^~NHC(NH)NH-C2-C 12-alkenyl,^"NHC(NH)NH- C2-C12-alkynyl,^~NHC(NH)NH-C3-C12-cycloalkyl,^~NHC(NH)NH-aryl,^"NHC(NH)NH- heteroaryl^" NHC(NH)NH-heterocycloalkyl, NHC(NH)-C 1 -C 12-alkyl,^~NHC(NH)-C2-C 12- alkenyi;NHC(NH)-C2-C12-alkynyl^~NHC(NH)-C3-C12-cycloalkyl,^~NHC(NH)-aryC NHC(NH)- heteroaryC NHC(NH)-heterocycloalkyl,^~ C(NH)NH-C 1 -C 12-alkyl^~ C(NH)NH-C2-C 12-alkenyl, ^" C(NH)NH-C2-C 12-alkynyl^~ C(NH)NH-C3 -C 12-cycloalkyl^~ C(NH)NH-aryl^~ C(NH)NH- heteroaryl^ C(NH)NH-heterocycloalkyC S(O)-C 1 -C 12-alkyC S(0)-C2-C 12-alkenyl^~ S(0)-C2- C12-alkynyl^~ S(O)-C3-C12-cycloalkyl,^~ S(O)-aryl^~ S(O)-heteroaryl^~ S(O)-heterocycloalkyl ^"SO2NH2^~SO2NH- Cl-C12-alkyl^~SO2NH- C2-C12-alkenyl,^" S 02NH- C2-C12-alkynyl, ^"S02NH- C3-C12-cycloalkyl^~SO2NH- aryl^~S02NH- heteroaryl^" S02NH- heterocycloalkyl, ^"NHS02-C 1 -C 12-alkyl,^~NHSO2-C2-C 12-alkenyl,^~ NHSO2-C2-C 12-alkynyl^~NHSO2-C3-C 12- cycloalkyl~NHSO2-aryl^~NHSO2-heteroaryCNHSO2-heterocycloalkyi;CH2NH2, ^"CH2SO2CH3^~aryl^~arylalkyl^~ heteroaryl,^" heteroarylalkyl^~heterocycloalkyl,^~C3-C12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, methoxymethoxy, methoxyethoxy, SH, S-Cl-C12-alkyl, S-C2-

C12-alkenyl^~S-C2-C12-alkynyl,^"S-C3-C12-cycloalkyl~S-aryl~S-heteroaryl^~S-heterocycloalkyl, or methylthiomethyl. It is understood that the aryls, heteroaryls, alkyls and the like can be further substituted.

[0073] Examples of compounds of the invention include but are not limited to the compounds of Formulas 1-8.

[0074] The compounds of the invention include any racemates, enantiomers, regioisomers, salts, esters or prodrugs thereof. The invention further provides pharmaceutical compositions comprising compounds of the invention.

[0075] Compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures. The compounds can be readily formulated to provide the pharmaceutical compositions of the invention. The pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form. This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.

[0076] The carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquefied form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.

[0077] Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference. Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, incorporated herein by reference. The active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.

[0078] For the treatment of conditions and diseases caused by infection, a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles. The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intramuscular, and intrasternal injection or infusion techniques. [0079] Dosage levels of the compounds of the invention are of the order from about 0.01 mg to about 50 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight per day. The dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient). In addition, the compounds of the invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals. [0080] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition. Dosage unit forms will generally contain from about 0.5 mg to about 500 nig of active ingredient. For external administration, the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight. [0081] It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought. Kits

[0082] The invention further provides kits for expressing isonitrile biosynthetic enzymes of the invention. Such kit comprises at least one isolated or recombinant polynucleotide encoding isnA and/or isnB or any functional portion thereof, or at least one recombinant vector or construct containing the polynucleotide sequence encoding isnA and/or isnB or a functional portion thereof. Said vector or construct may further comprise control elements such as a promoter operably linked to the polynucleotide encoding isnA and/or isnB. Such kits may be used in conjunction with heterologous host cells suitable for expressing the polynucleotides of the invention. Such kits may further comprise the antibodies of the invention for isolating and purifying the isonitrile biosynthetic enzymes encoded by the polynucleotides of the invention. [0083] In another embodiment, the kit contains isolated and purified biosynthetic enzymes of the invention or functional portions thereof for use, for example, in the production of isonitrile- containing pharmaceuticals or for use in biocatalysis of isonitriles. In one example, the kit may be useful for converting prodrugs to isonitrile-containing pharmaceuticals. [0084] A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the invention and shall not be construed as being a limitation on the scope of the invention or claims. Example 1

General Methodology

[0085] CSLGl 8, the eDNA clone that produces the compound of 1 was found in a cosmid library constructed from blunt ended, gel purified, high molecular weight eDNA extracted directly from soil collected in Boston, MA. The cosmid library was screened for antibacterially active clones using a top agar overlay containing Bacillus subtilis. (See; (a) S. F. Brady, J. Clardy, J. Am. Chem. Soc. 2000, 122, 12903; b) S. F. Brady, C. J. Chao, J. Handelsman, J. Clardy, Org. Lett. 2001, 3, 1981; c) S. F. Brady, C. J. Chao, J. Clardy, J. Am. Chem. Soc. 2002, 124, 9968; d) S. F. Brady, C. J. Chao, J. Clardy, Appl. Environ. Microbiol. 2004, 70, 6865; e) S. F. Brady, J. Clardy, J. Nat. Prod. 2004, 67, 1283; and Example 2 for library construction and screening methods. Clones that produced a zone of growth inhibition in the overlayed B. subtilis lawn were recovered from the assay plates and tested for the production of organic extractable antibacterial activities.

[0086] Bioassay guided fractionation of the antibacterially active ethyl acetate extract from cultures of CSLGl 8 led to the isolation of the antibiotic of 1, an isonitrile functionalized C3 substituted indole. The indole and trans olefin were easily inferred from standard ID and 2D NMR experiments. The triplet seen at 107.4 ppm in the 13C spectrum suggested the presence of the isonitrile, as 13 C-14N coupling between the isonitrile 's nitrogen and the adjacent carbon, results in a carbon triplet that is a hallmark of this functional group. The proposed structure of 1 was subsequently confirmed by single crystal X-ray diffraction. Although the cis isomer of 1 has been reported from cultured bacteria, to the best of our knowledge 1 has never been reported as a natural product ( see, (a) J. R. Evans, E. J. Napier, P. Yates, J. Antibiot. 1976, 29, 850; b) I. Hoppe, U. Schollkopf, Liebigs Ann. Chem. 1984, 600).

