Heterologous Hosts

Abstract

This invention is related to bacterial engineering and the heterologous expression of useful compounds. In particular, the invention relates to a heterologous host that has been engineered for expression of a gene which is capable of polyketide or non-ribosomal peptide synthesis. Methods of treating cancer are also disclosed.

Claims

1. An E. coli Nissle 1917 host cell comprising a genetic engineering that provides heterologous expression of one or more genes capable of polyketide synthesis (PKS), non-ribosomal peptide synthesis (NRPS), or hybrid polyketide-NRP synthesis.

2. The E. coli Nissle 1917 host cell according to claim 1, wherein the host cell is a heterologous species with respect to a species from which the one or more genes capable of polyketide, non-ribosomal peptide or hybrid polyketide-NRP synthesis is derived.

3. The E. coli Nissle 1917 host cell according to claim 1, wherein the polyketide, NRP, or hybrid polyketide-NRP synthesis is not naturally produced in said host cell.

4. The E. coli Nissle 1917 host cell according to claim 1, wherein the genetic engineering provides heterologous expression of all members of a gene cluster capable of polyketide, non-ribosomal peptide (NRP), or hybrid polyketide-NRP synthesis.

5. The E. coli Nissle 1917 host cell according to claim 1, wherein the genetic engineering comprises transformation of E. coli Nissle 1917 with a gene or gene cluster from one or more organisms selected from the group consisting of myxobacteria, Agrobacterium tumefaciens, Burkholderia and Stigmatella aurantiaca.

6. The E. coli Nissle 1917 host cell according to claim 5, wherein the myxobacteria is selected from Polyangium and Sorangium cellulosum.

7. The E. coli Nissle 1917 host cell according to claim 4, wherein the gene cluster is a glidobactin gene cluster, a epothilone gene cluster, a tubulysin gene cluster, a disorazol(e) gene cluster, a salinomycin gene cluster, a myxochromide S gene cluster, a myxothiazol gene cluster, or a combination of the genes comprising these gene clusters.

8. The E. coli Nissle 1917 host cell according to claim 7, wherein the host cell expresses glidobactin A, epothilone, myxochromide, colibactin, tubulysin, disorazol(e) or a functional derivative thereof.

9. The E. coli Nissle 1917 host cell according to claim 1, wherein the one or more genes are found in nature in the gene cluster.

10. The E. coli Nissle 1917 host cell according to claim 1, wherein the gene cluster comprises a genetic engineering.

11. The E. coli Nissle 1917 host cell according to claim 10, wherein the genetic engineering comprises inclusion of one or more genes or one or more domains of genes from a heterologous gene cluster in the gene cluster.

12. The E. coli Nissle 1917 host cell according to claim 1, wherein the genetic engineering comprises transformation of E. coli Nissle 1917 with a nucleic acid encoding a pPant transferase.

13. The E. coli Nissle 1917 host cell of claim 12, wherein the genetic engineering comprises transformation of E. coli Nissle 1917 with a nucleic acid encoding MtaA pPant transferase.

14. The E. coli Nissle 1917 host cell of claim 13, wherein expression of MtaA pPant transferase is regulated by a tetracycline inducible promoter.

15. A compound with a molecular weight selected from 572.2, 678.3 and 671.3 obtainable by expressing MtaA pPant transferase in E. coli Nissle 1917.

16. A method for generating a E. coli Nissle 1917 host cell comprising transforming E. coli Nissle 1917 with a heterologous gene or gene cluster capable of polyketide, non-ribosomal peptide (NRP), or hybrid polyketide-NRP synthesis.

17. A method of treatment comprising administering the E. coli Nissle 1917 host cell of claim 1 to a subject.

18. A method of treatment comprising administering the compound of claim 15 to a subject.

19. A method of treating cancer comprising administering the E. coli Nissle 1917 host cell of claim 1 to a tumor.

20. A method of treating or preventing cancer comprising administering the compound of claim 15 to a subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1. The structure of Glidobactin A.

[0081] FIG. 2. Glidobactin (glb) gene cluster.

[0082] FIG. 3. The final pGB-amp Ptet-glb plasmid for the expression of Glidobactin A.

[0083] FIG. 4. The pGB-15A-Tpase-neo-amp Ptet-syl plasmid comprising p15A origin and transposase cassette.

[0084] FIG. 5. The pGB-15A-Tpase-neo-amp Ptet-glb plasmid for integration into the chromosome of DSM7029 comprising p15A origin and transposase cassette.

[0085] FIGS. 6A, 6B, and 6C. Expression constructs for the expression of epothilone. The complete epothilone gene cluster was cloned between IRs for transposition. The transposase gene and oriT were cloned into the vector backbone. The top plasmid (FIG. 6A) has the constitutive Tn5 promoter. The middle plasmid (FIG. 6B) has the tetracycline inducible promoter Ptet. The lower plasmid (FIG. 6C) has the toluic acid inducible promoter.

[0086] FIGS. 7A, 7B, and 7C. HPLC/MS analysis of epothilone production by DSM7029 wildtype. Base peak chromatogram (m/z 100-1100); FIG. 7A) positive mode. FIG. 7B) Extracted-Ion-Chromatogram m/z 478 from Epothilone C. FIG. 7C) Extracted-Ion-Chromatogram m/z 492 from Epothilone D.

[0087] FIGS. 8A, 8B, and 8C. HPLC/MS analysis of epothilone production by DSM7029 kan1, with epothilone gene cluster. Base peak chromatogram (m/z 100-1100); FIG. 8A) positive mode. FIG. 8B) Extracted-Ion-Chromatogram m/z 478 from Epothilone C. FIG. 8C) Extracted-Ion-Chromatogram m/z 492 from Epothilone D.

[0088] FIGS. 9A, 9B, and 9C. FIG. 9A) HPLC/MS analysis of epothilone production by DSM7029 kan1, with epothilone gene cluster. FIG. 9B) MS/MS analysis from epothilone C fragmentation pattern for comparison to FIG. 9C) epothilone C from internal compound library.

[0089] FIGS. 10A, 10B, and 10C. FIG. 10A) HPLC/MS analysis of epothilone production by DSM7029 kan1, with epothilone gene cluster. FIG. 10B) MS/MS analysis from epothilone D fragmentation pattern for comparison to FIG. 10C) epothilone D from internal compound library.

[0090] FIG. 11A and FIG. 11B, respectively. pRK2-apra-km and pRK2-genta-km constructs comprising oriV, apramycin or gentamycin resistance and replicon (TrfA).

[0091] FIG. 12. pPm-mchS oriV-oripUS plasmid comprising mchS gene cluster and toluic acid inducible promoter.

[0092] FIG. 13. pPtet-mchS oriV-oripUC plasmid comprising mchS gene cluster and tetracycline inducible promoter.

[0093] FIGS. 14A and 14B. HPLC profiles from extracts of FIG. 14A) the A. tumefaciens C58::pPtet-mchS oriV-oripUS mutant strain and FIG. 14B) A. tumefaciens C58 wild-type. Diode array detection at 400 nm. Peaks are numbered as follows; 1Myxochromide S.sub.1, 2Myxochromide S.sub.2, 3Myxochromide S.sub.3. Peaks marked with an asterisk are assumed to be Myxochromide S derivatives as well.

[0094] FIG. 15. p15A-oriV-apra-Tn5-neo Epo epothilone expression plasmid with Tn5 promoter.

[0095] FIG. 16. Map of MtaA expression construct. The MtaA gene was cloned from Myxothiazol gene cluster in Stigmatella aurantiaca DW4/3-1 strain (Silakowski et al., 1999; Gaitatzis et al., 2001). It activates both PKS and NRPS enzymes. The sequence of the gene and encoded protein are given as SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

[0096] FIG. 17. In vivo activity of E. coli Nissle 1917 clones with or without over expression of MtaA. 10 nude mice bearing human neck and head tumors (UT-SCC-5) for each group were injected in tumor (i.t.) with 1.times.10.sup.7 bacterial cells in PBS in 5 l volume. PBS alone was used as negative control. Tumors were harvested after 3 weeks and tumor weight was measured. The colonization of bacterial cells in tissues (tumor, liver and kidney) was measured by plating the extract from homogenized tissues. Colonization data (not shown) is similar to the previously published data (Stritzker et al., 2007).

