Feedback regulation of doxorubicin biosynthesis in Streptomyces peucetius.

DnrO, one of three DNA binding regulatory proteins involved in daunorubicin biosynthesis in Streptomyces peucetius, has been purified as a maltose-binding protein-DnrO (MBP-DnrO) fusion protein. Gel mobility shift assays showed that it specifically bound to a DNA fragment containing both dnrN and dnrO promoters. In the presence of some low-molecular-weight compounds from the daunorubicin biosynthetic pathway, the DNA binding ability of MBP-DnrO was affected. Melanin production assays showed that both DnrO and DnrN were required for the increased activity of the dnrI promoter. Rhodomycin D (RHOD), one of the intermediates in the DNR and DXR biosynthetic pathways, had a positive effect on dnrI promoter activity only in the presence of both DnrO and DnrN proteins. The promoter activity of dnrO gene decreased in the presence of the DnrO protein, suggesting that dnrO gene was autoregulated. Repression could be relieved when RHOD was present in the culture, indicating that RHOD might directly interact with the DnrO protein.

Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602.

We illustrate the use of a PCR-based method by which the genomic DNA of a microorganism can be rapidly queried for the presence of type I modular polyketide synthase genes to clone and characterize, by sequence analysis and gene disruption, a major portion of the geldanamycin production gene cluster from Streptomyces hygroscopicus var. geldanus NRRL 3602.

Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues.

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.

Isolation and characterization of novel geldanamycin analogues.

New geldanamycin analogues with novel structures arising from direct microbial bioconversion and a genetically engineered geldanamycin producer were isolated and characterized. Three compounds, 15-hydroxygeldanamycin, a tricyclic geldanamycin analog (KOSN-1633), and methyl-geldanamycinate), were isolated after geldanamycin was added to a growing culture of the herbimycin producing strain-Streptomyces hygroscopicus AM-3672. Two related compounds, 17-formyl-17-demethoxy-18-O,-21-O-dihydrogeldanamycin and 17-hydroxymethyl-17-demethoxygeldanamycin were isolated from S. hygroscopicus NRRL 3602/pKOS279-78, a geldanamycin-producing strain containing various genes isolated from S. hygroscopicus AM-3672. Compared with geldanamycin, these five new compounds exhibited reduced cytotoxicity against SKBr3 cancer cells.

Characterization of a large, stable, high-copy-number Streptomyces plasmid that requires stability and transfer functions for heterologous polyketide overproduction.

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.

Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta.

Sponge-associated bacteria are thought to produce many novel bioactive compounds, including polyketides. PCR amplification of ketosynthase domains of type I modular polyketide synthases (PKS) from the microbial community of the marine sponge Discodermia dissoluta revealed great diversity and a novel group of sponge-specific PKS ketosynthase domains. Metagenomic libraries totaling more than four gigabases of bacterial genomes associated with this sponge were screened for type I modular PKS gene clusters. More than 90% of the clones in total sponge DNA libraries represented bacterial DNA inserts, and 0.7% harbored PKS genes. The majority of the PKS hybridizing clones carried small PKS clusters of one to three modules, although some clones encoded large multimodular PKSs (more than five modules). The most abundant large modular PKS appeared to be encoded by a bacterial symbiont that made up < 1% of the sponge community. Sequencing of this PKS revealed 14 modules that, if expressed and active, is predicted to produce a multimethyl-branched fatty acid reminiscent of mycobacterial lipid components. Metagenomic libraries made from fractions enriched for unicellular or filamentous bacteria differed significantly, with the latter containing numerous nonribosomal peptide synthetase (NRPS) and mixed NRPS-PKS gene clusters. The filamentous bacterial community of D. dissoluta consists mainly of Entotheonella spp., an unculturable sponge-specific taxon previously implicated in the biosynthesis of bioactive peptides.

Insights into the biosynthesis of the benzoquinone ansamycins geldanamycin and herbimycin, obtained by gene sequencing and disruption.

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.

Rapid engineering of the geldanamycin biosynthesis pathway by Red/ET recombination and gene complementation.

Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog.

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 dnrO gene encodes a DNA-binding protein that regulates daunorubicin production in Streptomyces peucetius by controlling expression of the dnrN pseudo response regulator gene.