[0087] The genes responsible for the biosynthesis of 1 were sought in pCSLGlδ.l, a 5.5 kB antibacterially active EcoRI subclone of the original eDNA cosmid, pCSLGlδ (GenBank accession number DQ084328). Two predicted open reading frames contained in pCSLGl 8.1 (Figure 1) were identified by transposon mutagenesis as necessary for the production of 1 and have been given the names isnA and isnB (isonitrile), respectively. The predicted translation product of isnA shows highest sequence identity to PvcA of pyoverdine chromophore biosynthesis (33%) and Ditl of yeast spore wall biosynthesis (21%). Neither PvcA nor Ditl has been functionally characterized, although both are thought to be involved in the biosynthesis of C-N bonds. (see, (a) P. Briza, M. Eckerstorfer, M. Breitenbach, Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 4524; (b) P. Briza, H. Kalchhauser, E. Pittenauer, G. Allmaier, M. Breitenbach, Eur. J. Biochem. 1996, 239, 124; (c) A. Stintzi, Z. Johnson, M. Stonehouse, U. Ochsner, J. M. Meyer, M. L. Vasil, K. Poole, J. Bacteriol. 1999, 181, 4118.)

[0088] The predicted translation product of isnB shows highest sequence identity to non- heme iron α-ketoglutarate dependent oxygenases including the oxygenase from clavaminate biosynthesis and PvcB, a second enzyme from pyoverdine chromophore biosynthesis, (see, (a) E. N. Marsh, M. D. Chang, C. A. Townsend, Biochemistry 1992, 31, 12648; (b) J. E. Hodgson, A. P. Fosberry, N. S. Rawlinson, H. N. Ross, R. J. Neal, J. C. Arnell, A. J. Earl, E. J. Lawlor, Gene 1995, 166, 49). Three additional ORFs (>100 amino acids) were found in CSLGl 8.1; ORFs 4 and 5 are related to hypothetical proteins of unknown function and ORF 3 is related to prenyltransferases.

[0089] The successful expression of isnA/B, the biosynthetic genes for 1, in E. coli allowed the biosynthesis of 1 and, more specifically, the isonitrile functional group to be studied in detail. As with many isonitrile containing natural products isolated from cultured bacteria and fungi, the structure of 1 suggests that the isonitrile nitrogen is likely derived from the amine of an amino acid. Feeding studies using native isonitrile producing organisms have; however, not always supported this hypothesis, (see, a) H. Achenbach, H. Grisebach, Z Naturforsch B 1965, 20, 137; b) V. Bornemann, G. M. L. Patterson, R. E. Moore, J. Am. Chem. Soc. 1988, 110, 2339; c) P. J. Scheuer, Ace. Chem. Res. 1992, 25, 433.)

[0090] The cloning and heterologous expression of isonitrile biosynthetic enzymes in E. coli allowed us to perform controlled feeding experiments which could not be carried out in less well-understood microbes and plants.

[0091] The role of tryptophan in the biosynthesis of 1 and, especially its connection with the isonitrile nitrogen, was investigated using isnA/B expressed in an E. coli host with a genetic background especially selected to eliminate the ambiguity seen in feeding experiments carried out in "wild-type" isonitrile producing organisms. To assess the role of tryptophan in the biosynthesis of 1, doubly labeled 15N-tryptophan was fed to a culture of a tryptophan auxotroph (trpC) and transaminase deficient (aspC, ilvE and tyrB) strain of E. coli (DL39W) transformed with isnA/B (see, D. M. LeMaster, F. M. Richards, Biochemistry 1988, 27, 142. See also, Example 5 for a detailed description of the methods used in these feeding experiments). Incorporation of the 15N label into compound 1 was determined by LCMS. The tryptophan auxotrophy ensures that the doubly labeled 15N-tryptophan is the only tryptophan present in the culture, and the transaminase deficiencies ensure that the 15N labeled amine of tryptophan is not lost during the feeding experiment. If both nitrogens are incorporated into 1 the observed m/z (M+H+) for 1 would be 171. If tryptophan is not used in the biosynthesis of 1 no 15N label will be incorporated into 1 resulting in an observed m/z of 169. If only one of the two 15N-nitrogens, either from the indole or the free amine, is incorporated into 1 the observed m/z for 1 would be 170.

[0092] In the presence of doubly labeled 15N-tryptophan the general transaminase and tryptophan deficient strain of E. coli transformed with isnA/B produces 1 with an observed m/z of 171. Both nitrogens in 1, including the isoiiitrile nitrogen are therefore derived from tryptophan (Figures 2 and 3). Almost all of 1 obtained from a wild type (transaminase proficient) E. coli contained a single 15N label (m/z=170). Labeling studies performed in wild- type isonitrile producing organisms would likely give the same result and could explain the ambiguous feeding results obtained from these organisms by previous workers. By using mutants that create an appropriately controlled metabolic background many of the drawbacks traditionally associated with feeding studies can be significantly reduced or completely eliminated in model microbial systems.

[0093] isnA and isnB individually overexpressed as glutathione-S-transferase fusion proteins (isnA-GST and isnB-GST, respectively) were used to elucidate the overall scheme for the biosynthesis of 1 (Table 1). Table 1

E. coli Culturing treatment E. coli 1

(strain 1) (strain 2) isnA + isnB + isnA-GST _ isnB-GST _ isnA-GST Co-cultured^[a] isnB-GST + isnA-GST Preconditioned^ isnB-GST + isnB-GST Preconditioned'-¹³-' isnA-GST isnB-GST N-methyl tryptophan^-¹ - isnB-GST N-formyl tryptophan^ - isnB-GST N-methyl tryptamine^M - isnB-GST N-formyl tryptamine¹^ - isnB-GST ₃M +

[0094] [c] Stains 1 and 2 were grown together in the same flask, [b] Strain 1 was removed by centrifugation from cultures that were grown for 12-14 h at 30 °C and the supernatant was then filter sterilized (0.2 μm). Strain 2 was grown in the filter-sterilized media, [c] lOOμg/mL. [0095] Cultures of E. coli that overexpressed either isnA-GST or isnB-GST individually did not accumulate 1. However, when these two strains were co-cultured in the same flask, 1 accumulated in the culture broth. isnA and isnB are therefore necessary and sufficient to confer the production of 1 to E. coli and one of the two enzymes must produce a diffusible intermediate that is converted to 1 by the other. isnA-GST grown in media preconditioned with the growth of isnB-GST does not lead to the accumulation of 1 in the culture broth; however, cultures of isnB- GST grown in media preconditioned with the growth of isnA-GST accumulate 1 in the culture broth. The diffusible intermediate is therefore produced by isnA and converted to 1 by isnB (Figure 7). All attempts to isolate this intermediate from cultures either overexpressing isnA- GST or transformed with isnA/B were unsuccessful.

