Duplication of partial spinosyn biosynthetic gene cluster in Saccharopolyspora spinosa enhances spinosyn production

Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. Most of the S. spinosa genes involved in spinosyn biosynthesis are found in a contiguous c. 74-kb cluster. To increase the spinosyn production through overexpression of their biosynthetic genes, part of its gene cluster (c. 18 kb) participating in the conversion of the cyclized polyketide to spinosyn was obtained by direct cloning via Red/ET recombination rather than by constructing and screening the genomic library. The resultant plasmid pUCAmT-spn was introduced into S. spinosa CCTCC M206084 from Escherichia coli S17-1 by conjugal transfer. The subsequent single-crossover homologous recombination caused a duplication of the partial gene cluster. Integration of this plasmid enhanced production of spinosyns with a total of 388 (± 25.0) mg L(-1) for spinosyns A and D in the exconjugant S. spinosa trans1 compared with 100 (± 7.7) mg L(-1) in the parental strain. Quantitative real time polymerase chain reaction analysis of three selected genes (spnH, spnI, and spnK) confirmed the positive effect of the overexpression of these genes on the spinosyn production. This study provides a simple avenue for enhancing spinosyn production. The strategies could also be used to improve the yield of other secondary metabolites.     

A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14-membered macrolides (D-desosamine) can be efficiently attached to a 16-membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers L-oleandrose, has recently been shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6-deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamnosyl-6-deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16-membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5-O-desosaminyl-tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.     

A new modular polyketide synthase in the erythromycin producer Saccharopolyspora erythraea

A previously unidentified set of genes encoding a modular polyketide synthase (PKS) has been sequenced in Saccharopolyspora erythraea, producer of the antibiotic erythromycin. This new PKS gene cluster (pke) contains four adjacent large open reading frames (ORFs) encoding eight extension modules, flanked by a number of other ORFs which can be plausibly assigned roles in polyketide biosynthesis. Disruption of the pke PKS genes gave S. erythraea mutant JC2::pSBKS6, whose growth characteristics and pattern of secondary metabolite production did not apparently differ from the parent strain under any of the growth conditions tested. However, the pke PKS loading module and individual pke acyltransferase domains were shown to be active when used in engineered hybrid PKSs, making it highly likely that under appropriate conditions these biosynthetic genes are indeed expressed and active, and synthesize a novel polyketide product.     

<Emphasis Type="Italic">SpnH</Emphasis> from <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> encodes a rhamnosyl 4′-<Emphasis Type="Italic">O</Emphasis>-methyltransferase for biosynthesis of the insecticidal macrolide,spinosyn A

Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.     

Improved bioconversion of 15-fluoro-6-deoxyerythronolide B to 15-fluoro-erythromycin A by overexpression of the eryK Gene in Saccharopolyspora erythraea

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S. erythraea K301-105B, compared to its parent. In shake flasks, K301-105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor.     

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21–carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.     

Biosynthesis of the anti-parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea

Megalomicin is a therapeutically diverse compound which possesses antiparasitic, antiviral and antibacterial properties. It is produced by Micromonospora megalomicea and differs from the well-known macrolide antibiotic erythromycin by the addition of a unique deoxyamino sugar, megosamine, to the C-6 hydroxyl. We have cloned and sequenced a 48 kb segment of the megalomicin (meg) biosynthetic gene cluster which contains the modular polyketide synthase (PKS) and the complete pathway for megosamine biosynthesis. The similarities and distinctions between the related megalomicin and erythromycin gene clusters are discussed. Heterologous expression of the megalomicin PKS in Streptomyces lividans led to production of 6-deoxyerythronolide B, the same macrolactone intermediate for erythromycin. A 12 kb fragment harbouring the putative megosamine pathway was expressed in Saccharopolyspora erythraea, resulting in the conversion of erythromycin to megalomicin. Considering the extensive knowledge surrounding the genetic engineering of the erythromycin PKS and the familiarity with genetic manipulation and fermentation of S. erythraea, the ability to produce megalomicin in this strain should allow the engineering of novel megalomicin analogues with potentially improved therapeutic activities.     

Distribution of oxoacyl synthase homology sequences within Streptomyces DNA

Abstract A genomic DNA sequence of Streptomyces strain ISP 5485 was cloned, sequenced and compared with corresponding information from nucleic acid data banks. The DNA sequence was unique, but showed homology to DNA coding for the condensing enzyme, 2-oxoacyl synthase, of the deoxyerythronolide B synthase complex (DEBS) from Saccharopolyspora erythraea NRRL 2338. A subfragment of the sequenced DNA was used to construct a gene-specific probe that formed part of the putative 2-oxoacyl synthase gene. The PCR-amplified and labelled probe was used in hybridization experiments involving 33 streptomycete strains that produced different classes of antibiotics. The probe showed widespread homology with DNA considered to be part of analogous genes within genomes of different polyketide producers. The implications of the probe homology to bacterial chromosomal DNA are discussed.     

Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea.

The ery A region of the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previously been shown to contain three large open reading frames (ORFs) that encode the components of 6-deoxyerythronolide B synthase (DEBS). Polyclonal antibodies were raised against recombinant proteins obtained by overexpression of 3' regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different high molecular weight protein in extracts of erythromycin-producing S. erythraea cells. These putative DEBS 2 and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the and DEBS 3 proteins were purified and subjected to N-terminal sequence analysis. The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences of ORFs 2 and 3. A third high molecular weight protein co-purified with DEBS 2 and DEBS 3 and had an N-terminal sequence that matched a protein sequence translated from the DNA sequence some 155 base pairs upstream from the previously proposed start codon of ORF1.     

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402. Received 31 May 2001/ Accepted in revised form 09 July 2001     

Mutation and cloning of eryG, the structural gene for erythromycin O-methyltransferase from Saccharopolyspora erythraea, and expression of eryG in Escherichia coli.

A mutant strain derived by chemical mutagenesis of Saccharopolyspora erythraea (formerly known as Streptomyces erythreus) was isolated that accumulated erythromycin C and, to a lesser extent, its precursor, erythromycin D, with little or no production of erythromycin A or erythromycin B (the 3"-O-methylation products of erythromycin C and D, respectively). This mutant lacked detectable erythromycin O-methyltransferase activity with erythromycin C, erythromycin D, or the analogs 2-norerythromycin C and 2-norerythromycin D as substrates. A 4.5-kilobase DNA fragment from S. erythraea originating approximately 5 kilobases from the erythromycin resistance gene ermE was identified that regenerated the parental phenotype and restored erythromycin O-methyltransferase activity when transformed into the erythromycin O-methyltransferase-negative mutant. Erythromycin O-methyltransferase activity was detected when the 4.5-kilobase fragment was fused to the lacZ promoter and introduced into Escherichia coli. The activity was dependent on the orientation of the DNA relative to lacZ. We have designated this genotype eryG in agreement with Weber et al. (J.M. Weber, B. Schoner, and R. Losick, Gene 75:235-241, 1989). It thus appears that a single enzyme catalyzes all of the 3"-O-methylation reactions of the erythromycin biosynthetic pathway in S. erythraea and that eryG codes for the structural gene of this enzyme.     

Identification of erythrobactin, a hydroxamate-type siderophore produced by Saccharopolyspora erythraea

AIMS: To investigate the production of siderophores by Saccharopolyspora erythraea SGT2 and how this production is affected by the inoculum. METHODS AND RESULTS: When grown in a low-iron, chemically defined medium (CDM), the soil dwelling actinomycete S. erythraea secretes a substance that is reactive in the nonspecific chrome azurol S (CAS) assay. Importantly, the production of CAS-reactive substance is highly reduced upon the addition of 0.925 micromol l(-1) iron to the cultures and has a peak of production in the late-log to early stationary growth phase. In addition, the culture supernatants tested were negative in the Arnow and Rioux assays but positive in the Csáky procedure. Interestingly, we also found evidence that the production of this CAS-reactive substance in CDM was highly reduced, when inoculated with cells that had been previously grown to late-stationary phase. Conversely, inocula derived from late-log to early stationary cultures presented high levels of CAS activity. CONCLUSIONS: These results indicate that S. erythraea produces a hydroxamate-type siderophore that we have generically designated as erythrobactin. Additionally, the inocula growth stage plays a key role in siderophore production in S. erythraea. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first evidence for siderophore synthesis in S. erythraea and one of the first examples of non-polyketide secondary metabolite production by this organism.     

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.     

Saccharopolyspora erythraea-catalyzed bioconversion of 6-deoxyerythronolide B analogs for production of novel erythromycins.

A method was developed for the large-scale bioconversion of novel 6-deoxyerythronolide B (6-dEB) analogs into erythromycin analogs. Erythromycin biosynthesis in Saccharopolyspora erythraea proceeds via the formation of a polyketide aglycone, 6-dEB, which is subsequently glycosylated, hydroxylated and methylated to yield the antibiotic erythromycin A. A modular polyketide synthase (PKS) directs 6-dEB synthesis using a dedicated set of active sites for the condensation of each of seven propionate units. Strategies based on genetic manipulation and precursor feeding are available for the efficient generation of novel 6-dEB analogs using a plasmid-based system in Streptomyces coelicolor. 6-dEB and 13-substituted 6-dEB analogs produced in this manner were fed to S. erythraea mutants which could not produce 6-dEB, yet retained their 6-dEB modification systems, and resulted in the generation of erythromycin A and 13-substituted erythromycin A analogs. Erythromycin B, C and D analogs were observed as intermediates of the process. Dissolved oxygen, temperature, the specific aglycone feed concentration, and pH were found to be important for obtaining a high yield of erythromycin A analogs. Cultivation conditions were identified which resulted in the efficient bioconversion of 6-dEB analogs into erythromycin A analogs, which this process demonstrated at the 100 l scale.     

Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis.

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.     

Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives.

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.     

Characterization of two polyketide methyltransferases involved in the biosynthesis of the antitumor drug mithramycin by Streptomyces argillaceus

A DNA chromosomal region of Streptomyces argillaceus ATCC 12596, the producer organism of the antitumor polyketide drug mithramycin, was cloned. Sequence analysis of this DNA region, located between four mithramycin glycosyltransferase genes, showed the presence of two genes (mtmMI and mtmMII) whose deduced products resembled S-adenosylmethionine-dependent methyltransferases. By independent insertional inactivation of both genes nonproducing mutants were generated that accumulated different mithramycin biosynthetic intermediates. The M3DeltaMI mutant (mtmMI-minus mutant) accumulated 4-demethylpremithramycinone (4-DPMC) which lacks the methyl groups at carbons 4 and 9. The M3DeltaM2 (mtmMII-minus mutant) accumulated 9-demethylpremithramycin A3 (9-DPMA3), premithramycin A1 (PMA1), and 7-demethylmithramycin, all of them containing the O-methyl group at C-4 and C-1', respectively, but lacking the methyl group at the aromatic position. Both genes were expressed in Streptomyces lividans TK21 under the control of the erythromycin resistance promoter (ermEp) of Saccharopolyspora erythraea. Cell-free extracts of these clones were precipitated with ammonium sulfate (90% saturation) and assayed for methylation activity using different mithramycin intermediates as substrates. Extracts of strains MJM1 (expressing the mtmMI gene) and MJM2 (expressing the mtmMII gene) catalyzed efficient transfer of tritium from [(3)H]S-adenosylmethionine into 4-DPMC and 9-DPMA3, respectively, being unable to methylate other intermediates at a detectable level. These results demonstrate that the mtmMI and mtmMII genes code for two S-adenosylmethionine-dependent methyltransferases responsible for the 4-O-methylation and 9-C-methylation steps of the biosynthetic precursors 4-DPMC and 9-DPMA3, respectively, of the antitumor drug mithramycin. A pathway is proposed for the last steps in the biosynthesis of mithramycin involving these methylation events.     

Precursor-directed production of erythromycin analogs by Saccharopolyspora erythraea.

Diketide N-acetylcysteamine (diketide NAC) thioester precursors were fed to 6-Deoxyerythronolide B synthase (DEBS) ketosynthase-1 inactivated (KS1 degree) Saccharopolyspora erythraea strains to produce 13-substituted erythromycin analogs. This direct feeding process potentially represents a simplified production process over the current analog production system. Titers of these analogs were observed to increase linearly with the diketide concentration up to a precursor-specific saturation level. However, the rate of product formation was lower and the rate of diketide consumption higher with S. erythraea than was previously observed with a recombinant strain of Streptomyces coelicolor. Several strategies were pursued to address the issue of these high diketide consumption rates: (1) elucidation of the locale of diketide degradation, (2) addition of beta-oxidation inhibitors to the cultures, and (3) addition of a sacrificial diketide enantiomer to occupy putative degradative enzymes. Additionally, repeated addition of diketide to an S. erythraea KS1 degrees culture indicated that the titer of these erythromycin analogs is also currently limited by a shorter production period than observed during erythromycin synthesis by the parent strain. These results indicate potential avenues for expanding the use of this precursor-directed system from the generation of limited quantities of erythromycin analogs to a large-scale production system for these compounds.     

Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus).

Genes that govern the formation of deoxysugars or their attachment to erythronolide B and 3 alpha-mycarosyl erythronolide B, intermediates of the biosynthesis of the 14-membered macrolide antibiotic erythromycin, were cloned from Saccharopolyspora erythraea (formerly Streptomyces erythreus). Segments of DNA that complement the eryB25, eryB26, eryB46, eryC1-60, and eryD24 mutations blocking the formation of erythronolide B or 3 alpha-mycarosyl erythronolide B, when cloned in Escherichia coli-Streptomyces shuttle cosmids or plasmid vectors that can transform S. erythraea, were located in a ca. 18-kilobase-pair region upstream of the erythromycin resistance (ermE) gene. The eryC1 gene lies just to the 5' side of ermE, and one (or possibly two) eryB gene is approximately 12 kilobase pairs farther upstream. Another eryB gene may be in the same region, while an additional eryB mutation appears to be located elsewhere. The eryD gene lies between the eryB and eryC1 genes and may regulate their function on the basis of the phenotype of an EryD- mutant.