[0097] FIGS. 18A and 18B. New compounds produced in E. coli Nissle 1917 after over expression of MtaA gene. Three main compounds with MW 572.2, 678.3 and 671.3 are present only in clones overexpressing MtaA. There are other new compounds produced in a relative smaller quantity (data not shown).

[0098] FIGS. 19A, 19B, and 19C. E. coli Nissle 1917 colibactin gene cluster and its engineering. FIG. 19A) The original gene cluster includes 16 genes that are a mixture of PKS and NRPS. FIG. 19B) To activate the pPant transferase gene, a constitutive promoter plus chloramphenicol resistance gene (cm) was inserted in front of the gene using homologous recombination. FIG. 19C) A tetracycline inducible promoter was added in front of the PKS gene cluster.

[0099] FIG. 20. In vivo activity of E. coli Nissle 1917 with activation of endogenous pPant transferase. The experiment was done as described in FIG. 17.

[0100] FIGS. 21A, 21B, 21C, 21D, 21E, 21F, and 21G. In vitro test of E. coli Nissle 1917 with additional exogenous pPant transferase MtaA. The co-incubation protocol for tumor cells and bacterial cells was followed with method described by Putze et al., 2009 but human U-2 OS osteosarcoma cells were used. FIGS. 21A-21F) Giemsa staining of tumor cells incubated with different bacterial strains and without bacterial cells. FIG. 21G) MTT assay of the tumor cells to show the growth inhibition.

[0101] FIG. 22. Glb gene cluster plasmid for expression E. coli. Nissle 1917-cm. The complete glb gene cluster was cloned under tetracycline inducible promoter using RecET homologous recombination. This plasmid can replicate in E. coli Nissle 1917 and the genes can be expressed following induction by tetracycline.

[0102] FIG. 23A. The enzyme progression in the methylmalonyl-CoA catabolic pathway present in E. coli. FIG. 23B. The methylmalonyl-CoA catabolic pathway present in E. coli.

[0103] FIG. 24. Pathways for (2S)-methylmalonyl-CoA and Propionyl-CoA synthesis.

[0104] FIGS. 25A, 25B, and 25C. Three illustrations of the L-threonine metabolic pathway to generate propionyl-CoA in E. coli.

[0105] FIG. 26. tcd operon in E. coli for L-threonine metabolism.

[0106] FIG. 27. pR6K-Sc MM-CoA pPantT expression plasmid for MM-CoA mutase/epimerase pathway.

[0107] FIG. 28. pR6K-Ec MM-CoA pPantT expression plasmid for MM-CoA mutase/epimerase pathway.

[0108] FIG. 29. pR6K-Sc MM-CoA Mx-PCC Ec-tcdBE expression plasmid for MM-CoA mutase/epimerase and PCC pathway.

[0109] FIG. 30. pR6K-Ec MM-CoA Mx-PCC Ec-tcdBE expression plasmid for MM-CoA mutase/epimerase and PCC pathway.

EXAMPLES

Example 1The Generation of Expression Plasmids for the Expression of Glidobactin in DSM7029

[0110] To allow the expression of glidobactin A in heterologous hosts, RecET system was used to clone the glidobactin (glb) gene cluster.

[0111] To clone the glb gene cluster, genomic DNA from DSM7.029 was prepared and digested with Sfi I. YZ2005, comprising RecE and RecT, was then transformed with this genomic preparation and a linear vector to utilise RecET to subclone the gene cluster into a plasmid (FIG. 3).

[0112] The construct was further modified to allow the expression of the metabolites in DSM7029 Burkholderia. ColE1 plasmids cannot replicate in DSM7029 so the construct was integrated into the chromosome of DSM7029. The p15A origin of replication and a transposase cassette (Tpase-bsd-oriT) from p15A-Tn5-neo-epo-Tpase-BSD-oriT was used to replace the colE1 origin (FIGS. 4 and 5).

[0113] Glidobactin A, B and C were produced from the endogenous glb gene cluster It is therefore demonstrated that such gene clusters can be successfully transferred to heterologous hosts for the heterologous expression of secondary metabolites.

[0114] The glb expression cluster is, therefore, suitable for transferal to a heterologous host for expression of glidobactin A and related molecules. Suitable heterologous hosts include Photorhabdus luminescens, Escherichia coli Nissle 1917, Agrobacterium tumefaciens and DSM7029 Burkholderia. Escherichia coli Nissle 1917 and Photorhabdus luminescens are particularly suitable heterologous hosts. Furthermore, there are great possibilities in generating novel compounds with useful properties such as anticancer compounds by swapping modules and domains between gene clusters.

Example 2Heterologous Expression of Epotholines in DSM7029

[0115] The epothilone gene cluster was engineered to carry the transposase (Tps) and invert sequences (IR) as described above. Using RecE, a 57-59 kb DNA fragment comprising the epothilone gene cluster under the Tn5 constitutive promoter was cloned and integrated into the chromosome of DSM 7029.

[0116] Transformation was performed according to the following protocol using ice-cold dH.sub.2O, and Eppendorfs and electroporation cuvettes cooled to 2 C.

[0117] 1. A hole was made in the lid of a 1.5 ml Eppendorf tube and a single colony was inoculated in 1.0 ml of CY medium. The cells were cultured overnight at 37 C. with shaking at 900 rpm. 2. A hole was made in the lid of a 2.0 ml Eppendorf tube and 1.9 ml of CY medium was inoculated with 50 l of the overnight culture. 3. The 2.0 ml tube was incubated at 37 C. with shaking at 1100 rpm for 5 hours. 4. The cells were centrifuged at 9000 rpm for 20 seconds at 2 C. The supernatant was discarded and placed on ice. 5. The cells were re-suspended in 1.6 ml of ice-cold dH.sub.2O. 6. The cells were centrifuged at 9000 rpm for 3 seconds. 7. The cells were re-suspended in 1.6 ml of ice-cold dH.sub.2O. 8. The cells were centrifuged at 9000 rpm for 3 seconds. 9. The supernatant was discarded using a 1 ml pipette and leaving around 20-30 l of solution (alternatively the supernatant can be discarded through swinging). 10. 1 g of plasmid DNA was added and the mixture was transferred to an ice-cold electroporation cuvette with a 1 mm gap. 11. The cells were electroporated at 1250V. 12. 1.5 ml of CY medium was added and the cells were transferred to an Eppendorf tube. 13. The cells were incubated at 37 C. for 2.5 hours with shaking at 1100 rpm. 14. 150 l of transformed cells was plated on LB agar plates containing a suitable antibiotic. 15. The plates were incubated at 37 C. for 48 hours to allow colonies to be visualised.

[0118] A small transpositional plasmid pTpase-lacZ (15.6 kb) and a large transpositional plasmid p15A-Tn5-neo-epo-Tpase-BSD-oriT (61 kb) (Fu J et al., 2008) were transferred into DSM7029 by performing the electroporation procedure as described above. The stably integrated recombinants were kanamycin resistant. Colonies on kanamycin (50 g/ml) plates were counted and the total number per transformation is shown below:

TABLE-US-00001 pTps-lacZ 4870 p15A-Tn5-neo-epo-Tpase-BSD-oriT 420

[0119] Compared to the conventional conjugation methods, this electroporation transformation method is efficient, simple and fast and is not dependent on helper DNA. Two positive clones were selected and were analysed for epothilone production. Cells and XAD were harvested via centrifugation at 10000 rpm for 10 minutes and extracted with 10 ml acetone and 30 ml methanol. The filtered extract was dried by vacuum electroporation, resuspended with 1 ml methanol and analysed by HPLC/MS and HPLC/MS/MS (HPLC: Agilent 1100 Series, CC 125/2 Nucleodur C18 Gravity, 3 m; MS: Bruker HCT Plus; 0 min-22 min 5%-95% acetonitrile with 0.1% formic acid) (FIGS. 7-10).