[0096] N-formyl and N~methyl compounds have been proposed to be likely isonitrile intermediates in what was believed to be a two-step isonitrile biosynthetic scheme. However N- formyl and N-methyl derivatives of tryptophan and tryptamine did not serve as substrates for the production of 1 when added to (0.1 mg/ml) cultures overexpressing isnB-GST. It seemed plausible that isnA might form the isonitrile in a single enzymatic step and that isnB's role would be the oxidative decarboxylation of a proposed isonitrile functionalized intermediate 1-2. In our hands the synthetic intermediate 1-2 was not stable and therefore could not be used in feeding experiments to test this hypothesis. However, we were able to synthesize the methyl ester of 1-2 (1-3) and thought that the slow cleavage of the methyl ester by promiscuous bacterial esterases might circumvent the instability problem, (see, (a) D. H. R. Barton, G. Bringmann, W. B. Motherwell, Synthesis 1980, 68; (b) N. Hashimoto, T. Aoyama, T. Shioiri, Chem. Pharm. Bull. 1981, 29, 1475). Upon the addition of 1-3 (0.1 mg/ml) to cultures expressing zmδ-GST, 1 accumulated in the culture broth, and when cultures of CSLGl 8.1 were spiked with 1-3 they showed increased production of 1 compared to control CSLGl 8.1 cultures. Compound 1 was not detected in ethyl acetate extracts from vector control cultures spiked with 1-3 or from isnA- GST cultures spiked with 1-3. These feeding studies suggest that the isonitrile seen in 1 is formed in a single enzymatic step on isnA and that the proposed isonitrile containing intermediate 2 is oxidatively decarboxylated by isnB to give 1 (Figure 7). Compound 1-2's lack of stability is likely responsible for our inability to isolate the proposed intermediate. [0097] The eDNA approach used in this study to discover antibiotic 1 directly couples natural products with their biosynthetic gene clusters, providing both a means of isolating previously inaccessible natural products from uncultured bacteria and a direct link to their biosynthetic enzymes. Characterization of the antibacterially active eDNA clone CLSGl 8 led to the identification of the isonitrile containing natural product antibiotic 1, and in turn to the first isonitrile synthase, isnA.

Example 2

General Methods for Library Construction and Screening [0098] A 250 gm soil sample from Boston, MA was resuspended in 250 mL of lysis buffer containing 5 mg/mL lysozyme. After 1 h at 37 ⁰C Proteinase K and SDS were added to final concentrations of 0.5 mg/mL and 0.5%, respectively, and the temperature was raised to 50 ⁰C. After 1 h at 50 °C the SDS concentration was raised to 5%, CTAB was added to give a final concentration of 1% and the temperature was raised to 70 ⁰C. After 1 h at 70 °C the sample was removed from the water bath, allowed to cool to room temperature and then ground in a mortar and pestle for 2 minutes. Crude eDNA sample was isopropanol precipitated from the centrifuge clarified soil extract and then collected by centrifugation. High molecular weight eDNA was purified from the resuspended crude eDNA pellet by preparative gel electrophoresis (1% agarose gel, 1 h 100 volts and then overnight at 20 volts). A gel slice containing the band of high molecular weight (HMW) eDNA was cut from the preparative gel and the HMW eDNA was electroeluted from the agarose gel slice (150 V for 2h). Purified HMW eDNA was then concentrated and rinsed 2X with TE in a centrifugal concentrator (Amicon Ultra 35 kDa). 2.5 μg aliquots of purified eDNA were blunt ended in 80 μl reactions using the End-It enzyme mixture from Epicentre. 250 ng aliquots of the blunt ended eDNA were then ligated with 500 ng of precut and dephosphorylated pWEB cosmid vector. Heat-treated (70 °C/10 min) ligation reactions were packaged into lambda phage packaging extracts and transfected into E. coli. The resulting eDNA library was screened for antibacterially active clones using a top agar overlay containing Bacillus subtilis. Clones that produce a zone of growth inhibition in the top agar overlay were recovered from the assay plates by restreaking on plates containing ampicillin to kill the B. subtilis assay strain.

Example 3

Isolation of 1 from E. coli Cultures Transformed with isnA/B:

[0099] Ethyl acetate extracts from cultures of CSLGl 8.1 grown overnight in LB at 30 °C were partitioned using normal phase flash chromatography developed with 60:40

CH₂Cl₂ :hexanes to give purified 1. Compound 1 was crystallized by slow evaporation from hexanes and methylene chloride.

Example 4

NMR Structure Solution for 1

[00100] ID-NMR experiments suggested that 1 contained 8 protons and 11 carbons. ¹H-¹H

RelayH experiments indicated that six of the eight protons seen in the ¹H spectrum were part of highly deshielded two- and four-proton spin systems. ¹H-¹³C HMQC indicated that one of the two remaining protons (H-I, δ 8.58) was not bound to a carbon. The indole in 1 was easily deduced from the four-proton spin system, the finely split proton at δ 7.38 and the broad, highly deshielded NH singlet at δ 8.58. Long-range ¹H-¹³C HMBC correlations join the trans olefin (J= 14Hz) that makes up the two-proton spin system to the indole at C-3. The carbon and nitrogen in the structure could either exist as an isonitrile or an isonitrile. The presence of an isonitrile was supported by the appearance of a triplet in the ¹³C spectrum (C-IO, 107.4, J=12.6Hz) which is indicative of the ¹³C-¹⁴N coupling observed between the quaternary amine of an isonitrile and the adjacent carbon.

Example 5

General Methods Used for Feeding Studies

[00101] Synthesis of 1-3 (Adapted from D. H. R. Barton, G. Bringmann, W. B. Motherwell, Synthesis 1980, 68; and N. Hashimoto, T. Aoyama, T. Shioiri, Chem. Pharm. Bull. 1981, 29, 1475.) N-formyltryptophan (0.5 mM) and a slight molar excess of TMSCHN₂ (2.0 M in hexanes) were added to 8 mL of MeOH:toluene (1 :7). After stirring for 1 h the solvent was removed under a stream of N₂ and the product purified by normal phase flash chromatography (95:5 CH2Cl₂:Me0H). The conversion of the resulting methyl ester to 3 was described previously (N. Hashimoto, T. Aoyama, T. Shioiri, Chem. Pharm. Bull. 1981, 29, 1475.) and was carried out as they reported. The methyl ester was dissolved in 400 μl CH₂Cl₂ and then 100 μl of TEA and 37 μl POCl₃ were added. After stirring for 4 h the reaction was diluted with ice cold aqueous NaHCO₃ and then extracted with chloroform. Compound 1-3 was obtained from the chloroform extract by normal phase flash chromatography (CH₂Cl₂) (68% yield over two steps). Attempts to further purify 3 resulted in decomposition and therefore 3 was used in feeding experiments as it was obtained from a single silica column.

Example 6

Cloning and Expression of isnA and isnB as Glutathione-S-transferase Fusion Proteins [00102] isnA and isnB were cloned in pGEX-3X (Pharmacia Biotech) as glutathione-S- transferase fusion proteins. isnA was amplified from pCSLGlS using primers 5'- GCGGGATCCCCATGTTCAAAAAATCTCTTGACAG-3' and 5'-

CGCGAATTCCACAGCTCCTATCGAAGCCATT-3'. isnB was amplified from pCSLG18 using primers 5'-GCGGGATCCCCATGACACACGCTACCTTGC-S' and 5'- CGCGAATTCCTATGTTCAGAGAGCGCATGG-3'. Gel purified PCR products were digested with BamHI and EcoRI, ligated into similarly digested pGEX-3X and transformed into E. coli to give plasmids pisnA-GST and pisnB-GST, respectively.

