The enzymology of clavam and carbapenem biosynthesis

The enzyme-catalysed reactions involved in formation of the bicyclic clavam and carbapenem nuclei, including beta-amino acid and beta-lactam formation, are discussed and compared with those involved in penicillin and cephalosporin biosynthesis. The common role of unusual oxidation reactions in the biosynthetic pathways and the lack of synthetic reagents available to effect them are highlighted.     

Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups, including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not, however, linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system, which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system, as well efforts to engineer the biosynthesis of novel natural products.     

Identification and characterization of xiamycin A and oxiamycin gene cluster reveals an oxidative cyclization strategy tailoring indolosesquiterpene biosynthesis

Xiamycin A (XMA) and oxiamycin (OXM) are bacterial indolosesquiterpenes featuring rare pentacyclic ring systems and are isolated from a marine-derived Streptomyces sp. SCSIO 02999. The putative biosynthetic gene cluster for XMA/OXM was identified by a partial genome sequencing approach. Eighteen genes were proposed to be involved in XMA/OXM biosynthesis, including five genes for terpene synthesis via a non-mevalonate pathway, eight genes encoding oxidoreductases, and five genes for regulation and resistance. Targeted disruptions of 13 genes within the xia gene cluster were carried out to probe their encoded functions in XMA/OXM biosynthesis. The disruption of xiaK, encoding an aromatic ring hydroxylase, led to a mutant producing indosespene and a minor amount of XMA. Feeding of indosespene to XMA/OXM nonproducing mutants revealed indosespene as a common precursor for XMA/OXM biosynthesis. Most notably, the flavin dependent oxygenase XiaI was biochemically characterized in vitro to convert indosespene to XMA, revealing an unusual oxidative cyclization strategy tailoring indolosesquiterpene biosynthesis.     

The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: characterisation of an aminotransferase involved in the formation of 2-deoxystreptamine

The biosynthetic gene cluster of the 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic neomycin has been cloned for the first time by screening of a cosmid library of Streptomyces fradiae NCIMB 8233. Sequence analysis has identified 21 putative open reading frames (ORFs) in the neomycin gene cluster (neo) with significant protein sequence similarity to gene products involved in the biosynthesis of other DOS-containing aminoglycosides, namely butirosin (btr), gentamycin (gnt), tobramycin (tbm) and kanamycin (kan). Located at the 5'-end of the neo gene cluster is the previously-characterised neomycin phosphotransferase gene (apH). Three genes unique to the neo and btr clusters have been revealed by comparison of the neo cluster to btr, gnt, tbm and kan clusters. This suggests that these three genes may be involved in the transfer of a ribose moiety to the DOS ring during the antibiotic biosynthesis. The product of the neo-6 gene is characterised here as the L-glutamine : 2-deoxy-scyllo-inosose aminotransferase responsible for the first transamination in DOS biosynthesis, which supports the assignment of the gene cluster.     

Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: cluster analysis and assignment of functions

The biosynthetic gene cluster for the angiogenesis inhibitor borrelidin has been cloned from Streptomyces parvulus Tü4055. Sequence analysis indicates that the macrolide ring of borrelidin is formed by a modular polyketide synthase (PKS) (borA1-A6), a result that was confirmed by disruption of borA3. The borrelidin PKS is striking because only seven rather than the nine modules expected for a nonaketide product are encoded by borA1-A6. The starter unit of the PKS has been verified as trans-cyclopentane-1,2-dicarboxylic acid (trans-1,2-CPDA), and the genes involved in its biosynthesis identified. Other genes responsible for biosynthesis of the nitrile moiety, regulation, and self-resistance were also identified.     

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway

BACKGROUND: The polyene macrolide antibiotic nystatin produced by Streptomyces noursei ATCC 11455 is an important antifungal agent. The nystatin molecule contains a polyketide moiety represented by a 38-membered macrolactone ring to which the deoxysugar mycosamine is attached. Molecular cloning and characterization of the genes governing the nystatin biosynthesis is of considerable interest because this information can be used for the generation of new antifungal antibiotics. RESULTS: A DNA region of 123,580 base pairs from the S. noursei ATCC 11455 genome was isolated, sequenced and shown by gene disruption to be involved in nystatin biosynthesis. Analysis of the DNA sequence resulted in identification of six genes encoding a modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport and regulatory proteins. One of the PKS-encoding genes, nysC, was found to encode the largest (11,096 amino acids long) modular PKS described to date. Analysis of the deduced gene products allowed us to propose a model for the nystatin biosynthetic pathway in S. noursei. CONCLUSIONS: A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.     

Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars

The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the A. kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-l-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase, a conserved set of enzymes involved in formation, attachment, and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar d-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-d-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitrosugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.     

A novel synthesis of the carbapen-2-em ring system

A new synthesis of the carbapenem ring system, as found in thienamycin and related natural products, has been developed. The key step involves a highly efficient carbene insertion reaction which produces the bicyclic ring system by forming the N3 bond.     

The biosynthetic gene cluster for a monocyclic beta-lactam antibiotic, nocardicin A

The monocyclic beta-lactam antibiotic nocardicin A is related structurally and biologically to the bicyclic beta-lactams comprised of penicillins/cephalosporins, clavams, and carbapenems. Biosynthetic gene clusters are known for each of the latter, but not for monocyclic beta-lactams. A previously cloned gene encoding an enzyme specific to the biosynthetic pathway was used to isolate the nocardicin A cluster from Nocardia uniformis. Sequence analysis revealed the presence of 14 open reading frames involved in antibiotic production, resistance, and export. Among these are a two-protein nonribosomal peptide synthetase system, p-hydroxyphenylglycine biosynthetic genes, an S-adenosylmethionine-dependent 3-amino-3-carboxypropyl transferase (Nat), and a cytochrome P450. Gene disruption mutants of Nat, as well as an activation domain of the NRPS system, led to loss of nocardicin A formation. Several enzymes involved in antibiotic biosynthesis were heterologously overproduced, and biochemical characterization confirmed their proposed activities.     

Carbapenem biosynthesis: confirmation of stereochemical assignments and the role of CarC in the ring stereoinversion process from L-proline

(5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring carbapenem beta-lactam antibiotics. It co-occurs with two saturated (3S,5S)- and (3S,5R)-carbapenam carboxylic acids. Confusion persists in the literature about the signs of rotation and absolute configurations of these compounds that is resolved in this paper. (3S,5S)-Carbapenam carboxylic acid was prepared from L-pyroglutamic acid to unambiguously establish its absolute configuration as identical to the natural product isolated from Serratia marcescens and from overexpression of the biosynthetic genes carAB in Escherichia coli. L-Proline labeled with deuterium or tritium at the diastereotopic C-5 methylene loci was shown to incorporate one label at the bridgehead of (3S,5S)-carbapenam carboxylic acid, but not into the "inverted" (3S,5R)-carbapenam carboxylic acid or the final carbapenem product. CarC, the third enzyme of the biosynthetic pathway required to synthesize the carbapenem, was demonstrated in cell-free studies to be dependent on alpha-ketoglutarate and ascorbate in keeping with weak sequence identities with other non-heme iron, alpha-ketoglutarate-dependent oxygenases. CarC mediated the stereoinversion of synthetic (3S,5S)-carbapenam carboxylic acid to the (5R)-carbapenem as judged by bioassay. These findings suggest that L-proline is desaturated to pyrroline-5-carboxylic acid prior to uptake into the biosynthetic pathway. The loss of the bridgehead hydrogen from the (3S,5S)-carbapenam during the ring inversion process to form the epimeric (3S,5R)-carbapenam and desaturation to the (5R)-carbapenem are proposed to be coupled by CarC to the reduction of dioxygen to drive the formation of these higher energy products, an unprecedented reaction for this enzyme class.     

Identification of a cluster of genes that directs desferrioxamine biosynthesis in Streptomyces coelicolor M145

Desferrioxamines are a structurally related family of tris-hydroxamate siderophores that form strong hexadentate complexes with ferric iron. Desferrioxamine B has been used clinically for the treatment of iron overload in man. We have unambiguously identified desferrioxamine E as the major desferrioxamine siderophore produced by Streptomyces coelicolor M145 and have identified a cluster of four genes (desA-D) that directs desferrioxamine biosynthesis in this model actinomycete. On the basis of comparative sequence analysis of the proteins encoded by these genes, we propose a plausible pathway for desferrioxamine biosynthesis. The desferrioxamine biosynthetic pathway belongs to a new and rapidly emerging family of pathways for siderophore biosynthesis, widely distributed across diverse species of bacteria, which is biochemically distinct from the better known nonribosomal peptide synthetase (NRPS) pathway used in many organisms for siderophore biosynthesis.     

Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids austinol and dehydroaustinol in Aspergillus nidulans

Meroterpenoids are a class of fungal natural products that are produced from polyketide and terpenoid precursors. An understanding of meroterpenoid biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has previously been found to produce two meroterpenoids, austinol and dehydroaustinol. Using targeted deletions that we created, we have determined that, surprisingly, two separate gene clusters are required for meroterpenoid biosynthesis. One is a cluster of four genes including a polyketide synthase gene, ausA. The second is a cluster of 10 additional genes including a prenyltransferase gene, ausN, located on a separate chromosome. Chemical analysis of mutant extracts enabled us to isolate 3,5-dimethylorsellinic acid and 10 additional meroterpenoids that are either intermediates or shunt products from the biosynthetic pathway. Six of them were identified as novel meroterpenoids in this study. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans meroterpenoids.     

Characterization of yatakemycin gene cluster revealing a radical S-adenosylmethionine dependent methyltransferase and highlighting spirocyclopropane biosynthesis

Yatakemycin (YTM), an antitumor natural product, represents the most potent member of a class of potent anticancer natural products including CC-1065 and duocarmycins. Herein we describe the biosynthetic gene cluster of YTM, which was identified by genome scanning of Streptomyces sp. TP-A0356. This cluster consists of 31 open reading frames (ORFs) and was localized to a 36 kb DNA segment. Moreover, its involvement in YTM biosynthesis was confirmed by cluster deletion, gene replacement, and complementation. Inactivation of ytkT, which encodes a radical S-adenosylmethionine (SAM) protein, created a mutant strain that failed to produce YTM but accumulated a new metabolite, which was structurally elucidated as a precursor that was related to the formation of the cyclopropane ring. More importantly, biochemical characterization of the radical SAM-dependent enzyme YtkT revealed that it is a novel C-methyltransferase and contributes to an advanced intermediate during formation of the cyclopropane ring through a radical mechanism in the YTM biosynthetic pathway. On the basis of in silico analysis, genetic experiments, structure elucidation of the novel intermediate, and biochemical characterization, a biosynthetic pathway for yatakemycin was proposed, which sets the stage to further investigate the novel enzymatic mechanisms and engineer the biosynthetic machinery for the production of novel analogues.     

Biosynthesis of the antitumor chromomycin A3 in Streptomyces griseus: analysis of the gene cluster and rational design of novel chromomycin analogs

The biosynthetic gene cluster of the aureolic acid type antitumor drug chromomycin A3 from S. griseus subsp. griseus has been identified and characterized. It spans 43 kb and contains 36 genes involved in polyketide biosynthesis and modification, deoxysugar biosynthesis and sugar transfer, pathway regulation and resistance. The organization of the cluster clearly differs from that of the closely related mithramycin. Involvement of the cluster in chromomycin A3 biosynthesis was demonstrated by disrupting the cmmWI gene encoding a polyketide reductase involved in side chain reduction. Three novel chromomycin derivatives were obtained, named chromomycin SK, chromomycin SA, and chromomycin SDK, which show antitumor activity and differ with respect to their 3-side chains. A pathway for the biosynthesis of chromomycin A3 and its deoxysugars is proposed.     

Insights into polyether biosynthesis from analysis of the nigericin biosynthetic gene cluster in Streptomyces sp. DSM4137

Nigericin was among the first polyether ionophores to be discovered, but its biosynthesis remains obscure. The biosynthetic gene cluster for nigericin has been serendipitously cloned from Streptomyces sp. DSM4137, and deletion of this gene cluster abolished the production of both nigericin and the closely related metabolite abierixin. Detailed comparison of the nigericin biosynthetic genes with their counterparts in the biosynthetic clusters for other polyketides has prompted a significant revision of the proposed common pathway for polyether biosynthesis. In particular, we present evidence that in nigericin, nanchangmycin, and monensin, an unusual ketosynthase-like protein, KSX, transfers the initially formed linear polyketide chain to a discrete acyl carrier protein, ACPX, for oxidative cyclization. Consistent with this, deletion of either monACPX or monKSX from the monensin gene cluster effectively abolished monensin A biosynthesis.     