[0120] The data shows that Epothilone C and Epothilone D were successfully produced by DSM7029 comprising the epothilone gene cluster under Tn5 inducible promoter.

[0121] Furthermore, additional constructs have been made using RecE to generate expression plasmids with the epothilone gene cluster under the control of alternative promoters such as Ptet and Pm (FIGS. 6A, 6B, and 6C).

TABLE-US-00002 TABLE 1 Epothilone production by 4 individual DSM7029 clones with the epothilone gene cluster. Quantification of Epothilones C and D in Polyangium (Burkholderia) DSM7029 Epothilon C Epothilon D [g/l culture] [g/l culture] Polyangium Kan 1 14, 2 4, 8 Polyangium Kan 3 8 3, 1 Polyangium Kan 5 10, 7 5, 9 Polyangium Kan 7 10, 1 4, 4

Example 3Reclassification of DSM7029

[0122] DSM7029 was previously classified as a myxobacterium. However, myxobacteria are characterised by a long doubling time and slow growth. In contrast, DSM7029 grows fast and its doubling time was estimated as 1 hour. Therefore, it was investigated whether DSM7029 is, in fact, a myxobacterium. 16S rRNA coding sequences from DSM7029 strains (wild-type and kan1) were amplified and sequenced.

TABLE-US-00003 Partialsequenceof16SrRNAgeneofDSM7029 wildtype (SEQIDNO:1) CCCTTATGACTACTTGTTACGACTTCACCCCAGTCACGAACCCTGCCGTG GTGATCGCCCTCCTTGCGGTTAGGCTAACCACTTCTGGCAGAACCCGCTC CCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCG GCATGCTGATCCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTG CAGACTGCGATCCGGACTACGACCGGTTTTCTGGGATTAGCTCCCCCTCG CGGGTTGGCAGCCCTCTGTACCGGCCATTGTATGACGTGTGTAGCCCTAC CCATAAGGGCCATGATGACCTGACGTCATCCCCACCTTCCTCCGGTTTGT CACCGGCAGTCTCATTAGAGTGCCCTTTCGTAGCAACTAATGACAAGGGT TGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGAC GGCCATGCAGCACCTGTGTCCAGGTTCTCTTTCGAGCACTCCCACATCTC TGCAGGATTCCTGGCATGTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATC GAATTAAACCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTT GAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTA GCTTCGTTACTGAACAGCAAGCCGTCCAACAACTAGTTGACATCGTTTAG GGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTG CATGAGCGTCAGTGCAGGCCCAGGAGATTGCCTTCGCCATCGGTGTTCCT CCGCATATCTACGCATTTCACTGCTACACGCGGAATTCCATCTCCCTCTG CCGCACTCTAGCCGTGCAGTCACAAATGCAGTTCCCAGGTTGAGCCCGGG GATTTCACATCTGTCTTGCACAACCGCCTGCGCACGCTTTACGCCCAGTA ATTCCGATTAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTA GTTAGGCGGGTGCTTATTCTTCAGGTACCGTCATCGGCTCCGGGGTATAG CCCAGAACTTTTCTTCCCTGACAAAAGCGGTTTACACCCGGACGTCTTCT TCCCGCACGCGGCATGGCTGGATCAGGCTGCGCCATGGTCAAAACTCCCC ACTGCTGCCTCCGTAGGAGTCTGGCGTGTCCTCAGTCCCAGGTGTGGCTT GTCGTCCTCTCAGACAGCTACGATCGTCGCATGTAGCTACCCACACACTA GCTAATCTGACTCGGGCGATCAAATAGGCGCGAGCCTTGGCAATTCTGT Partialsequenceof16SrRNAgeneofDsm7029Kan1 (SEQIDNO:2) TCGTAATTGCTCATTGTTACGACTTCACCCCAGTCACGAACCCTGCCGTG GTGATCGCCCTCCTTGCGGTTAGGCTAACCACTTCTGGCAGAACCCGCTC CCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCG GCATGCTGATCCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTG CAGACTGCGATCCGGACTACGACCGGTTTTCTGGGATTAGCTCCCCCTCG CGGGTTGGCAGCCCTCTGTACCGGCCATTGTATGACGTGTGTAGCCCTAC CCATAAGGGCCATGATGACCTGACGTCATCCCCACCTTCCTCCGGTTTGT CACCGGCAGTCTCATTAGAGTGCCCTTTCGTAGCAACTAATGACAAGGGT TGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGAC GGCCATGCAGCACCTGTGTCCAGGTTCTCTTTCGAGCACTCCCACATCTC TGCAGGATTCCTGGCATGTCAAGGGTAGGTAAGGTTTTTCGCGTTGCATC GAATTAAACCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTT GAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTA GCTTCGTTACTGAACAGCAAGCCGTCCAACAACTAGTTGACATCGTTTAG GGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTG CATGAGCGTCAGTGCAGGCCCAGGAGATTGCCTTCGCCATCGGTGTTCCT CCGCATATCTACGCATTTCACTGCTACACGCGGAATTCCATCTCTCTCTG CCGCACTCTAGCCGTGCAGTCACAAATGCAGTTCCCAGGTTGAGCCCGGG GATTTCACATCTGTCTTGCACAACCGCCTGCGCACGCTTTACGCCCAGTA TTTCCGATTAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTA GTTAGCCGGTGCTTATTCTTCAGGTACCGTCATCGCTCCGGGGGTATTAG CCCAGATCTTTTCTTCCCTGACAAAGCGGTTTACACCCGGACGTCTTCTT CCGGCACGGCGGCATGGCTGGATCAGGCTTGCGCCGATGATCCAAACTCC CCACTGCTGCCTCAGTAGGAGTCTGGGGCCGTGTCTCAGTCCCAGGGTGG CTGTCGTCCTCTCGACTAGCTACGAATCGCTCGCTTGTAGCATACCACCA ACTAGCTAATCTGACATCGACGTTCATTAGGCGGGAGGCTGTGGCAAATC

[0123] These sequences were analysed with Blast and except for one hit which was Polyangium brachysporum (itself), all other similar sequences that were identified were Burkholderia strains. To investigate further the 16S rRNA sequences, the sequences from DSM7029 were compared to those from various different strains of myxobacterium and Burkholderia.

[0124] It was found that there was a high sequence similarity between DSM7029 16S rRNA sequences and those from Burkholderia strains. In contrast, there was low sequence similarity between DSM7029 16S rRNA sequences and those from myxobacteria strains, despite low variance between different myxobacteria strains.

[0125] Therefore, this evidence strongly suggests that DSM7029 is, in fact, a Burkholderia and not a myxobacteria.