Example 7

Natural Products from the Heterologous Expression of New Gene Clusters [00103] Using the sequence of the eDNA derived isonitrile synthase (isnA) in a BLAST search of fully sequenced bacterial genomes we identified six potential natural product biosynthetic pathways that contained isnA homologs and therefore might also produce isonitrile containing natural products (Fig. 4). None of these pathways had been previously annotated as producing isonitriles and no isonitrile containing natural products had been previously isolated from any of the bacteria from which the isnA homologs were derived. This collection of previously unstudied biosynthetic pathways is therefore representative of the pathways we might find using the DNA based screens of eDNA libraries.

[00104] The six novel pathways containing isnA homologs were investigated for their ability to confer the production of novel natural products to E. coli. A detailed analysis of the isnA containing gene clusters indicated that five of the six gene clusters contain LysR-like transcription factors (Fig. 4). The vast majority of LysR transcription factors that have been characterized to date are positive activators of transcription and depend on the binding of a small molecule activator for the induction of the promoters they control (see, Schell, M. A., Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol, 1993. 47: p. 597- 626). To circumvent this transcriptional control each gene cluster was cloned under the control of the P_tac promoter, thereby removing it from the control of its native promoter. Ethyl acetate extracts from E. coli cultures transformed with these P_tac regulated constructs were examined by TLC and LCMS for the presence of clone specific small molecules.

[00105] To date, we have isolated six novel natural products (Fig. 4) from the extracts of E. coli cultures expressing the six isnA containing biosynthetic gene clusters. In addition to an isonitrile containing tyrosine derivative (3) that is related to the tryptophan-based isonitrile (1) we originally isolated, we have now characterized N-formyl (4),isonitrile (5), and rhamnosamine (6) derivatized small molecules from these clones. The two compounds with additional rings (7, 8) contain novel heterocycles that have not yet been reported as natural products or synthetic compounds. Although the biosynthetic origin of these ring systems is not yet clear we believe that they likely arise from isonitrile containing intermediates that are produced by the isnA homologs found in these gene clusters. [00106] Although the gene clusters used in this study were derived from cultured bacteria, the same general strategy can be used to study natural product gene clusters uncovered from the screening of large eDNA libraries in sequence based screens. The work with the isnA containing pathways strongly suggests that the heterologous expression of previously unstudied gene clusters (whether from cultured or uncultured bacteria) will be a rewarding and effective strategy for the discovery of new biologically active natural products.

Example 8

Systemic Investigation of the Escherichia coli Metabolome for the Biosynthetic Origin of the Isonitrile Carbon of Formula 1

[00107] In vivo studies on the biosynthesis of natural products are often hampered by the inability to predictably control the metabolome of the producing organism. In contrast, when biosynthetic pathways are expressed in well-understood model organisms, the ability to predictably control metabolism through the use of mutant strains, allows pathways to be examined with a rigor that cannot be achieved in the wild type producing organism. The ability to systematically control Escherichia coli metabolism through the mutagenesis of genes coding for primary metabolic enzymes has enabled us to elucidate the biosynthetic origin of the isonitrile functional group found in Formula 1. Almost fifty years after the discovery of the first isonitrile functionalized natural product, xanthocillin, no consensus view of isonitrile biosynthesis has emerged from the extensive feeding studies using isonitrile-producing organisms. The origin of the isonitrile carbon found in Formula 1 is presented here. [00108] In most biosynthetic studies a hint as to the origin of a fragment can be seen in its structure, but the lone carbon of an isonitrile contains no such clue. In a normal feeding study, labeled precursors are added to an unlabeled background to decipher the origin of atoms in a molecule; however, because of the abundance of possible isonitrile precursors, we chose to use an "inverse labeling" strategy to study the origin of the isonitrile carbon. In this approach ¹²C precursors are added to a ¹³C background, eliminating the need to synthesize a ¹³C (or ¹⁴C) sample of every precursor.

[00109] Feeding studies using native isonitrile producing organisms suggested that the isonitrile carbon might be derived from an amino acid. See: R. B. Herbert, J. Mann, Tetrahedron Lett. 1984, 25, 4263; b) R. B. Herbert, J. Mann, J. Chein. Soc. Chem. Comm. 1984, 1474; c) M. S. Puar, H. Munayyer, V. Hegde, B. K. Lee, J. A. Waitz, J. Antϊbiot. 1985, 38, 530; d) J. E. Baldwin, H. S. Bansal, J. Chondrogianni, L. D. Field, A. A. Taha, V. Thaller, Tetrahedron 1985, 41, 1931; e) V. Bornemann, G. M. L. Patterson, R. E. Moore, J. Am. Chem. Soc. 1988, 110, 2339; f) C. W. J. Chang, P. J, Scheuer, Comp. Biochem. Physiol B 1990, 97, 227; g) P. J. Scheuer, Ace. Chem. Res. 1992, 25, 433; h) K. M. Cable, R. B. Herbert, A. R. Knaggs, J. Mann, J. Chem. Soc. Perkin Trans. 1 1991, 595. To test this hypothesis we carried out feeding studies using isnAJB, the biosynthetic pathway for 1 , expressed in E. coli amino acid auxotrophs. Strains used in the amino acid feeding studies are referenced as follows: strain name (E. coli genetic stock center (CGSC) number) reference, a) Strain AB 1359 (CGSC 1359) A. L. Taylor, M. S. Thoman, Genetics 1964, 50, 659; b) Strain S1228 (CGSC 6432) A. del Campillo- Campbell, A. Campbell, J. Bacteriol. 1982, 149, 469; c) Strain DG30 (CGSC 5799) D. H. Gelfand, R. A. Steinberg, J. Bacteriol. 1977, 130, 429; d) Strain KL285 (CGSC 4310) S. J. Clarke, B. Low, W. Konigsberg, J. Bacteriol. 1973, 113, 1096. Each auxotroph was grown in minimal media containing ³C-glucose, ¹²C-tryptophan and the appropriate ¹²C-amino acids needed to compensate for the auxotrophies. Compound 1 isolated from these cultures always had an observed m/z of 170, which is consistent with its containing 10 carbons from ¹²C? tryptophan; one carbon, the isonitrile carbon, from ¹³C-glucose; and no carbons from the other

