Evelyn Wendt-Pienkowski

Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production

The nucleotide sequence on both sides of the eryA polyketide synthase genes of the erythromycin-producing bacterium Saccharopolyspora erythraea reveals the presence of ten genes that are involved in L-mycarose (eryB) and D-desosamine (eryC) biosynthesis or attachment. Mutant strains carrying targeted lesions in eight of these genes indicate that three (eryBIV, eryBV and eryBVI) act in L-mycarose biosynthesis or attachment, while the other five (eryCII, eryCIII, eryCIV, eryCV and eryCVI) are devoted to D-desosamine biosynthesis or attachment. The remaining two genes (eryBII and eryBVII) appear to function in L-mycarose biosynthesis based on computer analysis and earlier genetic data. Three of these genes, eryBII, eryCIII and eryCII, lie between the eryAIII and eryG genes on one side of the polyketide synthase genes, while the remaining seven, eryBIV, eryBV, eryCVI, eryBVI, eryCIV, eryCV and eryBVII lie upstream of the eryAI gene on the other side of the gene cluster. The deduced products of these genes show similarities to: aldohexose 4-ketoreductases (eryBIV), aldoketo reductases (eryBII), aldohexose 5-epimerases (eryBVII), the dnmT gene of the daunomycin biosynthetic pathway of Streptomyces peucetius (eryBVI), glycosyltransferases (eryBV and eryCIII), the AscC 3,4-dehydratase from the ascarylose biosynthetic pathway of Yersinia pseudotuberculosis (eryCIV), and mammalian N-methyltransferases (eryCVI). The eryCII gene resembles a cytochrome P450, but lacks the conserved cysteine residue responsible for coordination of the haem iron, while the eryCV gene displays no meaningful similarity to other known sequences. From the predicted function of these and other known eryB and eryC genes, pathways for the biosynthesis of L-mycarose and D-desosamine have been deduced.

Nosiheptide Biosynthesis Featuring a Unique Indole Side Ring Formation on the Characteristic Thiopeptide Framework

Nosiheptide (NOS), belonging to the e series of thiopeptide antibiotics that exhibit potent activity against various bacterial pathogens, bears a unique indole side ring system and regiospecific hydroxyl groups on the characteristic macrocyclic core. Here, cloning, sequencing, and characterization of the nos gene cluster from Streptomyces actuosus ATCC 25421 as a model for this series of thiopeptides has unveiled new insights into their biosynthesis. Bioinformatics-based sequence analysis and in vivo investigation into the gene functions show that NOS biosynthesis shares a common strategy with recently characterized b or c series thiopeptides for forming the characteristic macrocyclic core, which features a ribosomally synthesized precursor peptide with conserved posttranslational modifications. However, it apparently proceeds via a different route for tailoring the thiopeptide framework, allowing the final product to exhibit the distinct structural characteristics of e series thiopeptides, such as the indole side ring system. Chemical complementation supports the notion that the S-adenosylmethionine-dependent protein NosL may play a central role in converting tryptophan to the key 3-methylindole moiety by an unusual carbon side chain rearrangement, most likely via a radical-initiated mechanism. Characterization of the indole side ring-opened analogue of NOS from the nosN mutant strain is consistent with the proposed methyltransferase activity of its encoded protein, shedding light into the timing of the individual steps for indole side ring biosynthesis. These results also suggest the feasibility of engineering novel thiopeptides for drug discovery by manipulating the NOS biosynthetic machinery.

Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model

A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodology to clone remaining prototypical genes from producers of structurally determined enediynes in both family types are presented. A phylogenetic analysis of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes/molecular scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.

Identification and Utility of FdmR1 as a Streptomyces Antibiotic Regulatory Protein Activator for Fredericamycin Production in Streptomyces griseus ATCC 49344 and Heterologous Hosts

The fredericamycin (FDM) A biosynthetic gene cluster, cloned previously from Streptomyces griseus ATCC 49344, contains three putative regulatory genes, fdmR, fdmR1, and fdmR2. Their deduced gene products show high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family (FdmR1) or to MarR-like regulators (FdmR and FdmR2). Here we provide experimental data supporting FdmR1 as a SARP-type activator. Inactivation of fdmR1 abolished FDM biosynthesis, and FDM production could be restored to the fdmR1::aac(3)IV mutant by expressing fdmR1 in trans. Reverse transcription-PCR transcriptional analyses revealed that up to 26 of the 28 genes within the fdm gene cluster, with the exception of fdmR and fdmT2, were under the positive control of FdmR1, directly or indirectly. Overexpression of fdmR1 in S. griseus improved the FDM titer 5.6-fold (to about 1.36 g/liter) relative to that of wild-type S. griseus. Cloning of the complete fdm cluster into an integrative plasmid and subsequent expression in heterologous hosts revealed that considerable amounts of FDMs could be produced in Streptomyces albus but not in Streptomyces lividans. However, the S. lividans host could be engineered to produce FDMs via constitutive expression of fdmR1; FDM production in S. lividans could be enhanced further by overexpressing fdmC, encoding a putative ketoreductase, concomitantly with fdmR1. Taken together, these studies demonstrate the viability of engineering FDM biosynthesis and improving FDM titers in both the native producer S. griseus and heterologous hosts, such as S. albus and S. lividans. The approach taken capitalizes on FdmR1, a key activator of the FDM biosynthetic machinery.

Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway for diterpenoid biosynthesis. The PTM and PTN biosynthetic machineries provide a rare glimpse at how secondary metabolic pathway evolution increases natural product structural diversity and support the wisdom of applying combinatorial biosynthesis methods for the generation of novel PTM and/or PTN analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds.