TABLE-US-00004 TABLE2 Partialcomparisonof16SrRNAgenesfromDSM7029andmyxobacterium GTCGAGTTGCAGACTG-----CGATCCG-GACTACGACCGGTTTT-CTGGGATTAG--C- ACCAAGGCAACGACGGGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGAACTGAGACA ACCAAGGCGACGACGGGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGAACTGAGACA ACCAAGGCAACGACGGGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGAACTGAGACA ACCAAGGCTACGACGGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACA ACCAAGGCTACGACGGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACA ACCAAGGCGACGACGGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACA ACCAAGGCGACGACGGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACA ACCAAGGCGACGACGGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACA ---TCC---CCCTCGCGGG---------TTGGCAGCCCTCTGTACCGG--------CCAT CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTGCGCAATGGGCGAAAGCCTGA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGCGCAATGGGCGAAAGCCTGA CGGTCCAGACTC-TACGGGAGGCAGCAGTGGGGAATCTTGCGCAATGGGCGAAAGCCTGA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGAAAGCCTGA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGAAAGCCTGA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTGCGCAATGGGCGAAAGCCTGA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTGCGCAATGGGCGAAAGCCTGA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTGCGCAATGGGCGAAAGCCTGA TGTA----TGACGTGTGT CGCAGCAACGCCGCGTGT CGCAGCAACGCCGCGIGT CGCAGCAACGCCGCGTGT CGCAGCAACGCCGCGTGT CGCAGCAACGCCGCGTGT CGCAGCAACGCCGCGTGT CGCAGCAACGCCGCGTGT CGCAGCAACGCCGCGTGT

[0126] From top to the bottom: DSM7029; Anaeromyxobacter dehalogenans strain 2CP-1; Corallococcus coralloides strain Cc9736; Myxobacterium KC; Myxobacterium SHI-1; Myxobacterium SMH-27-4; Polyangium cellulosum So ce56; Polyangium cellulosum So ce90; Polyangium cellulosum So0089-1.

TABLE-US-00005 TABLE-US-00005 TABLE3Partialcomparisonof16SrRNAgenesfromDSM7029 andBurkholderiastrains gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt GCGTTGCATCGAATTAAACCACATCATCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTT gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gcgttgcatcgaattaatccacatcatccaccgcttgtgcgggtccccgt caattccttt gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gccacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta GAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTCACGCGTTAGCTTCGTTA gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta gagttttaatcttgcgaccgtactccccaggcggtcaacttcacgcgtta gctacgtta

[0127] From top to bottom: Burkholderia mallei strain NCTC 10260; Burkholderia pseudomallei BCC215; Burkholderia gladioli strain R1879; Burkholderia gladioli strain R406; DSM7029; Burkholderia mallei strain ATCC 23344; Burkholderia mallei strain NCTC 10260; Burkholderia mallei strain 2000031063; Burkholderia cepacia strain 2Pe38; Burkholderia cepacia strain 1-3b; Burkholderia cepacia strain ESR87; Burkholderia cepacia strain NE9; Burkholderia mallei strain AE9.

Example 4The Expression of Myxochromide Heterologously in Agrobacterium tumefaciens

[0128] The mchS gene cluster with the Pm promoter has the necessary components for Pseudomonas expression but cannot be expressed in A. tumefaciens C58. The plasmid was modified to make it possible to express it in A. tumefaciens C58. Two constructs were made based on a shuttle vector pBC301 with kanamycin resistance and either apramycin resistance or gentamycin resistance. Vitally, they were made with oriV cassettes to allow them to replicate in A. tumefaciens C58 (FIGS. 11A and 11B).

[0129] The cassette trfA-apra-oriV was generated by PCR with two homology arms at its ends by using the template generated above (FIG. 3) and inserted into the vector backbone used for Pseudomonas expression to form pPm-mchS oriV-oripUC (FIG. 12). Myxochromide gene cluster (mchS) is under Pm inducible promoter in this construct.

[0130] The ability of different promoters to drive myxochromide was investigated. Constructs were generated carrying the toluic acid inducible promoter (Pm) and the tetracycline inducible promoter (Ptet) with the mchS gene cluster. To test the Ptet inducible promoter, one cassette carrying the hygromycin resistance gene, the regulator gene tetR and the tetracycline inducible promoter (Ptet) was inserted in front of mchS gene cluster to form pPtet-mchS oriV-oripUC (FIG. 13). Constructs with the Pm and Ptet inducible promoters were transformed then into A. tumefaciens C58 by electroporation. Plasmid DNA prepared from the transformants was verified by restriction analysis. The plasmids are able to stably propagate under selection pressure.

[0131] A. tumefaciens C58 carrying the constructs was cultured in LB medium. Myxochromide compounds were then extracted and detected. As shown in the HPLC profiles from the extract, the pPtet promoter was able to drive expression and successful synthesis of Myxochromide S.sub.1, Myxochromide S.sub.2, and Myxochromide S.sub.3 (FIG. 14A). The Pm promoter was similarly able to drive successfully synthesis (data not shown).

Example 5Expression of Epothilone Gene Cluster in Agrobacterium tumefaciens

[0132] The trfA-apra-oriV cassette was inserted in the epothilone cluster construct used for Myxococcus xanthus expression by Fu et al. 2008 to form p15A-oriV-apra-Tn5-neo-epo (FIG. 15). The epothilone cluster was driven by Tn5 promoter in this construct.

[0133] Following transformation, kanamycin resistant clones were identified and it was verified by restriction analysis that they were carrying the epothilone gene cluster.

[0134] To make this construct capable of successfully expressing epothilone, the methlymalonyl-coA mutase pathway must be integrated. This will then allow the synthesis of this useful compound from A. tumefaciens.

Example 6Expression of Colibactin-Anticancer Compounds in E. coli Nissle 1917

[0135] 4 phosphopantetheinyl (pPant) transferase is a component required to activate the PKS/NRPS enzymes. An over-expression plasmid of a known universal pPant transferase gene called MtaA (pSUMtaA) (Gaitatzis et al., 2001) (FIG. 16) was transformed into E. coli Nissle 1917. This pPant transferase has advantage of activating both PKS and NRPS enzymes which are presented in most of the gene clusters including colibactin, glidobactin and epothilone. When the transformants expressing exogenous pPant transferase were tested in vivo, they showed activity to against cancer (FIG. 17). As shown by previous publication (Stritzker et al., 2007), the original E. coli Nissle 1917 has no effect on the tumor growth in vivo. LC-MS data shows several new compounds are produced after overexpression of MtaA gene in E. coli Nissle 1917 (FIGS. 18A and B). To be able to control the expression of colibactin gene cluster, MtaA gene was cloned under a tetracycline inducible promoter and it has been transformed into E. coli Nissle 1917.

[0136] Upstream of the colibactin PKS/NRPS gene cluster is a predicted endogenous pPant transferase gene (FIG. 19 A). To test whether this component is functional, a chloroamphenicol (Cm) resistance gene and a ribosomal binding site were inserted in front of the pPant transferase gene (FIG. 19 B). Endogenous pPant transferase gene is under the control of a Cm promoter and is expressed constitutively. The transformed strain showed activity against cancer in vivo (FIG. 20). A cassette containing hyg-tetR-Ptet was then inserted in front of the PKS/NRPS gene cluster (FIG. 19 C) to regulate colibactin expression. The pPant transferase gene is predicted to be activated constitutively and the colibactin PKS/NRPS gene cluster is activated by the addition of tetracycline.

[0137] Following tetracycline induction, secondary metabolites were extracted and detected. The new compounds produced after activation of endogenous pPant transferase are the same as additional exogenous pPant transferase (FIGS. 18A and B).

[0138] Data from both experiments indicate that over expression of a pPant transferase gene in E. coli Nissle 1917 can activate anticancer compounds and also improve the heterologous production of polyketides and non-ribosomal peptides.

Example 7In Vitro Test of Expression of Colibactin in E. coli Nissle 1917

[0139] Colibactin has cytopathic activity to against malignant mammalian cells (Nougayrede et al., 2006; Homburg et al., 2007). E. coli Nissle 1917 has been shown to have cytopathic activity in vitro but has no effect on tumor growth in vivo (Putze et al., 2009; Stritzker et al., 2007). To investigate the in vitro activity of endogenous colibactin PKS/NRPS gene cluster activated by exogenous pPant transferase (pSUMtaA), human U-2 OS osteosarcoma cells were co-incubated with bacterial cells for 4 hours. Giemsa staining and MTT (thiazolyl blue tetrazolium bromide) assay were performed on the tumor cells after additional 3 days culturing without bacterial cells. Giemsa staining shows that LB medium negative control (FIG. 21A) and E. coli K12 strain GB2005 (FIG. 21B) have no cytopathic activity on tumor cells, but all of E. coli Nissle 1917 strains have cytopathic activity. E. coli Nissle 1917 transformed by control plasmid pACYC184 (FIG. 21C) shows much weaker activity than E. coli Nissle 1917 transformed by pSUMtaA (FIGS. 21D, E, and F). It is interesting that MTT assay data shows E. coli Nissle 1917+pACYC184 has no effect on tumor cell growth but E. coli Nissle 1917+pSUMtaA has strong inhibitory effect on tumor cell growth (FIG. 21B). This data fits with the in vivo data described above in FIG. 17 in Experiment 6. We have shown here that additional exogenous pPant transferase can increase the inhibitory effect of E. coli Nissle 1917 on tumor growth in vitro and in vivo.