C-amino acids added to the media. All feeding experiments were carried out in the presence of ¹²C-tryptophan, the tryptophan portion of 1 is therefore always derived from a ¹²C-carbon source (Figure 5). See Example 5 for a more detailed description of the feeding experiment methods. Using this strategy the certain amino acids in the overview of the E. coli metabolome shown in Figure 6 were ruled out as precursors in the biosynthesis of the isonitrile. [00110] When none of the amino acids tested labeled the isonitrile carbon, we chose to systematically examine the remainder of the E. coli metabolome for the source of the isonitrile carbon. Figure 6 summarizes the differential labeling of 1 in a variety of E. coli strains deficient in key primary metabolic enzymes. Each strain was transformed with isnAJB and grown in an isotopically defined mixture of carbon sources such that unique regions of the metabolome were labeled in isotopically distinct manners. This approach allowed us to rapidly investigate the entire E. coli metabolome for the source of the isonitrile carbon. When the carbon flow between early and late glycolysis is blocked by a mutation in glyceraldehyde-3P dehydrogenase (GAPDH, gapA) the E. coli metabolome is dissected into two distinct carbon pools (Figure 6). Compound 1 isolated from a gapA mutant transformed with isnAJB and grown in ¹³C-glucose, ¹²C-malate from the TCA cycle and ¹²C-tryptophan has an observed m/z of 170. The isonitrile carbon is therefore derived from a descendant of ¹³C-glucose before the conversion of glyceraldehyde-3P to 1,3-bis-phosphoglycerate by GAPDH.

[00111] The pentose phosphate pathway is central to the remaining portion of the E. coli metabolome from which the isonitrile carbon must be derived (Figure 6). We tested major metabolic branches that arise from the pentose phosphate pathway as possible sources of the isonitrile carbon. G. A. Sprenger, Arch. Microbiol. 1995, 164, 324. E. coli strains containing mutations in genes that represent the metabolic entrance points into the following pentose pathway branch points (G. A. Sprenger, Arch. Microbiol. 1995, 164, 324): glycerol derived metabolites (fructose- 1,6-bisphosphate aldolase, /δα), sugar phosphates (mannose-6-phosphate isomerase, manA and L-glutamine:D-fructose-6-phosphate aminotransferase, glmS), nucleic acids (ribose-phosphate diphosphokinase,/»ra and aspartatetranscarbamylase, pyrB) and glucose derived metabolites (phosphoglucose isomerase, pgi and glucose 6-phosphate-l -dehydrogenase, zwf), were transformed with isnA/B (Figure 6). Strains used in the differential labeling studies are referenced as follows: relevant mutation(s), strain name (CGSC number) reference, a) gapA, DSl 12 (CGSC 7563) F. D. Seta, S. Boschi-Muller, M. L. Vignais, G. Branlant, J Bacteriol. 1997, 179, 5218; b)fba, JM2087 (CGSC 6806) E. O. Davis, M. C. Jones-Mortimer, P. J. Henderson, J. Biol Chem. 1984, 259, 1520; c) manA, JE5511 (CGSC 5505) Y. Hirota, H. Suzuki, Y. Nishimura, S. Yasuda, Proc. Natl. Acad. Set U. S. A. 1977, 74, 1417; d) manA, F500/GMS724 (CGSC 6673) M. Novel, G. Novel, J. Bacteriol. 1976, 127, 406; e) glmS, El 11 (CGSC 5393) H. C. Wu, T. C. Wu, J. Bacteriol. 1971, 105, 455; f) zwf and pgi - DF2000 (CGSC 4873) D. G. Fraenkel, S. Banerjee, Genetics 1972, 71, 481; g)prs HO733 B. Hove- Jensen, J. Bacteriol. 1996, 178, 714; h)pyrB, Hfr 3000 pyr (CGSC 6851) J. R. Beckwith, A. B. Pardee, R. Austrian, F. Jacob, J. MoI. Biol. 1962, 5, 618; i) tpi, AA200 (CGSC 5570) A. Anderson, R. A. Cooper, J. Gen. Microbiol. 1970, 62, 329; j) zwf, K10-15-16 (CGSC 4848) Each strain was grown in C-tryptophan, C-glucose and a C-carbon source(s) that would supply an isotopically distinct carbon pool to the blocked region of metabolism (Figure 6). In each feeding experiment we observed that the isonitrile carbon was derived from the carbon source that is able to flux into the pentose phosphate pathway and not the blocked region of metabolism. In the case of manA and glmS, each of which block the exit of metabolites from the pentose phosphate pathway but not the entrance, we detected a mixture of labeled and unlabeled isonitriles (Figure 6). As with the other feeding studies this suggests that the isonitrile carbon must be derived from the carbon source that is able to flux into the pentose phosphate pathway and not the blocked region of metabolism. Glycerol metabolites, fba, sugar phosphates, manA and glmS, glucose metabolites, pgi/zwf and nucleic acids, prs oxpyrB in Figure 6 could therefore now also be excluded as sources of the isonitrile carbon. Taken together these feeding studies indicate that the isonitrile carbon is likely derived from one of the eight sugars directly involved in the pentose phosphate shunt (Figure 6, black), the major region of metabolism that was not eliminated by our feeding experiments. Metabolites downstream of xylulose-5P and ribulose-5P were not ruled out with these experiments.

[00112] Additional feeding experiments were performed to determine which of the carbons present in early glycolytic hexose intermediates feeds directly into the isonitrile carbon. In a triose phosphate isomerase (tpϊ) mutant the C1-C2-C3 and C4-C5-C6 carbons of early glycolytic intermediates can be differentially labeled using glycerol and glucuronic acid. Compound 1 isolated from cultures of a tpi knockout transformed with isnA/B and grown in media containing ¹³C-glycerol and ¹²C-glucuronic acid (Figure 6, brown) has an observed m/z of 170. The isonitrile carbon must therefore arise from ¹³C-glycerol which corresponds to the C1-C2-C3 portion of early glycolytic hexose intermediates (Figure 6, black). The glucuronic acid derived carbons, which are colored brown in Figure 6 could therefore also be eliminated as a possible source of the isonitrile carbon.

[00113] A glucose-6P dehydrogenase (zwf) deficient strain of E. coli, which helps prevent the scrambling of labeled sugars in the pentose phosphate shunt by blocking the decarboxylation of 6P-gluconate, was used to determine which of the three remaining carbons is incorporated into the isonitrile. The zw/knockout strain grown on Cl labeled ¹³C-glucose did not produce ¹³C labeled 1 while cultures grown on either universally labeled or C2 labeled ¹³C-glucose produced almost exclusively ¹³C labeled 1 (Figure 7). In this system the C2 carbon of glucose specifically labels only six major sugars that were not excluded as a source of the isonitrile carbon by our other feeding studies. The six remaining metabolites (fructose-6P, fructose- 1,6-bis-P, ribulose- 5P, ribose-5P, xylulose-5P and sedoheptulose-7P) are shown in Figure 6 and the C2 position of each is highlighted in green (C). Five of these six sugar phosphates are commercially available and were therefore tested in in vitro reconstitution experiments. Xylulose-5P was not tested because it was not commercially available. The in vitro reconstitution experiments using purified isnA, isnB, tryptophan, α-ketoglutarate and both ribulose-5P and ribose-5P were found to produce a compound with the same retention time and mass as naturally occurring 1. Other commercially available C4 to C7 sugars and sugar phosphates were tested in the same reconstitution system and only arabinose-5P was found to result in isonitrile production. The following sugars were tested in reconstitution experiments: ribulose-5-P, ribose-5-P, arabinose- 5P₅ erythrose-4P, ribulose, ribose, arabinose, xylose, xylulose, glucose-6P, glucosamine-6P, 6P- gluconate, fructose, fructose-6P, fructose- l :6-bis-P, mannose, mannose-6P₅ allose, sedoheptulose-7P, 2-deoxyribose-5P. Only a small quantity of 1 is produced in the reconstitution system. Experiments are being performed to look for possible cofactor and Fe requirements. See other Examples information for cloning, protein expression and reconstitution procedures.