Comparative analysis of the biosynthetic gene clusters and pathways for three structurally related antitumor antibiotics: bleomycin, tallysomycin, and zorbamycin.

The biosynthetic gene clusters for the glycopeptide antitumor antibiotics bleomycin (BLM), tallysomycin (TLM), and zorbamycin (ZBM) have been recently cloned and characterized from Streptomyces verticillus ATCC15003, Streptoalloteichus hindustanus E465-94 ATCC31158, and Streptomyces flavoviridis ATCC21892, respectively. The striking similarities and differences among the biosynthetic gene clusters for the three structurally related glycopeptide antitumor antibiotics prompted us to compare and contrast their respective biosynthetic pathways and to investigate various enzymatic elements. The presence of different numbers of isolated nonribosomal peptide synthetase (NRPS) domains in all three clusters does not result in major structural differences of the respective compounds. The seemingly identical domain organization of the NRPS modules responsible for heterocycle formation, on the other hand, is contrasted by the biosynthesis of two different structural entities, bithiazole and thiazolinyl-thiazole, for BLM/TLM and ZBM, respectively. Variations in sugar biosynthesis apparently dictate the glycosylation patterns distinct for each of the BLM, TLM, and ZBM glycopeptide scaffolds. These observations demonstrate natures ingenuity and flexibility in achieving structural differences and similarities via various mechanisms and will surely inspire combinatorial biosynthesis efforts to expand on natural product structural diversity.

Oxazolomycin Biosynthesis in Streptomyces albus JA3453 Featuring an “Acyltransferase-less” Type I Polyketide Synthase That Incorporates Two Distinct Extender Units

The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species that show diverse and important antibacterial, antitumor, and anti-human immunodeficiency virus activity. Oxazolomycin A is a peptide-polyketide hybrid compound containing a unique spiro-linked β-lactone/γ-lactam, a 5-substituted oxazole ring. The oxazolomycin biosynthetic gene cluster (ozm) was identified from Streptomyces albus JA3453 and localized to 79.5-kb DNA, consisting of 20 open reading frames that encode non-ribosomal peptide synthases, polyketide synthases (PKSs), hybrid non-ribosomal peptide synthase-PKS, trans-acyltransferases (trans-ATs), enzymes for methoxymalonyl-acyl carrier protein (ACP) synthesis, putative resistance genes, and hypothetical regulation genes. In contrast to classical type I polyketide or fatty acid biosynthases, all 10 PKS modules in the gene cluster lack cognate ATs. Instead, discrete ATs OzmM (with tandem domains OzmM-AT1 and OzmM-AT2) and OzmC were equipped to carry out all of the loading functions of both malonyl-CoA and methoxymalonyl-ACP extender units. Strikingly, only OzmM-AT2 is required for OzmM activity for OZM biosynthesis, whereas OzmM-AT1 seemed to be a cryptic AT domain. The above findings, together with previous results using isotope-labeled precursor feeding assays, are assembled for the OZM biosynthesis model to be proposed. The incorporation of both malonyl-CoA (by OzmM-AT2) and methoxymalonyl-ACP (by OzmC) extender units seemed to be unprecedented for this class of trans-AT type I PKSs, which might be fruitfully manipulated to create structurally diverse novel compounds.

Iterative type II polyketide synthases, cyclases and ketoreductases exhibit context-dependent behavior in the biosynthesis of linear and angular decapolyketides

BACKGROUND Iterative type II polyketide synthases (PKSs) produce polyketide chains of variable but defined length from a specific starter unit and a number of extender units. They also specify the initial regiospecific folding and cyclization pattern of nascent polyketides either through the action of a cyclase (CYC) subunit or through the combined action of site-specific ketoreductase (KR) and CYC subunits. Additional CYCs and other modifications may be necessary to produce linear aromatic polyketides. The principles of the assembly of the linear aromatic polyketides, several of which are medically important, are well understood, but it is not clear whether the assembly of the angular aromatic (angucyclic) polyketides follows the same rules. RESULTS We performed an in vivo evaluation of the subunits of the PKS responsible for the production of the angucyclic polyketide jadomycin (jad), in comparison with their counterparts from the daunorubicin (dps) and tetracenomycin (tcm) PKSs which produce linear aromatic polyketides. No matter which minimal PKS was used to produce the initial polyketide chain, the JadD and DpsF CYCs produced the same two polyketides, in the same ratio; neither product was angularly fused. The set of jadABCED PKS plus putative jadl CYC genes behaved similarly. Furthermore, no angular polyketides were isolated when the entire set of jad PKS enzymes and Jadl or the jad minimal PKS, Jadl and the TcmN CYC were present. The DpsE KR was able to reduce decaketides but not octaketides; in contrast, the KRs from the jad PKS (JadE) or the actinorhodin PKS (ActIII) could reduce octaketide chains, giving three distinct products. CONCLUSIONS It appears that the biosynthesis of angucyclic polyketides cannot be simply accomplished by expressing the known PKS subunits from artificial gene cassettes under the control of a non-native promoter. The characteristic structure of the angucycline ring system may arise from a kinked precursor during later cyclization reactions involving additional, but so far unknown, components of the extended decaketide PKS. Our results also suggest that some KRs have a minimal chain length requirement and that CYC enzymes may act aberrantly as first-ring aromatases that are unable to perform all of the sequential cyclization steps. Both of these characteristics may limit the widespread application of CYC or KR enzymes in the synthesis of novel polyketides.

In Vivo Manipulation of the Bleomycin Biosynthetic Gene Cluster in Streptomyces verticillus ATCC15003 Revealing New Insights into Its Biosynthetic Pathway

Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by λRED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.

The tallysomycin biosynthetic gene cluster from streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.