Example 8Heterologous Expression of Glidobactin PKS/NRPS Gene Cluster in E. coli Nissle 1917

[0140] The plasmid pGB-Ptet-glb was transformed into E. coli Nissle 1917-cm (FIG. 19B), which the cm gene in front of endogenous pPant transferase gene to drive its expression constitutively (FIG. 22). This plasmid carries the glidobactin gene cluster under tetracycline inducible promoter (FIG. 22). Following tetracycline induction, compounds were extracted and detected to monitor glidobactin production. Table 4 shows that glidobactin compounds were successfully synthesized.

TABLE-US-00006 TABLE 4 Compounds produced by E. coli Nissle 1917-cm wild-type and E. coli Nissle 1917-cm carrying the glidobactin gene cluster. Strain Compound E. coli Nissie 1917-cm wild-type E. coli Nissle 1917-cm + pGB-Ptet-glb glidobactin A, B, C

Example 9Development of E. coli Nissle 1917 and K12 as Heterologous Hosts for Epothilone, Tubulysin and Disorazol Production within Tumours

[0141] In addition to expressing the glidobactin gene cluster in E. coli Nissle 1917, it will be useful to express other compounds such as epothilone, tubulysin, disorazol and related compounds in E. coli Nissle 1917 and other cells. It will be especially useful to develop systems for expressing secondary metabolites in vivo, for example by bacteria targeted to tumors.

[0142] Current methods for the expression of epothilone in E. coli are not suitable for in vivo expression because they require propionate feeding in the culturing medium, IPTG induction or arabinose induction. New E. coli strains for epothilone, tubulysin or disorazole expression under anaerobic (or in vivo) conditions, at 37 C. and without feeding of propionate need to be generated for in vivo experimentation and use. The protocol below allows the expression of epothilone, tubulysin and disorazol and related compounds in E. coli Nissle 1917 and K12.

[0143] 4-phosphopantetheinyl transferase (pPant transferase) for posttranslational modification of the PKS proteins and substrates for starter units and extending units are required for PKS gene cluster expression in E. coli. Substrates like methylmalonyl-CoA (MM-CoA) are not synthesized in E. coli. Therefore, an E. coli strain was engineered to synthesize methylmalonyl-CoA. (2S)-methylmalonyl-CoA is a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases like epothilone and salinomycin synthases. There are 3 ways in which (2S)-methylmalonyl-CoA can be produced in E. coli.

[0144] (1) methylmalonyl-CoA mutase/epimerase pathwayB12 or hydroxocobalamin (B12 precursor) must be fed. This will work without B12 due to endogenous production but at low rate.

[0145] (2) propionyl-CoA carboxylase (PCC) pathwayrequires that propionate is fed.

[0146] (3) malonyl/methylmalonyl-CoA ligase (matB) pathwaymethylmalonate must be fed and produce most of the (2S,2R)-methylmalonyl-CoA (47% of CoA pool), but produce trace amount 6dEB (6-Deoxyerythronolide) from DEBS (erythromycin gene cluster). This pathway will not be discussed here.

[0147] The Methylmalonyl-CoA mutase/epimerase pathway is a pathway that produces methylmalonyl-CoA and propionyl-CoA in E. coli (FIG. 23B). Although E. coli possesses a gene (sbm) that encodes a putative methylmalonyl-CoA mutase, neither mutase activity nor methylmalonyl-CoA is detectable in cell extracts. This gene is tightly controlled. Even if the gene sbm is expressed, the product (methylmalonyl-CoA mutase from E. coli) only catalyzes the isomerization of the (R)-diastereomer of methylmalonyl CoA (L-methylmalonyl-CoA or (2R)-methylmalonyl-CoA) and succinyl CoA. E. coli K12 has no methylmalonyl-CoA epimerase which converts (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the active substrate for PKS. Therefore, methylmalonyl-CoA epimerase was introduced into E. coli for (2S)-methylmalonyl-CoA synthesis. For accumulation of methylmalonyl-CoA, the ygfG gene was removed. YgfG is methylmalonyl-CoA decarboxylase and it can convert (2S)- and (2R)-methylmalonyl-CoA into propionyl-CoA. To accumulate propionyl-CoA, ygfH was removed. YgfH is propionyl CoA:succinate CoA transferase which catalyzes the conversion of propionyl-CoA to propionate.

[0148] Additionally, E. coli does not contain coenzyme B12. Methylmalonyl-CoA mutase apo-enzymes require reconstitution with coenzyme B12 to form the active holo-enzyme.

[0149] Methylmalonyl-CoA mutase/epimerase genes from other species have been expressed in E. coli. Propionibacteria shennanii methylmalonyl-CoA mutaseAB/epimerase encoding genes have been integrated into the yfgG locus in E. coli. The Mutase/epimerase genes are under T7 promoter (T7prom-mutAB-T7prom-epi-T7). They were also introduced in E. coli in an expression plasmid. In the absence of hydroxocobalamin (a precursor of B12), mutase activity was undetectable unless extracts were supplemented with coenzyme B12, indicating exclusive expression of the apo-enzyme. 10% of total CoA, corresponding to .about.40 M, is methylmalonyl-CoA from expression of Propionibacteria shermanii methylmalonyl-CoA mutaseAB or E. coli methylmalonyl-CoA mutase. There is no propionyl-CoA.

[0150] To allow this pathway to function, B12 or its precursor was fed for active mutase expression and ygfG gene was removed to accumulate MM-CoA.

[0151] There are no propionyl-CoA carboxylase (PCC) genes for the Propionyl-CoA carboxylase pathway in E. coli. Propionyl CoA is carboxylated by propionyl CoA carboxylase to form the (2S)-methylmalonyl-CoA (FIG. 24). In mammalian cells and many bacterial cells, propionyl-CoA can be metabolized into (2S)-methylmalonyl-CoA directly by propionyl-CoA carboxylase (PCC) which has two subunits; A and B. Two genes, pccA and pccB, encode PCC. Biotin is a cofactor essential for PCC activity. Human pccA and B, Streptomyces coelicolor pccA and B, or Streptomyces coelicolor accA (acetyl-CoA carboxylase) and pccB gene pairs have been expressed in E. coli. The activity of the biotinylated subunit (pccA) can be enhanced upon coexpression of the E. coli birA biotin ligase gene. There is no or very little propionyl-CoA synthesis in E. coli. To produce and accumulate propionyl-CoA in E. coli, 3 steps have been accomplished. 1, propionate was fed for E. coli culture. 2, the prp operon, which is putatively responsible for propionate catabolism in E. coli, was deleted. 3, PrpE is CoA transferase and it is thought to convert propionate into propionyl-CoA.

[0152] The important innovations that allow the function of this pathway are: 1, propionate feeding; 2, co-expression of birA gene; 3, propionyl-CoA pathway is better than methylmalonyl-CoA mutase/epimerase for 6-dEB production from DEBS gene cluster expression; 4, Production rate of polyketides in E. coli at lower than 30 C. (22 C. for 6dEB and 15 C. for Epothilones).