[00114] To investigate the source of the isonitrile carbon in vitro, Cl and C2 labeled ¹³C- ribose were phosphorylated with recombinant E. coli ribokinase (rbsK) and then each of the products was used for the in vitro biosynthesis of 1. See, a) J. N. Hope, A. W. Bell, M. A. Hermodson, J. M. Groarke, J, Biol. Chem. 1986, 261, 7663; b) C. E. Andersson, S. L. Mowbray, J. MoI. Biol. 2002, 315, 409. As suggested by the in vivo feeding experiments, the C2 carbon of the sugar phosphate is used in the enzymatic synthesis of 1. Cl labeled ribose leads to the production of unlabeled 1 (w/z=^:169) while C2 labeled ribose leads to a ¹³C labeled isonitrile (m/z=l70). The tautomerization of ribose-5P and arabinose-5P to ribulose-5P either in solution or in an enzyme-assisted fashion could explain the use of all three sugars in the in vitro system (Figure 4). 2-deoxyriobose-5P, which can not tautomerize to the equivalent keto sugar, does not serve as a substrate for the enzymatic synthesis of 1 using this system. [00115] The ability to control E. coli metabolism through the systematic use of strains carrying mutations in primary metabolic pathways allowed us to identify the origin of the isonitrile carbon in 1 as the C2 carbon of ribulose-5P or a tautomerically equivalent sugar. As shown previously the isonitrile nitrogen can be traced to the amine of tryptophan. The isonitrile present in many known microbial natural products can also theoretically be traced to the free amine of an amino acid. The proposed biosynthetic scheme for 1 may therefore be general for the biosynthesis of microbial derived isonitriles. The differential labeling of natural products produced by microorganisms carrying mutations in primary metabolic genes that lead to predictable changes in the microbial metabolome can now be carried out in a systematic fashion in many model microorganisms and could be a generally useful strategy for studying the biosynthetic origin of individual atoms found in microbial secondary metabolites. [00116] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. AU published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[00117] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. SEQUENCE LISTING SEQ ID NO: 1

