Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering

Natural products of microbial origin are widely used as pharmaceuticals and in agrochemistry. These compounds are often biosynthesized by multifunctional megasynthetases whose genetic engineering and heterologous expression offer considerable promise, especially if the natural hosts are genetically difficult to handle, slow growing, unculturable, or even unknown. We describe a straightforward strategy that combines the power of advanced DNA engineering (recombiogenic cloning) in Escherichia coli with the utility of pseudomonads as the heterologous host for the analysis and mutagenesis of known and unknown secondary metabolite pathways. The myxochromide S biosynthetic gene cluster from Stigmatella aurantiaca was rebuilt and engineered in E. coli to contain the elements required for expression in pseudomonads. The successful production in Pseudomonas putida, at unprecedented levels, demonstrates the feasibility of the new approach to the analysis and mutagenesis of these important pathways.     

Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster

Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans. Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4, fomA-D. A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered approximately 13 kb away from the cluster and was named fomR. The genes fomE and fomF, previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.     

Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa

BACKGROUND: Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS: We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS: Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.     

Cloning, sequencing, analysis, and heterologous expression of the fredericamycin biosynthetic gene cluster from Streptomyces griseus

Fredericamycin (FDM) A, a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique chiral spiro carbon center, exhibits potent cytotoxicity and has been studied as a new type of anticancer drug lead because of its novel molecular architecture. The fdm gene cluster was localized to 33-kb DNA segment of Streptomyces griseus ATCC 49344, and its involvement in FDM A biosynthesis was proven by gene inactivation, complementation, and heterologous expression experiments. The fdm cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS) and tailoring enzymes as well as several regulatory and resistance proteins. The FDM PKS features a KSalpha subunit with heretofore unseen tandem cysteines at its active site, a KSbeta subunit that is distinct phylogenetically from KSbeta of hexa-, octa-, or decaketide PKSs, and a dedicated phosphopantetheinyl transferase. Further study of the FDM PKS could provide new insight into how a type II PKS controls chain length in aromatic polyketide biosynthesis. The availability of the fdm genes, in vivo characterization of the fdm cluster in S. griseus, and heterologous expression of the fdm cluster in Streptomyces albus set the stage to investigate FDM A biosynthesis and engineer the FDM biosynthetic machinery for the production of novel FDM A analogues.     

The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster

BACKGROUND: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. RESULTS: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. CONCLUSIONS: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.     

Functional analysis of the validamycin biosynthetic gene cluster and engineered production of validoxylamine A

A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.     

Molecular analysis of the benastatin biosynthetic pathway and genetic engineering of altered fatty acid-polyketide hybrids

The entire gene locus encoding the biosynthesis of the potent glutathione-S-transferase inhibitors and apoptosis inducers benastatin A and B has been cloned and sequenced. The cluster identity was unequivocally proven by deletion of flanking regions and heterologous expression in S. albus and S. lividans. Inactivation and complementation experiments revealed that a KSIII component (BenQ) similar to FabH is crucial for providing and selecting the rare hexanoate PKS starter unit. In the absence of BenQ, several novel penta- and hexacyclic benastatin derivatives with antiproliferative activities are formed. In total, five new compounds were isolated and fully characterized, and the chemical analysis was confirmed by derivatization. The most intriguing observation is that the ben PKS can utilize typical straight and branched fatty acid synthase primers. If shorter straight-chain starters are utilized, the length of the polyketide backbone is increased, resulting in the formation of an extended, hexacyclic ring system reminiscent of proposed intermediates in the griseorhodin and fredericamycin pathways. Analysis and manipulation of the hybrid fatty acid polyketide pathway provides strong support for the hypothesis that the number of chain elongations is dependent on the total size of the polyketide chain that is accommodated in the PKS enzyme cavity. Our results also further substantiate the potential of metabolic engineering toward polyphenols with altered substituents and ring systems.     