[0153] Further to the pathways discussed above, there is an additional silent pathway that produces propionyl-CoA. Studies have shown that propionyl-CoA carboxylase (PCC) pathway is better than MM-CoA mutase/epimerase pathway for 6-dEB production. There are two reasons to be considered: 1, PCC pathway coverts propionyl-CoA to (2S)-methylmalonyl-CoA only which can be directly used by PKS; 2, MM-CoA mutase catalyzes (2R)-methylmalonyl-CoA to succinyl-CoA and its reverse activity is converting succinyl-CoA to (2R)-methylmalonyl-CoA. (2R)-MM-CoA cannot be used by PKS and it must be epimerized to (2S)-form by epimerase for PKS. Epimerase activity is bi-directional. From these points, the synthesis of (2S)-MM-CoA by mutase/epimerase is not more efficient than PCC pathway.

[0154] The involvement of 2-ketobutyrate as a precursor in L-isoleucine biosynthesis has been well studied, however, the route of anaerobic 2-ketobutyrate catabolism in E. coli is less well understood. Working with Salmonella typhimurium, (Van Dyk and LaRossa 1987) demonstrated that phosphotransacetylase (Pta) and acetate kinase are involved in the aerobic degradation of 2-ketobutyrate, indicating that propionyl-CoA is an intermediate. (Hesslinger et al., 1998) proposed an anaerobic pathway in E. coli that degrades L-threonine to propionate (FIG. 25A). FIGS. 25A and 25C shows that propionyl-CoA can be generated via L-threonine metabolism.

[0155] As discussed above, there is no propionyl-CoA produced in E. coli under normal culture conditions. This pathway must be silent in aerobic conditions. The tdc operon is shown in FIG. 26. Expression of the tdc operon is controlled directly by a number of positive transcriptional regulators, including CRP (cyclic-AMP-catabolite gene activator protein complex), IHF (integration host factor), TdcA and TdcR (encoded in the tcd operon), and is controlled indirectly by FNR. FNR is a dimeric, class II transcription factor, which activates gene expression when E. coli grows anaerobically.

[0156] FIG. 26 shows a detail of the L-threonine metabolism pathway. To produce propionyl-CoA in E. coli without the feeding of propionate as in current methods, tcdB (threonine deaminase or dehydratase) and tcdE (2-ketobutyrate formate-lyase) were expressed. To accumulate propionyl-CoA, Pta (phosphotransacetylase) was removed.

[0157] The significant innovations that allow this pathway to be activated are: 1, activation of tcdB and tcdE genes to synthesize propionyl-CoA; 2, deletion of Pta or Pta plus tcdD to accumulate propionyl-CoA.

[0158] Finally, to generate E. coli strains suitable for heterologous expression of epothilone, tubulysin and disorazol gene clusters, the following developments were made. Pta and ygfG genes in Nissle and K12 were removed Pta gene and ygfG gene using loxP* strategy. Super plasmids to express necessary components in Nissle and K12 were generated. First of all, pPant transferase genes from Agrobacterium and Myxococcus xanthus were integrated into the plasmid although there is a pPant transferase gene presented in front of PKS gene cluster in E. coli Nissle 1917. This is because it is not known whether the pPant transferase gene E. coli Nissle 1917 is functional, but it is known that the production of PKS gene cluster is functional in Agrobacterium and Myxococcus xanthus. Their pPant transferase genes are under a constitutive promoter (FIGS. 27-30).

[0159] To produce propinoyl-CoA in E. coli as substrate for propionyl-CoA carboxylase to synthesize (2S)-methylmalonyl-CoA without the feeding of propionate, tcdB and tcdE were expressed under a tetracycline promoter in case they are not functional in E. coli (FIGS. 29 and 30).

[0160] MM-CoA mutase/epimerase pathway and PCC pathway are under a constitutive promoter. BirA gene is under a constitutive promoter as well (FIGS. 27 and 28).

[0161] Super plasmids were generated to contain tcdB, tcdE, birA, MM-coA mutase genes from E. coli, MM-CoA epimerase from M. xanthus and pcc (propionyl-CoA carboxylase) operon from M. xanthus, or MM-CoA mutase/epimerase genes from So. ce56 (FIGS. 29 and 30). Lipid A modified (msbB.sup.-), auxotrophic (purl.sup.-), yersiniabactin gene cluster (ybt) and lacIZ were also deleted to reduce the bacterial toxicity. This plasmid is, therefore suitable for the expression of epothilone, tubulysin and disorazol gene clusters in E. coli Nissle 1917 and K12.

[0162] It will be understood that the invention has been described above by way of example only and that modifications in detail may be made within the scope of the invention.