ATGTTCAAAAAATCTCTTGACAGACAGCCGAAGTCGCGCTATACTATTTTAGAACAT

GTTCTAAAATTTCAATCGGTTCGCGAAGAGCATGTTTCGAGAGAGGACGCAGGACG

AATGAACACCTGGAATGCACCGCTGCCGCAGTCTGACTGGAAACACTCCGCATGGG

GGTTGCCGGATCTGTCTATCGGGCGAAGGCCTGGTTGGAATCCGCAACAGAGCCCC

GGTGCTGAAACAAAGACAGACGGTACTGCCAGCGAACTGGCGCGCGAAGTGCTCCA

ATGTCTGTTCCAACATCGGCGTCTGCTCCCGGAGGCAGGCGCCTGCGCCGATACACC

CTGTTCTCTCTGTCTGGCGCCGCATCTGTCGAAAGTGCGCCGCGCCATCGAGCGCCG

GGAACCGATCCACCTGGTTTTGCCTGCCTTTCCTGCCAAGTCGCCCAGCAGACGCAA

GACCTTCGGGCCGTTGCCGGACAAGGCCGAAGAGCTGGCATTGGAGTCTCTGCAAA

GCCTGTGCAATGCTATACAGCTCCTCTATGCGCCGGGAGCCCGCCTCACCCTCTGCT

CTGATGGGCGCGTCTTCAGCGATCTGGTCGGCGTCACCGAAGAGGATGTCACACGCT

ACGGCCAGGAAATCCGCACCATGATCCGCCGTCTGGGCCTGCGCTCGCTGGATACCT

TCCATCTGGAAGACCGCTTTACCGGCGACACCTTCCAGGAGATGCGAGACCTGATG

GAAACTCAATACGCCGAACCCTATGCGCAATTTGCTGCCCGCGTGCAGAAAGACAA

CCCGATCCTGCTCAACGGGATCGAACGCTTCCTGCTCGAAGAGCGTTCGCCCGACGC

CCCGGCGCTGAGCAAAACGCAGGCCCGTAAGGAGGCGCGAACGCGAGCCATCGAA

GTCGTTCGCCGCAGTGAGGCATGGGGACGACTGATCGCCGAGCGTTTTCCTTCTGCG

CTCCGCCTCTCCATCCATCCCCAGATGCCGCACTCCCCCAAAATCGGGCTGAGAATG

GGAGAACAGGGCAAAGATCTCTGGATCACCCCCTGGCATGGCGTCGCAGTGCAGGA

AGCCGGAGGGTGGACCTTGCGCAAACGGGAAGAAGCGGAAGCTCTGGGAGCGCAA

CTCGTAGAGCGCGAAGGGCGCCCTTACTATTACACGCTGGAGGTAGGACGGTGA

SEQ ID NO: 2 AMINO ACID SEQUENCE OF isnA

MFKKSLDRQPKSRYTILEHVLKFQSVREEHVSREDAGRMNTWNAPLPQSDWKHSAWG

LPDLSIGRRPGWNPQQSPGAETKTDGTASELAREVLQCLFQHRRLLPEAGACADTPCSL

CLAPHLSKVRRAIERREPIHLVLPAFPAKSPSRRKTFGPLPDKAEELALESLQSLCNAIQLL

YAPGARLTLCSDGRVFSDLVGVTEEDVTRYGQEIRTMIRRLGLRSLDTFHLEDRFTGDTF

QEMRDLMETQYAEPYAQFAARVQKDNPILLNGIERFLLEERSPDAPALSKTQARKEART

RAIEVVRRSEAWGRLIAERFPSALRLSIHPQMPHSPKIGLRMGEQGKDLWITPWHGVAV

QEAGGWTLRKREEAEALGAQLVEREGRPYYYTLEVGR

SEQ ID NO: 3

ATGACACACGCTACCTTGCAAAGCAACACAACCACGGAACGCTGGCAGCAGCAGTT

GCTGCCCACTTTTGGAATGCTGGTTCATGCTCGCGAGGCCGGTACGCCTCTCTCCTCC

CTGCCCGCCGACACCCTGCGCGCCTGGGCGGAAGCGGAGAGCCTGGTGATCCTGCG

CGGCTTCGCTCCTCCGGAAGGTGATGCGTTACCCTCCTACTGTCGCGGACTGGGCGA

TCTGCTCGAATGGGATTTCGGCGCGATCAACAACCTCCAGGCGCAATCGGAAGCGA

AAAACTACCTCTTCACCAATCGGGCCGTTCCCTTTCACTGGGATGGAGCGTTCGTCG GGCGCATCCCGCACTGGATCTTCTTCCACTGCGCCTCGGCCCCCGAGGAGAATACCG

GCGGCGAAACGCTCTTCTGCCACACCCCGCTCCTCCTGGAAGCGGTCTCCGCTGCCG

GGCGAGCGCAATGGGAGAACATCTCCATCCGCTACTCCACGGAGAAACTGGCGCAC

TACGGAGGCAGTTTTACCTCCCCGCTCCTCGCCGCCCATCCCATTCACGGCCAGACC

ATCCTGCGCTATGCCGAGCCGGTGAACGACCTCAACCCGGTCCATCTGGAGATCCAG

GGCCTACCCGAGGAGAGCCATACCGCCTTTCTGGAAGGGATGCACACGCGCCTCTA

CGACCCTGCCGTCTGTTATGCCCATGCCTGGCAGACCGGCGACATCGTCATCGCCGA

CAACTTCACGCTGCTCCATGGCCGACGCGCGTTTCTCCGCCCGGAGAGCCGCCATCT

GCGCCGCGTCAACATTCTGTAA

SEQ ID NO: 4 AMINO ACID SEQUENCE OF isnB

MTHATLQSNTTTERWQQQLLPTFGMLVHAREAGTPLSSLPADTLRAWAEAESLVILRGF APPEGDALPSYCRGLGDLLEWDFGAINNLQAQSEAKNYLFTNRAVPFHWDGAFVGRIP HWIFFHCASAPEENTGGETLFCHTPLLLEAVSAAGRAQWENISIRYSTEKLAHYGGSFTS PLLAAHPIHGQTILRYAEPVNDLNPVHLEIQGLPEESHTAFLEGMHTRL YDP AVCYAHA WQTGDIVIADNFTLLHGRRAFLRPESRHLRRVNIL*