The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: genetic and biochemical evidence for the roles of two glycosyltransferases and a deacetylase

An efficient protocol has been developed for the genetic manipulation of Streptomyces fradiae NCIMB 8233, which produces the 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic neomycin. This has allowed the in vivo analysis of the respective roles of the glycosyltransferases Neo8 and Neo15, and of the deacetylase Neo16 in neomycin biosynthesis. Specific deletion of each of the neo8, neo15 and neo16 genes confirmed that they are all essential for neomycin biosynthesis. The pattern of metabolites produced by feeding putative pathway intermediates to these mutants provided unambiguous support for a scheme in which Neo8 and Neo15, whose three-dimensional structures are predicted to be highly similar, have distinct roles: Neo8 catalyses transfer of N-acetylglucosamine to 2-DOS early in the pathway, while Neo15 catalyses transfer of the same aminosugar to ribostamycin later in the pathway. The in vitro substrate specificity of Neo15, purified from recombinant Escherichia coli, was fully consistent with these findings. The in vitro activity of Neo16, the only deacetylase so far recognised in the neo gene cluster, showed that it is capable of acting in tandem with both Neo8 and Neo15 as previously proposed. However, the deacetylation of N-acetylglucosaminylribostamycin was still observed in a strain deleted of the neo16 gene and fed with suitable pathway precursors, providing evidence for the existence of a second enzyme in S. fradiae with this activity.     

The biosynthetic gene cluster for the antitumor rebeccamycin: characterization and generation of indolocarbazole derivatives

Rebeccamycin, a halogenated natural product of the indolocarbazole family, is produced by Saccharothrix aerocolonigenes ATCC39243. Several rebeccamycin analogues, which target DNA topoisomerase I or II, have already entered clinical trials as anticancer drugs. Using as a probe an internal fragment of ngt, a Saccharothrix aerocolonigenes gene encoding an indolocarbazole N-glycosyltransferase, we isolated a DNA region that directed the biosynthesis of rebeccamycin when introduced into Streptomyces albus. Sequence analysis of 25.6 kb revealed genes for indolocarbazole core formation, halogenation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. Heterologous expression of subsets of these genes resulted in production of deschloro-rebeccamycin, 4'-demethyldeschloro-rebeccamycin, and deschloro-rebeccamycin aglycone. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and set the stage for the generation of novel indolocarbazole analogues by genetic engineering.     

Identification and analysis of the core biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer activity

Myxobacteria are well known for their biosynthetic potential, especially for the production of cytotoxic compounds with potential anticancer activities. The tubulysins are currently in preclinical development. They are produced in very low quantities, and genetic manipulation of producing strains has never been accomplished. We report the development of a mariner-based transposon mutagenesis system for Angiococcus disciformis An d48. Extracts from a library of 1200 mutants were analyzed for the presence of tubulysin by a microscopic cell nucleus fragmentation bioassay. The transposition sites of four tubulysin-negative mutants were identified by vector recovery, which led to the identification and the sequencing of the corresponding core biosynthetic gene locus. Sequence analysis of more than 80,000 bp reveals an unusual multimodular hybrid polyketide synthase/peptide synthetase assembly line with a variety of unprecedented features.     

Molecular analysis of the kirromycin biosynthetic gene cluster revealed beta-alanine as precursor of the pyridone moiety

Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus Tü 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.     

Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase

The lyngbyatoxins are potent skin irritants produced by Lyngbya majuscula and cause a condition known as "Swimmer's Itch" off Honolulu, HI. Reported is the molecular cloning of the lyngbyatoxin (ltx) biosynthetic gene cluster from L. majuscula using a strategy based on its predicted nonribosomal peptide synthetase (NRPS) assembly. The biosynthetic gene cluster spans 11.3 kilobase pairs and encodes for a two-module NRPS (LtxA), a P450 monooxygenase (LtxB), an aromatic prenyltransferase (LtxC), and an oxidase/reductase protein (LtxD). LtxC was heterologously produced and purified from E. coli and shown to catalyze the transfer of a geranyl group to (-)-indolactam V as the final step in the biosynthesis of lyngbyatoxin A.     