REFERENCES

[0163] Konishi, et al. (Sep. 8, 1987) U.S. Pat. No. 4,692,510. [0164] Oka, et al. (May 3, 1988) U.S. Pat. No. 4,742,047. [0165] Oka, et al. (Oct. 11, 1988) U.S. Pat. No. 4,777,160. [0166] Oka, et al. (Dec. 6, 1988) U.S. Pat. No. 4,789,731. [0167] Knoishi, et al. (May 23, 1989) U.S. Pat. No. 4,833,076. [0168] Abril M A, Michan C, Timmis K N, Ramos J L. (1989) Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol. December; 171 (12):6782-90. [0169] Agrawal N, Bettegowda C, Cheong I, Geschwind J F, Drake C G, Hipkiss E L, Tatsumi M, Dang L H, Diaz L A Jr, Pomper M, Abusedera M, Wahl R L, Kinzler K W, Zhou S, Huso D L, Vogelstein B. (2004) Bacteriolytic therapy can generate a potent immune response against experimental tumors. Proc Natl Acad Sci USA. 101 (42):15172-7 [0170] Altenhoefer A, Oswald S, Sonnenborn U, Enders C, Schulze J, Hacker J, Oelschlaeger T A. (2004) The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol Med Microbiol. 40 (3):223-9. [0171] Amrein, H. et al. (2004) Functional analysis of genes involved in the synthesis of syringolin A by Pseudomonas syringae pv. syringae B301D-R. Mol. Plant Microbe Interact 17: 90-97. [0172] Barbara, S., Laurent, B., and Robert, D. (2007) Identification of genes involved in the biosynthesis of the cytotoxic compound glidobactin from a soil bacterium. Environmental Microbiology 9: 1640-1. [0173] Binns, A. N. and Thomashow M. F. (1988) Cell biology of Agrobacterium infection and transformation of plants. Annual Review of Microbiology 42: 575-606. [0174] Brown, J. M., Giaccia, A. J. (1998) The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 58: 1408-16. [0175] Bryan, J., and Sanjay, S. (2002) Heterologous expression of epothilone biosynthetic genes in Myxococcus xanthus. Antimicrob. Agents. Chemother 46: 2772-2778. [0176] Carroll C L, Johnston J V, Kekec A, Brown J D, Parry E, Cajica J, Medina I, Cook K M, Corral R, Pan P S, McAlpine S R. (2005) Synthesis and cytotoxicity of novel sansalvamide A derivatives. Org Lett. 7 (16):3481-4. [0177] Christopher, N. B., Kinya, H., Martha, L. T., R. Edward, W., and Chaitan, K. (2004) Precursor-Directed Biosynthesis of Epothilone in Escherichia coli. J. AM. CHEM. SOC 126: 7436-7437. [0178] Clark, A. J. et al. (1984) Genes of the RecE and RecF pathways of conjugational recombination in Escherichia coli. Cold Spring Harb. Symp. Quant. Biol 49: 453-462. [0179] Coleman, C. S. et al. Syringolin A, (2006) a new plant elicitor from the phytopathogenic bacterium Pseudomonas syringae pv. syringae, inhibits the proliferation of neuroblastoma and ovarian cancer cells and induces apoptosis. Cell Prolif. 39: 599-609. [0180] Ditta, G., Stanfield, S., Corbin, D., Helinski, D. R. (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci 77: 7347-51. [0181] Fisher B, Brown A, Wolmark N, Fisher E R, Redmond C, Wickerham D L, Margolese R, Dimitrov N, Pilch Y, Glass A, et al. (1990) Evaluation of the worth of corynebacterium parvum in conjunction with chemotherapy as adjuvant treatment for primary breast cancer. Eight-year results from the National Surgical Adjuvant Breast and Bowel Project B-10. Cancer. 15; 66 (2):220-7. [0182] Frary, A., Hamilton, C. M. (2001) Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato. Transgenic Res 10: 121-132. [0183] Fu, J., Wenzel, S. C., Perlova, O., Wang, J., Gross, F., Tang, Z., Yin, Y., Stewart, A. F., Muller, R. & Zhang, Y. (2008). Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res. 36:e113. [0184] Gaitatzis N, Hans A, Muller R, and Beyer S. (2001) The mtaA gene of the myxothiazol biosynthetic gene cluster from Stigmatella aurantiaca DW4/3-1 encodes a phosphopantetheinyl transferase that activates polyketide synthases and polypeptide synthetases. J. Biochem. 129 (1):119-24 [0185] Gerth K, Bedorf N, Hofle G, Irschik H, Reichenbach H. (1996) Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties. J Antibiot (Tokyo). 49 (6):560-3. [0186] Gilbert N. Belofsky, Paul R. Jensen, and William Fenical. Sansalvamide. (1999) A New Cytotoxic Cyclic Depsipeptide Produced by a Marine Fungus of the Genus Fusarium. Tetrahedron Letters 40:2913-2916. [0187] Gross, F., Ring, M. W., Perlova, O., Fu, J., Schneider, S., Gerth, K., Kuhlmann, S., Stewart, A. F., Zhang, Y. and Muller, R. (2006) Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation. Chem. Biol 13: 1-13. [0188] Gust et al., (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. PNAS USA (2003) 100:1541-1546) [0189] Hamilton, C. M. (1997) A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene 200:107-116. [0190] Hamilton, C. M., Frary, A., Lewis, C., Tanksley, S. D., (1996) Stable transfer of intact high molecular weight DNA into plant chromosome. Proc. Natl. Acad. Sci 93: 9975-9979. [0191] Hesslinger C, Fairhurst S A, Sawers G. (1998) Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate. Mol Microbiol. 27 (2):477-92. [0192] Homburg S, Oswald E, Hacker J, Dobrindt U. (2007) Expression analysis of the colibactin gene cluster coding for a novel polyketide in Escherichia coli. FEMS Microbiol Lett. 275 (2):255-62. [0193] Jean-Philippe, N., Stefan, H. Fre'de'ric, T., Miche{grave over ( )}le, Boury, Elzbieta, B., Gerhard, G., Carmen, B., Jo{umlaut over ( )}rg, H., Ulrich, D., Eric, Oswald. (2006) Escherichia coli Induces DNA Double-Strand Breaks in Eukaryotic Cells. Science 313: 848-851. [0194] Jia L J, Xu H M, Ma D Y, Hu Q G, Huang X F, Jiang W H, Li S F, Jia K Z, Huang Q L, Hua Z C. (2005) Enhanced therapeutic effect by combination of tumor-targeting Salmonella and endostatin in murine melanoma model. Cancer Biol Ther. 4 (8):840-5. [0195] Jochen, S., Stephanie, W. C., Philip, J. H., Tobias, A. O., Werner, G., Aladar, A. S. (2007) Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. J Medical Microbiology 297: 151-162. [0196] Kang, Y., Son, M. S., Hoang, T. T. (2007) One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system. Protein Expr Purif 55:325-33. [0197] Kealey et al., (1998) Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts, PNAS USA, 95:505-509 [0198] Kohwi, Y., Imai, K., Tamura, Z., Hashimoto, Y. (1978) Antitumor effect of Bifidobacterium infantis in mice. Gann 69: 613-8. [0199] Lee C H, Wu C L, Shiau A L. (2004) Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models. J Gene Med. 6 (12):1382-93. [0200] Lee C H, Wu C L, Shiau A L. (2005a) Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther. (2):175-84. [0201] Lee C H, Wu C L, Shiau A L. (2005b) Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther. 12 (2):175-84. [0202] LiJun, J., HanMei, X., DingYuan, M., QinGang, H., XiaoFeng, H., WenHui, J., ShuFeng, L., KunZhi, Jia., QiLai, H., ZiChun, H. (2005) Enhanced Therapeutic Effect by Combination of Tumor-Targeting Salmonella and Endostatin in Murine Melanoma Model. Cancer Biology & Therapy 4: 840-845. [0203] Lodinova-Zadnikova R, Sonnenborn U. (1997) Effect of preventive administration of a nonpathogenic Escherichia coli strain on the colonization of the intestine with microbial pathogens in newborn infants. Biol Neonate. 71 (4):224-32. [0204] Loeffler M, Le'Negrate G, Krajewska M, Reed J C. (2007) Attenuated Salmonella engineered to produce human cytokine LIGHT inhibit tumor growth. Proc Natl Acad Sci USA. 104 (31):12879-83 [0205] Long, H. D., Chetan, B., David, L. H., Kenneth, W. K., and Bert, V. (2001) Combination bacteriolytic therapy for the treatment of experimental tumors. PNAS 98: 15155-15160. [0206] Low, K. B., Ittensohn, M., Le, T., et al. (1999) Lipid A mutant Salmonella with suppressed virulence and TNFa induction retain tumor-targeting in vivo. Nat Biotechnol 17: 37-41. [0207] Malmgren R A, Flanigan C C. (1955) Localization of the vegetative form of Clostridium tetani in mouse tumors following intravenous spore administration. Cancer Res. 15 (7):473-8. [0208] Mark, M., Gregory, J. P., and Anath D. (1990) Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 90: 149-151. [0209] Markus, L., Gaelle, L., Maryla, K., and John, C. R. (2007) Attenuated Salmonella engineered to produce human cytokine LIGHT inhibit tumor growth. PNAS 104: 12879-12883. [0210] Matthysse, A. G. (1986) Initial interactions of Agrobacterium tumefaciens with plant host cells. Critical Reviews in Microbiology 13: 281-307. [0211] Michel, K., Abderhalden, O., Bruggmann, R., and Dudler, R. (2006) Transcriptional