The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90.

BACKGROUND: Epothilones are produced by the myxobacterium Sorangium cellulosum So ce90, and, like paclitaxel (Taxol((R))), they inhibit microtubule depolymerisation and arrest the cell cycle at the G2-M phase. They are effective against P-glycoprotein-expressing multiple-drug-resistant tumor cell lines and are more water soluble than paclitaxel. The total synthesis of epothilones has been achieved, but has not provided an economically viable alternative to fermentation. We set out to clone, sequence and analyze the gene cluster responsible for the biosynthesis of the epothilones in S. cellulosum So ce90. RESULTS: A cluster of 22 open reading frames spanning 68,750 base pairs of the S. cellulosum So ce90 genome has been sequenced and found to encode nine modules of a polyketide synthase (PKS), one module of a nonribosomal peptide synthetase (NRPS), a cytochrome P450, and two putative antibiotic transport proteins. Disruptions in the genes encoding the PKS abolished epothilone production. The first PKS module and the NRPS module are proposed to co-operate in forming the thiazole heterocycle of epothilone from an acetate and a cysteine by condensation, cyclodehydration and subsequent dehydrogenation. The remaining eight PKS modules are responsible for the elaboration of the rest of the epothilone carbon skeleton. CONCLUSIONS: The overall architecture of the gene cluster responsible for epothilone biosynthesis has been determined. The availability of the cluster should facilitate the generation of designer epothilones by combinatorial biosynthesis approaches, and the heterologous expression of epothilones in surrogate microbial hosts.

Conservation of the pyrrolnitrin biosynthetic gene cluster among six pyrrolnitrin-producing strains.

The prnABCD gene cluster from Pseudomonas fluorescens encodes the biosynthetic pathway for pyrrolnitrin, a secondary metabolite derived from tryptophan which has strong anti-fungal activity. We used the prn genes from P. fluorescens strain BL915 as a probe to clone and sequence homologous genes from three other Pseudomonas strains, Burkholderia cepacia and Myxococcus fulvus. With the exception of the prnA gene from M. fulvus59% similar among the strains, indicating that the biochemical pathway for pyrrolnitrin biosynthesis is highly conserved. The prnA gene from M. fulvus is about 45% similar to prnA from the other strains and contains regions which are highly conserved among all six strains.

Functions encoded by pyrrolnitrin biosynthetic genes from Pseudomonas fluorescens.

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of L-tryptophan to form 7-chloro-L-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-L-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.

Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin.

Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of pyrrolnitrin. Deletion mutations in any of the four genes resulted in a pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-pyrrolnitrin gene fusion was shown to complement the pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for pyrrolnitrin biosynthesis.

The non-haem chloroperoxidase from Pseudomonas fluorescens and its relationship to pyrrolnitrin biosynthesis.

The non-haem chloroperoxidase gene (cpoF) from the pyrrolnitrin producer Pseudomonas fluorescens BL914 was cloned using an oligonucleotide derived from part of the N-terminal amino acid sequence of chloroperoxidase (CPO-P) from Pseudomonas pyrrocina as a probe. Based on the overexpression of cpoF in Escherichia coli and the stability of CPO-F against higher temperatures and proteases, the enzyme was purified to homogeneity. Partial characterization of the enzyme showed that it belongs to the class of bacterial non-haem CPOs. To investigate the role of CPO-F in pyrrolnitrin biosynthesis, the cpoF gene was inactivated by insertion of a kanamycin cassette. Exchange of the chromosomal cpoF gene against the disrupted copy had no influence on pyrrolnitrin production demonstrating that CPO-F was not involved in pyrrolnitrin biosynthesis.

A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes.

A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I.

Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease.

A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N' -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.

Physical and genetic characterization of linear DNA plasmids from the heterothallic yeast Saccharomycopsis crataegensis.

Five strains of the heterothallic yeast Saccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al. 1987). Three DNA plasmids, designated pScrl-1, -2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904. DNA plasmids were not identified in S. crataegensis strains Y-5910 or YB-192. Four S. crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al. 1987). Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3. Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3. This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al. (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902. The pScrl plasmids show no homology to the dsRNA molecules of S. crataegensis, the 2 microM circular DNA of Staccharomyces cerevisiae, the 'killer' plasmids of Kluyveromyces lactis, or the linear DNA plasmids of Pichia inositovora. In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered. Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.

Physical and biological characterization of linear DNA plasmids of the yeast Pichia inositovora.

Three cryptic DNA plasmids have been identified in a strain of the yeast Pichia inositovora that are 18, 13, and 10 kbp in size. All are sensitive to digestion by DNase I, restriction endonucleases, and exonuclease III, but are resistant to the activities of RNase A and lambda exonuclease. These results indicate that each plasmid is a linear DNA molecule whose 5' ends are protected. A restriction map has been developed for each of the plasmids, demonstrating that each is unique and confirming their linear nature. The plasmids are a major constituent of DNA prepared from whole cells, but are absent from DNA preparations of purified mitochondria and nuclei, indicating that the plasmids are located in the cytoplasm. These plasmids share many of the physical characteristics described for the linear plasmids of the yeasts Kluyveromyces lactis and Saccharomycopsis crataegensis. Unlike the linear plasmids of K. lactis, however, they appear not to be capable of killer toxin production.

Isolation and Characterization of Frankia sp. Strain FaC1 Genes Involved in Nitrogen Fixation.

Genomic DNA was isolated from Frankia sp. strain FaC1, an Alnus root nodule endophyte, and used to construct a genomic library in the cosmid vector pHC79. The genomic library was screened by in situ colony hybridization to identify clones of Frankia nitrogenase (nif) genes based on DNA sequence homology to structural nitrogenase genes from Klebsiella pneumoniae. Several Frankia nif clones were isolated, and hybridization with individual structural nitrogenase gene fragments (nifH, nifD, and nifK) from K. pneumoniae revealed that they all contain the nifD and nifK genes, but lack the nifH gene. Restriction endonuclease mapping of the nifD and nifK hybridizing region from one clone revealed that the nifD and nifK genes in Frankia sp. are contiguous, while the nifH gene is absent from a large region of DNA on either side of the nifDK gene cluster. Additional hybridizations with gene fragments derived from K. pneumoniae as probes and containing other genes involved in nitrogen fixation demonstrated that the Frankia nifE and nifN genes, which play a role in the biosynthesis of the iron-molybdenum cofactor, are located adjacent to the nifDK gene cluster.