The dnrO gene is located adjacent to and divergently transcribed from the response regulator gene, dnrN, that activates the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius. Gene disruption and replacement of dnrO produced the dnrO::aphII mutant strain and resulted in the complete loss of daunorubicin biosynthesis. Suppression of the dnrO::aphII mutation by the introduction of dnrN or dnrI on a plasmid suggested that DnrO is required for the transcription of dnrN, whose product is known to be required for dnrI expression. These conclusions were supported by the effects of the dnrO mutation on expression of dnrO, dnrN and dnrI, as viewed by melC fusions to each of these regulatory genes. DnrO was overexpressed in Escherichia coli and the cell-free extract was used to conduct mobility shift DNA-binding assays. The results showed that DnrO binds specifically to the overlapping dnrN/dnrO(p1) promoter region. Thus, DnrO may regulate the expression of both the dnrN and dnrO genes.

Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI.

Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR-binding domain as a template, DnrI was mapped by truncation and site-directed mutagenesis. Several highly conserved residues within the N-terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the -35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG-dpsABCD promoter were performed by site-directed mutagenesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG-dpsABCD operon.

A host-vector system for analysis and manipulation of rifamycin polyketide biosynthesis in Amycolatopsis mediterranei.

Modular polyketide synthases (PKSs) are a large family of multifunctional enzymes responsible for the biosynthesis of numerous bacterial natural products such as erythromycin and rifamycin. Advanced genetic analysis of these remarkable systems is often seriously hampered by the large size (>40 kb) of PKS gene clusters, and, notwithstanding their considerable fundamental and biotechnological significance, by the lack of suitable methods for engineering non-selectable modifications in chromosomally encoded PKS genes. The development of a facile host-vector strategy for genetic engineering of the rifamycin PKS in the producing organism, Amycolatopsis mediterranei S699, is described here. The genes encoding all 10 modules of the rifamycin PKS were replaced with a hygromycin-resistance marker gene. In a similar construction, only the first six modules of the PKS were replaced. The deletion hosts retained the ability to synthesize the primer unit 3-amino-5-hydroxybenzoic acid (AHBA), as judged by co-synthesis experiments with a mutant strain lacking AHBA synthase activity. Suicide plasmids carrying a short fragment from the 5' flanking end of the engineered deletion, an apramycin-resistance marker gene, and suitably engineered PKS genes could be introduced via electroporation into the deletion hosts, resulting in the integration of PKS genes and biosynthesis of a reporter polyketide in quantities comparable to those produced by the wild-type organism. Since this strategy for engineering recombinant PKSs in A. mediterranei requires only a selectable single crossover and eliminates the need for tedious non-selectable double-crossover experiments, it makes rifamycin PKS an attractive target for extensive genetic manipulation.

Identification of domains within megalomicin and erythromycin polyketide synthase modules responsible for differences in polyketide production levels in Escherichia coli.

The megalomicin and erythromycin polyketide synthases (PKSs) produce the same aglycon product, 6-deoxyerythronolide B (6-dEB). Both PKSs were examined in an Escherichia coli strain metabolically engineered to support complex polyketide biosynthesis. Production of 6-dEB in shake flask fermentations was undetectable by mass spectrometry in the strain expressing the megalomicin (Meg) PKS genes, whereas 31 mg/L 6-dEB was produced by the strain with the erythromycin (DEBS) PKS. The genes for each of the three subunits comprising the PKSs were expressed in different combinations from three compatible expression vectors (e.g., DEBS1, DEBS2, and MegA3) to identify two Meg PKS subunits, MegA1 and MegA3, which conferred lower 6-dEB titers than their DEBS counterparts. Comparison of protein expression levels and 6-dEB titers by engineered hybrid DEBS/Meg PKS genes further defined regions within modules 2 and 6 of MegA1 and MegA3, respectively, which limit protein expression and 6-dEB production in E. coli. Meg module 2 + TE (M2 + TE) and a hybrid DEBS M2/Meg M2 + TE protein were engineered and purified for in vitro comparisons with DEBS M2 + TE. The specific activity of the hybrid M2 + TE was approximately 16-fold lower than DEBS M2 + TE and only twice as high as the Meg M2 + TE enzyme in diketide elongation assays. Since the hybrid M2 worked comparably to DEBS M2 in vivo, this suggests that boosting subunit concentration could serve as a useful approach to overcome enzyme deficiencies in heterologous polyketide production.

Enhanced heterologous polyketide production in Streptomyces by exploiting plasmid co-integration.