Fungal indole alkaloid biosynthesis: genetic and biochemical investigation of the tryptoquialanine pathway in Penicillium aethiopicum

Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline (2) that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6-5-5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P. aethiopicum that is involved in the biosynthesis of tryptoquialanine (1), which is structurally similar to 2. The pathway has been confirmed to go through an intermediate common to the fumiquinazoline pathway, fumiquinazoline F, which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). By systematically inactivating every biosynthetic gene in the cluster, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of the pathway. An unusual oxidative opening of the pyrazinone ring by an FAD-dependent berberine bridge enzyme-like oxidoreductase has been proposed based on genetic knockout studies. Notably, a 2-aminoisobutyric acid (AIB)-utilizing NRPS module has been identified and reconstituted in vitro, along with two putative enzymes of unknown functions that are involved in the synthesis of the unnatural amino acid by genetic analysis. This work provides new genetic and biochemical insights into the biosynthesis of this group of fungal alkaloids, including the tremorgens related to 2.     

Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone. Using targeted deletions that we created, we determined that a cluster of 10 genes including a polyketide synthase gene, mdpG, is required for prenyl xanthone biosynthesis. mdpG was shown to be required for the synthesis of the anthraquinone emodin, monodictyphenone, and related compounds, and our data indicate that emodin and monodictyphenone are precursors of prenyl xanthones. Isolation of intermediate compounds from the deletion strains provided valuable clues as to the biosynthetic pathway, but no genes accounting for the prenylations were located within the cluster. To find the genes responsible for prenylation, we identified and deleted seven putative prenyltransferases in the A. nidulans genome. We found that two prenyltransferase genes, distant from the cluster, were necessary for prenyl xanthone synthesis. These genes belong to the fungal indole prenyltransferase family that had previously been shown to be responsible for the prenylation of amino acid derivatives. In addition, another prenyl xanthone biosynthesis gene is proximal to one of the prenyltransferase genes. Our data, in aggregate, allow us to propose a complete biosynthetic pathway for the A. nidulans xanthones.     

Deciphering deazapurine biosynthesis: pathway for pyrrolopyrimidine nucleosides toyocamycin and sangivamycin

Pyrrolopyrimidine nucleosides analogs, collectively referred to as deazapurines, are an important class of structurally diverse compounds found in a wide variety of biological niches. In this report, a cluster of genes from Streptomyces rimosus (ATCC 14673) involved in production of the deazapurine antibiotics sangivamycin and toyocamycin was identified. The cluster includes toyocamycin nitrile hydratase, an enzyme that catalyzes the conversion of toyocamycin to sangivamycin. In addition to this rare nitrile hydratase, the cluster encodes a GTP cyclohydrolase I, linking the biosynthesis of deazapurines to folate biosynthesis, and a set of purine salvage/biosynthesis genes, which presumably convert the guanine moiety from GTP to the adenine-like deazapurine base found in toyocamycin and sangivamycin. The gene cluster presented here could potentially serve as a model to allow identification of deazapurine biosynthetic pathways in other bacterial species.     

Role of the cytochrome P450 NocL in nocardicin A biosynthesis

The nocardicins are members of a class of monocyclic beta-lactam antibiotics produced by the actinomycete Nocardia uniformis subsp. tsuyamanesis (ATCC 21806). The oxime moiety in nocardicin A is required for full biological activity and is rare among natural products. A putative gene cluster encoding the enzymes of the nocardicin biosynthetic pathway has been identified. Among these, NocL shows similarity to cytochromes P450. We describe its heterologous expression and demonstrate its role in the formation of this unusual structural feature in nocardicin A. This is the first gene from this pathway to be expressed and its function established, and it is the first example of oxime formation from a primary amine in bacteria unambiguously linked to a cytochrome P450.     

The hedamycin locus implicates a novel aromatic PKS priming mechanism

The biosynthetic gene cluster for the pluramycin-type antitumor antibiotic hedamycin has been cloned from Streptomyces griseoruber. Sequence analysis of the 45.6 kb region revealed a variety of unique features such as a fabH homolog (KSIII), an acyltransferase (AT) gene, a set of type I polyketide synthase (PKS) genes, and two putative C-glycosyltransferase genes. As the first report of the cloning of the biosynthetic gene cluster for the pluramycin antibiotics, this work suggests that the biosynthesis of pluramycins utilize an iterative type I PKS system for the generation of a novel starter unit that subsequently primes the type II PKS system. It also implicates the involvement of a second catalytic ketosynthase (KSIII) to regulate this unusual priming step. Gene disruption is used to confirm the importance of both type I and II PKS genes for the biosynthesis of hedamycin.