SEQ ID NO: 5 THE COMBINED SEQUENCE OF isnA AND isnB coding regions

ATGTTCAAAAAATCTCTTGACAGACAGCCGAAGTCGCGCTATACTATTTTAGAACAT

GTTCTAAAATTTCAATCGGTTCGCGAAGAGCATGTTTCGAGAGAGGACGCAGGACG

AATGAACACCTGGAATGCACCGCTGCCGCAGTCTGACTGGAAACACTCCGCATGGG

GGTTGCCGGATCTGTCTATCGGGCGAAGGCCTGGTTGGAATCCGCAACAGAGCCCC

GGTGCTGAAACAAAGACAGACGGTACTGCCAGCGAACTGGCGCGCGAAGTGCTCCA

ATGTCTGTTCCAACATCGGCGTCTGCTCCCGGAGGCAGGCGCCTGCGCCGATACACC

CTGTTCTCTCTGTCTGGCGCCGCATCTGTCGAAAGTGCGCCGCGCCATCGAGCGCCG

GGAACCGATCCACCTGGTTTTGCCTGCCTTTCCTGCCAAGTCGCCCAGCAGACGCAA

GACCTTCGGGCCGTTGCCGGACAAGGCCGAAGAGCTGGCATTGGAGTCTCTGCAAA

GCCTGTGCAATGCTATACAGCTCCTCTATGCGCCGGGAGCCCGCCTCACCCTCTGCT

CTGATGGGCGCGTCTTCAGCGATCTGGTCGGCGTCACCGAAGAGGATGTCACACGCT

ACGGCCAGGAAATCCGCACCATGATCCGCCGTCTGGGCCTGCGCTCGCTGGATACCT

TCCATCTGGAAGACCGCTTTACCGGCGACACCTTCCAGGAGATGCGAGACCTGATG

GAAACTCAATACGCCGAACCCTATGCGCAATTTGCTGCCCGCGTGCAGAAAGACAA

CCCGATCCTGCTCAACGGGATCGAACGCTTCCTGCTCGAAGAGCGTTCGCCCGACGC

CCCGGCGCTGAGCAAAACGCAGGCCCGTAAGGAGGCGCGAACGCGAGCCATCGAA

GTCGTTCGCCGCAGTGAGGCATGGGGACGACTGATCGCCGAGCGTTTTCCTTCTGCG

CTCCGCCTCTCCATCCATCCCCAGATGCCGCACTCCCCCAAAATCGGGCTGAGAATG

GGAGAACAGGGCAAAGATCTCTGGATCACCCCCTGGCATGGCGTCGCAGTGCAGGA

AGCCGGAGGGTGGACCTTGCGCAAACGGGAAGAAGCGGAAGCTCTGGGAGCGCAA

CTCGTAGAGCGCGAAGGGCGCCCTTACTATTACACGCTGGAGGTAGGACGGTGAGG

GAGCAGAGCGTTCGCGAGGCGCGTGACCGTTTTCTGGCGCGCAATGGCTTCGATAG

GAGCTGTTACGGCGCGCCCCTGCTTCCCCTGCACATCGGGCGATGGACCTGGAACGT

GCGGAATCCCGGCCTGTTGCACTGGCACGACCTGCACCATGTCGCCACCGGATTTAA TACCACCTTATGGGGTGAGGCGGCCATCAGCGCCTTTGAACTGCGCGCCGGATGCCC

CAACCGCACGGTCTTCTGGCTCTGCCTGGGCGCCCTGACGCTGGGGATGCTGCGTAT

GCCGCGCACCATGCTCCGCATCTGGCGCAGTGCGGCCGGAGCACGCACGCTCTACA

CCGATTCGCCCGACTATGAGACCCTGCTTACCATGACGGTGAGCGAACTGCGCGATT

ACCTGCGCCTGCCCCCCGAAGAGCTTGCCGCATTTCTGATTCAGGAGAAACAAAATG

ACACACGCTACCTTGCAAAGCAACACAACCACGGAACGCTGGCAGCAGCAGTTGCT

GCCCACTTTTGGAATGCTGGTTCATGCTCGCGAGGCCGGTACGCCTCTCTCCTCCCTG

CCCGCCGACACCCTGCGCGCCTGGGCGGAAGCGGAGAGCCTGGTGATCCTGCGCGG

CTTCGCTCCTCCGGAAGGTGATGCGTTACCCTCCTACTGTCGCGGACTGGGCGATCT

GCTCGAATGGGATTTCGGCGCGATCAACAACCTCCAGGCGCAATCGGAAGCGAAAA

ACTACCTCTTCACCAATCGGGCCGTTCCCTTTCACTGGGATGGAGCGTTCGTCGGGC

GCATCCCGCACTGGATCTTCTTCCACTGCGCCTCGGCCCCCGAGGAGAATACCGGCG

GCGAAACGCTCTTCTGCCACACCCCGCTCCTCCTGGAAGCGGTCTCCGCTGCCGGGC

GAGCGCAATGGGAGAACATCTCCATCCGCTACTCCACGGAGAAACTGGCGCACTAC

GGAGGCAGTTTTACCTCCCCGCTCCTCGCCGCCCATCCCATTCACGGCCAGACCATC

CTGCGCTATGCCGAGCCGGTGAACGACCTCAACCCGGTCCATCTGGAGATCCAGGG

CCTACCCGAGGAGAGCCATACCGCCTTTCTGGAAGGGATGCACACGCGCCTCTACG

ACCCTGCCGTCTGTTATGCCCATGCCTGGCAGACCGGCGACATCGTCATCGCCGACA

ACTTCACGCTGCTCCATGGCCGACGCGCGTTTCTCCGCCCGGAGAGCCGCCATCTGC

GCCGCGTCAACATTCTGTAAGGA

Claims

CLAIMSWhat is claimed is:

1. An isolated or recombinant polynucleotide sequence comprising at least one open reading frame which encodes one or more enzymes, or a functional portion thereof having catalytic activity, that catalyze one or more steps in the conversion of an amine to an isonitrile.

2. An isolated or recombinant polynucleotide sequence of Claim 1 encoding an isonitrile synthase A or a functional portion thereof having catalytic activity.

3. An isolated or recombinant polynucleotide sequence of Claim 2 substantially homologous to an isonitrile synthase polynucleotide sequence of SEQ ID NO: 1.

4. An isolated or recombinant polynucleotide sequence of Claim 1 encoding an isonitrile synthase substantially homologous to the sequence of SEQ ID NO: 2, or a functional portion thereof having catalytic activity.

5. An isolated or recombinant polynucleotide sequence of Claim 1 encoding an isonitrile synthase having the sequence of SEQ ID NO:2.

6. An isolated or recombinant polynucleotide sequence of Claim 1 having the sequence of SEQ ID NO: 1.

7. A recombinant vector comprising a polynucleotide sequence of Claim 2 and a control sequence operably linked to express said polynucleotide sequence.

8. A host cell transformed with a vector of Claim 7 or progeny of said host cell.

9. The host cell of Claim 8 wherein the host cell is selected from E. coli, the genus Pseudomonas, the genus Erwinia, or the genus Streptomyces.

10. A method for producing a polypeptide comprising an isonitrile synthase comprising the amino acid sequence of SEQ ID NO: 2 or a functional portion or homologs thereof having catalytic activity, comprising maintaining a host cell of Claim 8 under conditions suitable for expression of the nucleic acid whereby the encoded polypeptide is produced.

11. The method of Claim 10 further comprising the step of isolating the polypeptide.

12. An isolated or recombinant polypeptide that catalyze one or more steps in the conversion of tryptophan to an 3-(isonitrileethylene)indole.

13. An isolated or recombinant polypeptide of Claim 12 that is an isonitrile synthase A or a functional portion thereof having catalytic activity.

14. An isolated or recombinant polypeptide of Claim 12 that is an isonitrile synthase that is substantially homologous to the sequence of SEQ ID NO:2, or a functional portion thereof having catalytic activity.

15. An isolated or recombinant polypeptide of Claim 12 that is an isonitrile synthase of SEQ ID NO:2.

16. An isolated or recombinant polynucleotide sequence of Claim 1 encoding an isonitrile synthase B or a functional portion thereof having catalytic activity.

17. An isolated or recombinant polynucleotide sequence of Claim 16 substantially homologous to an oxidative decarboxylase polynucleotide sequence of SEQ ID NO: 3.

18. A recombinant vector comprising a polynucleotide sequence of Claim 17 and a control sequence operably linked to express said polynucleotide sequence.

19. A host cell transformed with a vector of Claim 18 or progeny of said host cell.

20. The host cell of Claim 19 wherein the host cell is selected from E. coli the genus Pseudomonas, the genus Erwinia or the genus Streptomyces.

21. A method for producing a polypeptide comprising an oxidative decarboxylase (isnB) comprising the amino acid sequence of SEQ ID NO: 4 or a functional portion or homologs thereof having catalytic activity, comprising maintaining a host cell of Claim 20 under conditions suitable for expression of the nucleic acid whereby the encoded polypeptide is produced.

22. The method of Claim 20 further comprising the step of isolating the polypeptide.

23. An isolated or recombinant polypeptide of Claim 12 that is an oxidative decarboxylase or a functional portion thereof having catalytic activity.

24. An isolated or recombinant polypeptide of Claim 12 that is an isonitrile synthase that is substantially homologous to the sequence of SEQ ID NO:4, or a functional portion thereof having catalytic activity.

25. An isolated or recombinant polypeptide of Claim 12 that is an isonitrile synthase of SEQ ID NO:4.

26. An isolated or recombinant polynucleotide of claim 1 comprising an isnA (SEQ ID NO: 1) gene and isnB (SEQ ID NO: 3) gene and one or more control sequences positioned to express such genes.

27. A recombinant vector comprising a polynucleotide of Claim 26 and one or more control sequences positioned to express such genes.

28. The recombinant vector of Claim 27 comprising functional portions of the isnA (SEQ ID NO: 1) and isnB (SEQ ID NO: 3) genes, said functional portions having catalytic activity.

29. A host cell comprising the vector of Claim 27.

30. An isolated or recombinant polynucleotide sequence comprising at least one open reading frame which encodes one or more enzymes, or a functional portion thereof having catalytic activity that catalyze one or more steps is the formation of an isonitrile- containing compound from an amine-containing compound.

31. A vector comprising the polynucleotide sequence of Claim 30 and at least one control sequence positioned to express said polynucleotide sequence.

32. The vector of Claim 31 wherein said at least one control sequence is a promoter.

33. A host cell comprising the vector of Claim 32, or progeny of said host cell.

34. A host cell of claim 33 wherein said host cell produces an isonitrile-containing compound and wherein said host cell, in its naturally occurring non-recombinant state can not produce an isonitrile containing compound.

35. A method of producing an isonitrile-containing compound comprising the steps of: a) providing a cell culture comprising recombinant host cells according to Claim 34; and b) maintaining said culture under conditions in which isonitrile-containing compounds are biosynthetically produced.

36. The method of Claim 35 further comprising the step of isolating and purifying the isonitrile-containing compounds of step (b).

37. A method for identifying bioactive, isonitrile-containing natural products comprising the steps of: a) providing a cell culture comprising recombinant host cells according to Claim 19; b) maintaining said culture under conditions in which isonitrile-containing compounds are biosynthetically produced; and c) assaying the culture for the presence of bioactive compounds.

38. A method of synthesizing an isonitrile -containing compound comprising the step of contacting an enzyme of Claim 12 with an amine-containing substrate under conditions in which the isonitrile-containing compound is produced.