A plasmid named pSMALL was discovered in a Streptomyces coelicolor strain that significantly enhanced the levels of production of 15-methyl-6-deoxyerythronolide B, a polyketide lactone normally produced in low amounts by engineered polyketide synthase (PKS) genes. It is a co-integrate between a conventional SCP2*-derived Streptomyces expression plasmid, pJRJ2, and SCP2@, a variant of the parental SCP2* plasmid. SCP2@ has a 45-bp deletion 35 bp upstream of the start codon of the repI gene in the replication region; and this correlated with an enhanced plasmid copy number and polyketide overproduction by its derivatives. This discovery was exploited to construct pBOOST, a high-copy-number cloning vector that can be used to achieve greatly elevated (at least 25-fold), stable metabolite production by PKS genes cloned in SCP2*-derived vectors by forming co-integrates with them.

Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains.

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms- Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively-to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site ( attB) for phiC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.

Transformations of cyclic nonaketides by Aspergillus terreus mutants blocked for lovastatin biosynthesis at the lovA and lovC genes.

Two mutants of Aspergillus terreus with either the lovC or lovA genes disrupted were examined for their ability to transform nonaketides into lovastatin 1, a cholesterol-lowering drug. The lovC disruptant was able to efficiently convert dihydromonacolin L 5 or monacolin J 9 into 1, and could also transform desmethylmonacolin J 15 into compactin 3. In contrast, the lovA mutant has an unexpectedly active beta-oxidation system and gives only small amounts of 1 upon addition of the immediate precursor 9, with most of the added nonaketide being degraded to heptaketide 22. Similarly, the lovA mutant does not accumulate the polyketide synthase product 5 and rapidly degrades any 5 added as a precursor via two cycles of beta-oxidation and hydroxylation at C-6 to give 20. The possible involvement of epoxides 21a and 21b in the biosynthesis of 1 was also examined, but their instability in fermentation media and fungal cells will require purified enzymes to establish their role.

Structural and functional analysis of tetracenomycin F2 cyclase from Streptomyces glaucescens. A type II polyketide cyclase.

Tetracenomycin F2 cyclase (tcmI gene product), catalyzes an aromatic rearrangement in the biosynthetic pathway for tetracenomycin C in Streptomyces glaucescens. The x-ray structure of this small enzyme has been determined to 1.9-A resolution together with an analysis of site-directed mutants of potential catalytic residues. The protein exhibits a dimeric betaalphabeta ferredoxin-like fold that utilizes strand swapping between subunits in its assembly. The fold is dominated by four strands of antiparallel sheet and a layer of alpha-helices, which creates a cavity that is proposed to be the active site. This type of secondary structural arrangement has been previously observed in polyketide monooxygenases and suggests an evolutionary relationship between enzymes that catalyze adjacent steps in these biosynthetic pathways. Mutational analysis of all of the obvious catalytic bases within the active site suggests that the enzyme functions to steer the chemical outcome of the cyclization rather than providing a specific catalytic group. Together, the structure and functional analysis provide insight into the structural framework necessary to perform the complex rearrangements catalyzed by this class of polyketide cyclases.

Reverse engineering of industrial pharmaceutical-producing actinomycete strains using DNA microarrays.

Transcript levels in production cultures of wildtype and classically improved strains of the actinomycete bacteria Saccharopolyspora erythraea and Streptomyces fradiae were monitored using microarrays of the sequenced actinomycete S. coelicolor. Sac. erythraea and S. fradiae synthesize the polyketide antibiotics erythromycin and tylosin, respectively, and the classically improved strains contain unknown overproduction mutations. The Sac. erythraea overproducer was found to express the entire 56-kb erythromycin gene cluster several days longer than the wildtype strain. In contrast, the S. fradiae wildtype and overproducer strains expressed the 85-kb tylosin biosynthetic gene cluster similarly, while they expressed several tens of other S. fradiae genes and S. coelicolor homologs differently, including the acyl-CoA dehydrogenase gene aco and the S. coelicolor isobutyryl-CoA mutase homolog icmA. These observations indicated that overproduction mechanisms in classically improved strains can affect both the timing and rate of antibiotic synthesis, and alter the regulation of antibiotic biosynthetic enzymes and enzymes involved in precursor metabolism.

Gene disruption and replacement in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253.

A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel forms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the phi C31 phage derivative KC515 (C+ attp) carrying a thiostrepton or viomycin resistance gene along with segments of the S. hygroscopicus chromosome. Each of the cloned segments also contained the aphll neomycin/kanamycin resistance gene to enable gene replacement by loss of the phage-derived DNA. Specific deletion of the entire polyketide synthase (PKS) believed to govern rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode four PKS open reading frames, or another region predicted to encode another PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no effect on the production of rapamycin or nigericin, a polyether antibiotic also produced by S. hygroscopicus. Therefore, S. hygroscopicus may have the capacity to produce polyketides additional to rapamycin and nigericin.