Heterologous expression, biosynthesis, and mutagenesis of type II lantibiotics from Bacillus licheniformis in Escherichia coli

Lichenicidin is a class II two-component lantibiotic produced by Bacillus licheniformis. It is composed of the two peptides Bliα and Bliβ, which act synergistically against various Gram-positive bacteria. The lichenicidin gene cluster was successfully expressed in Escherichia coli, thus constituting the first report to our knowledge of a full reconstitution of a lantibiotic biosynthetic pathway in?vivo by a Gram-negative host. This system was further exploited to characterize and assign the function of proteins encoded in the biosynthetic gene cluster in the maturation of lichenicidin peptides. Moreover, a trans complementation system was developed for expression of Bliα and Bliβ variants in?vivo. This contribution will spur future studies in the heterologous expression and engineering of lantibiotics.     

Identification and characterization of the lysobactin biosynthetic gene cluster reveals mechanistic insights into an unusual termination module architecture

Lysobactin (katanosin B) is a macrocyclic depsipeptide, displaying high antibacterial activity against human pathogens. In this work, we have identified and characterized the entire biosynthetic gene cluster responsible for lysobactin assembly. Sequential analysis of the Lysobacter sp. ATCC 53042 genome revealed the lysobactin gene cluster to encode two multimodular nonribosomal peptide synthetases. As the number of modules found within the synthetases LybA and LybB directly correlates with the primary sequence of lysobactin, a linear logic of lysobactin biosynthesis is proposed. Investigation of adenylation domain specificities in?vitro confirmed the direct association between the synthetases and lysobactin biosynthesis. Furthermore, an unusual tandem thioesterase architecture of the LybB termination module was identified. Biochemical characterization of the individual thioesterases in?vitro provides evidence that solely penultimate thioesterase domain mediates the cyclization and simultaneous release of lysobactin.     

Characterization of tiacumicin B biosynthetic gene cluster affording diversified tiacumicin analogues and revealing a tailoring dihalogenase

The RNA polymerase inhibitor tiacumicin B is currently undergoing phase III clinical trial for treatment of Clostridium difficile associated diarrhea with great promise. To understand the biosynthetic logic and to lay a foundation for generating structural analogues via pathway engineering, the tiacumicin B biosynthetic gene cluster was identified and characterized from the producer Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085. Sequence analysis of a 110,633 bp DNA region revealed the presence of 50 open reading frames (orfs). Functional investigations of 11 orfs by in vivo inactivation experiments, preliminarily outlined the boundaries of the tia-gene cluster and suggested that 31 orfs were putatively involved in tiacumicin B biosynthesis. Functions of a halogenase (TiaM), two glycosyltransferases (TiaG1 and TiaG2), a sugar C-methyltransferase (TiaS2), an acyltransferase (TiaS6), and two cytochrome P450s (TiaP1 and TiaP2) were elucidated by isolation and structural characterization of the metabolites from the corresponding gene-inactivation mutants. Accumulation of 18 tiacumicin B analogues from 7 mutants not only provided experimental evidence to confirm the proposed functions of individual biosynthetic enzymes, but also set an example of accessing microbial natural product diversity via genetic approach. More importantly, biochemical characterization of the FAD-dependent halogenase TiaM reveals a sequentially acting dihalogenation step tailoring tiacumicin B biosynthesis.     

Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin

Background: Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron. Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion. The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively.Results: A ten-gene cluster spanning 24 kilobases of the M. tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis. The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, Mbtl an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase. An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation. Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP.Conclusions: Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.     

Spiroketal polyketide formation in Sorangium: identification and analysis of the biosynthetic gene cluster for the highly cytotoxic spirangienes

Natural products constitute important lead structures in drug discovery. In bacteria, they are often synthesized by large, modular multienzyme complexes. Detailed analysis of the biosynthetic machinery should enable its directed engineering and production of desirable analogs. The myxobacterium Sorangium cellulosum So ce90 produces the cytotoxic spiroketal polyketide spirangien, for which we describe the identification and functional analysis of the biosynthetic pathway. The gene cluster spans 88 kb and encodes 7 type I polyketide synthases and additional enzymes such as a stand-alone thioesterase and 2 methyltransferases. Inactivation of two cytochrome P(450) monooxygenase genes resulted in the production of acyclic spirangien derivatives, providing direct evidence for the involvement of these enzymes in spiroketal formation. The presence of large DNA repeats is consistent with multiple rounds of gene duplication during the evolution of the biosynthetic gene locus.