changes in powdery mildew infected wheat and Arabidopsis leaves undergoing syringolin triggered hypersensitive cell death at infection sites. Plant Mol Biol 62: 561-578. [0212] Moese, J. R., Moese, G. (1964) Oncolysis by clostridia. I. Activity of Clostridium butyricum (m-55) and other non-pathogenic clostridia against the ehrlich carcinoma. Cancer Res 24: 212-6. [0213] Mutka, S. C., Carney, J. R., Liu, Y., and Kennedy, J. (2006) Heterologous production of epothilone C and D in Escherichia coli. Biochemistry 45: 1321-30. [0214] Muyrers et al., 1999 (Rapid modification of bacterial artificial chromosomes by ET-recombination, Nucleic Acid Res., 27, 1555-1557) [0215] Muyrers, J. P. P. et al., 2000a ET-Cloning: Think Recombination First. Genetic Eng., vol. 22, 77-98. [0216] Muyrers J. P et al., 2000b Point mutation of bacterial artificial chromosomes by ET recombination, EMBO Reports, 1, 239-243 [0217] Muyrers J. P et al., 2000c RecE/RecT and Red.alpha.a/Red.beta.a initiate double-stranded break repair by specifically interacting with their respective partners, Genes Dev., 14 1971-1982 [0218] Muyrers, J. P. P et al., 2001 Techniques: Recombinogenic engineeringnew options for cloning and manipulating DNA, Trends in Biochem. Sci., 26, 325-31 [0219] Narayanan K. et al., (1999) Efficient and precise engineering of a 200 kb .beta.-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system, Gene Therapy, 6, 442-447 [0220] Nester, E. W., Gordon, M. P., Amasino, R. M. and Yanofsky, M. F. (1984) Crown gall: a molecular and physiological analysis. Annual Review of Plant Physiology 35: 387-413. [0221] Newman, J. R., Fuqua, C. (1999) Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227:197-203. [0222] Nougayrede J P, Homburg S, Taieb F, Boury M, Brzuszkiewicz E, Gottschalk G, Buchrieser C, Hacker J, Dobrindt U, and Oswald E. (2006) Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science. 313 (5788):848-51. [0223] Oka, M., Nishiyama, Y., Ohta, S., Kamei, H., Konishi, M., Miyaki, T., et al. (1988a) Glidobactins A, B and C, new antitumor antibiotics. I. Production, isolation, chemical properties and biological activity. J Antibiot 41: 1331-1337. [0224] Oka, M., Yaginuma, K., Numata, K., Konishi, M., Oki, T., and Kawaguchi, H. (1988b) Glidobactins A, B and C, new antitumor antibiotics. II. Structure elucidation. J Antibiot 41: 1338-1350. [0225] Oka, M., Ohkuma, H., Kamei, H., Konishi, M., Oki, T., and Kawaguchi, H. (1988c) Glidobactins D, E, F, G and H; minor components of the antitumor antibiotic glidobactin. J Antibiot 41: 1906-1909. [0226] Parker R C, Plummer H C, et al. (1947) Effect of histolyticus infection and toxin on transplantable mouse tumors. Proc Soc Exp Biol Med. 66 (2):461-7. [0227] Pawelek J M, Low K B, Bermudes D. (1997) Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res. 57 (20):4537-44. [0228] Pawelek J M, Sodi S, Chakraborty A K, Platt J T, Miller S, Holden D W, Hensel M, Low K B. (2002) Salmonella pathogenicity island-2 and anticancer activity in mice. Cancer Gene Ther. 9 (10):813-8. [0229] Pawelek J M, Low K B, Bermudes D. (2003) Bacteria as tumour-targeting vectors. Lancet Oncol.; 4 (9):548-56. [0230] Pawelek J M. (2005) Tumour-cell fusion as a source of myeloid traits in cancer. Lancet Oncol. 6 (12):988-93. [0231] Perlova, O., Fu, J., Kuhlmann, S., Krug, D., Stewart, A. F., Zhang, Y., and Muller, R. (2006) Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus. Appl Environ Microbiol, 72: 7485-7494. [0232] Pfeifer et al., (2001) Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291: 1790-1792 [0233] Putze J, Hennequin C, Nougayrede J P, Zhang W, Homburg S, Karch H, Bringer M A, Fayolle C, Carniel E, Rabsch W, Oelschlaeger T A, Oswald E, Forestier C, Hacker J, Dobrindt U. (2009) Genetic structure and distribution of the colibactin genomic island among members of the family Enterobacteriaceae. Infect Immun. 77 (11):4696-703. Epub 2009 Aug. 31. [0234] Rembacken B J, Snelling A M, Hawkey P M, Chalmers D M, Axon A T. (1999) Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet. August 21; 354 (9179):635-9. [0235] Rondon M R, Ballering K S, Thomas M G. (2004) Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58. Microbiol 150 (Pt 11):3857-66 [0236] Schellenberg, B., Bigler, L., and Dudler, R. (2007) Identification of genes involved in the biosynthesis of the cytotoxic compound glidobactin from a soil bacterium. Environ. Microbiol 9: 1640-1650. [0237] Schneiker S., et at. (2007) Complete genome sequence of the myxobacterium Sorangium cellulosum. Nat Biotechnol 25: 1281-9. [0238] Smith, E. F. and Towsend, C. O. (1907) A plant tumour of bacterial origin. Science 25: 671-673. [0239] Shoji, J., Hinoo, H., Kato, T., Hattori, T., Hirooka, K., Tawara, K., et al. (1990) Isolation of cepafungins I, II and III from Pseudomonas species. J Antibiot 43: 783-787. [0240] Shuanglin, X., Johannes, F., and Chiang, J. Li. (2006) Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nature biotechnology 24: 6. [0241] Silakowski B, Schairer H U, Ehret H, Kunze B, Weinig S, Nordsiek G, Brandt P, Blocker H, Hofle G, Beyer S, and Muller R. (1999) New lessons for combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of Stigmatella aurantiaca DW4/3-1. J Biol Chem. 274 (52):37391-9. [0242] Stefan, H., Eric, O.2, Jo{umlaut over ( )}rg, H. and Ulrich, D. (2007) Expression analysis of the colibactin gene cluster coding for a novel polyketide in Escherichia coli. FEMS Microbiol Lett: 1-8. [0243] Stritzker J, Weibel S, Hill P J, Oelschlaeger T A, Goebel W, Szalay A A. (2007) Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. Int J Med Microbiol. 297 (3):151-62. Epub 2007 Apr. 19. [0244] Sznol, M., Lin, S. L., Bermudes, D., Zheng, L. M., King, I. (2000) Use of preferentially replicating bacteria for the treatment of cancer. J Clin Invest 105: 1027-30. [0245] Tang, L., Sanjay, S., Loleta, C., John, C., Leonard, K., Chaitan, K., and Bryan, J. (2000) Cloning and heterologous expression of the epothilone gene cluster. Science 287: 640-642. [0246] Van Dyk T K, LaRossa R A. (1987) Involvement of ack-pta operon products in alpha-ketobutyrate metabolism by Salmonella typhimurium. Mol Gen Genet. 207 (2-3):435-40. [0247] Vlasak, J., Ondre, j M. (1992) Construction and use of Agrobacterium tumefaciens binary vectors with A. tumefaciens C58 T-DNA genes. Folia Microbiol (Praha) 37:227-30. [0248] Waspi, U., Blanc, D., Winkler, T., Ruedi, P., and Dudler, R. (1998) Syringolin, a novel peptide elicitor from Pseudomonas syringae pv. syringae that induces resistance to Pyricularia oryzae in rice. Mol Plant Microbe Interact 11: 727-733. [0249] Waspi, U., Hassa, P., Staempfli, A., Molleyres, L.-P., Winkler, T., and Dudler, R. (1999) Identification and structure of a family of syringolin variants: unusual cyclic peptides from Pseudomonas syringae pv. syringae that elicit defense responses in rice. Microbiol Res 154: 1-5. [0250] Waspi, U., Schweizer, P., and Dudler, R. (2001) Syringolin reprograms wheat to undergo hypersensitive cell death in a compatible interaction with powdery mildew. Plant Cell 13:153-161. [0251] Wenzel, S., Gross, F., Zhang, Y., Fu, J., Stewart, F. and Muller, R. (2005) Heterologous expression of a myxobacterial natural products assembly line in Pseudomonads via Red/ET recombineering. Chem. Biol., 12: 349-356. [0252] Yi C, Huang Y, Guo Z Y, Wang S R. (2005) Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma. Acta Pharmacol Sin. 26 (5):629-34. [0253] Yu Q, Stamenkovic I. (2004) Transforming growth factor-beta facilitates breast carcinoma metastasis by promoting tumor cell survival. Clin Exp Metastasis. 21 (3):235-42. [0254] Zhang Y. et al., (1998) A new logic for DNA engineering using recombination in Escherichia coli, Nat. Genet., 20, 123-128 [0255] Zhang, Y., Muyrers, J. P., Testa, G., and Stewart, A. F. (2000) DNA cloning by homologous recombination in Escherichia coli. Nature Biotechnology 18: 1314-1317. [0256] Zhang, Y. et al., 2003 (BMC Mol Biol. 2003 Jan. 16; 4 (1):1)) [0257] Zhao M, Yang M, Li X M, Jiang P, Baranov E, Li S, Xu M, Penman S, Hoffman R M. (2005) Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA. 102 (3):755-60. [0258] Zhao M, Yang M, Ma H, Li X, Tan X, Li S, Yang Z, Hoffman R M. (2006) Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice. Cancer Res. 66 (15):7647-52.