Biosynthesis of Phenylnannolone A,a Multidrug Resistance Reversal Agent from the Halotolerant Myxobacterium Nannocystis pusilla B150

The myxobacterial strain Nannocystis pusilla B150 synthesizes the structurally new polyketides phenylnannolone A–C. Apart from some common volatiles and siderophores, these are the first natural products from the genus Nannocystis. Phenylnannolone A shows inhibitory activity towards the ABCB1 gene product P‐glycoprotein and reverses daunorubicin resistance in cancer cells. To decipher the biochemical reactions leading to the formation of phenylnannolone A, the putative biosynthetic genes were identified (phn1, phn2). Phn2 is a polyketide synthase (PKS) with an NRPS‐like loading module, and its domain order is consistent with the phenylnannolone A structure. The functionality and substrate selectivity of the loading module were determined by means of a γ‐¹⁸O₄‐ATP pyrophosphate exchange and a phosphopantetheine ejection assay. A specific activation of cinnamic acid by the AMP‐ligase was detected. Phn1 is a putative butyryl‐CoA carboxylase (BCC), providing ethylmalonyl‐CoA for the formation of the ethyl‐substituted part of phenylnannolone A. Phn1 is the first BCC found in biosynthetic genes for an ethyl‐substituted natural compound. Biosynthesis of phenylnannolone A, putatively encoded by phn1 and phn2, thus utilizes the first biosynthetic machinery in which both a BCC and a PKS are involved.     

Exploring Chemical Diversity of α‐Pyrone Antibiotics: Molecular Basis of Myxopyronin Biosynthesis

Myxopyronins and corallopyronins are structurally related α‐pyrone antibiotics from myxobacteria. They are thought to represent a highly promising compound class for the development of broad‐spectrum antibacterial therapeutic agents, because of their ability to inhibit RNA polymerase through interaction with the “switch region”, a recently identified novel drug target. Here we describe the identification and characterization of the myxopyronin biosynthetic pathway from Myxococcus fulvus Mx f50. A detailed comparison with the recently identified corallopyronin biosynthetic pathway revealed the genetic and biochemical basis, thus explaining the observed structural differences between the two natural product families. Directed mutagenesis procedures for M. fulvus Mx f50 were developed to enable functional studies and pathway modifications. Our work provided new insights into myxopyronin biosynthesis and led to the production of a novel and unexpected myxopyronin derivative.     

Biosynthesis of volatiles by the myxobacterium Myxococcus xanthus

The volatiles emitted from cell cultures of myxobacterium Myxococcus xanthus were collected by use of a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Two new natural products, (S)-9-methyldecan-3-ol ((S)-1) and 9-methyldecan-3-one (2), were identified and synthesized, together with other aliphatic ketones and alcohols, and terpenes. Biosynthesis of the two main components (S)-1 and 2 was examined in feeding experiments carried out with the wild-type strain DK1622 and two mutant strains JD300 and DK11017, which are impaired in the degradation pathway from leucine to isovaleryl-SCoA. Isovaleryl-SCoA is used as a starter, followed by chain elongation with two malonate units. Subsequent use of methyl malonate and decarboxylation leads to (S)-1 and 2. Furthermore, 3,3-dimethylacrylic acid (DMAA) can be used by the mutant strain to form isovaleryl-SCoA, which corroborates recent data on the detection of a novel variety of the mevalonate pathway giving rise to isovaleryl-SCoA from HMGCoA.     

Mutasynthesis-derived myxalamids and origin of the isobutyryl-CoA starter unit of myxalamid B

Myxalamids are potent inhibitors of the eukaryotic electron transport chain produced by different myxobacteria. Here, we describe the identification of the myxalamid biosynthesis gene cluster from Myxococcus xanthus. Additionally, new myxalamids (5-13) have been obtained by mutasynthesis from bkd mutants of M. xanthus and Stigmatella aurantiaca. Moreover, as these bkd mutants are still able to produce myxalamid B (2), the origin of the isobutyryl-CoA (IB-CoA) starter unit required for its biosynthesis has been determined. In a M. xanthus bkd mutant, IB-CoA originates from valine, but in S. aurantiaca this starter unit is derived from alpha-oxidation of iso-odd fatty acids, thereby connecting primary and secondary metabolism.     

Parallel Post‐Polyketide Synthase Modification Mechanism Involved in FD‐891 Biosynthesis in Streptomyces graminofaciens A‐8890

To isolate a key polyketide biosynthetic intermediate for the 16‐membered macrolide FD‐891 ( 1 ), we inactivated two biosynthetic genes coding for post‐polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD‐892 ( 2 ), which lacks the epoxide moiety at C8–C9, the hydroxy group at C10, and the O‐methyl group at O‐25 of FD‐891, was isolated from the gfsF/gfsG double‐knockout mutant. In addition, 25‐O‐methyl‐FD‐892 ( 3 ) and 25‐O‐demethyl‐FD‐891 ( 4 ) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8‐C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1 , respectively. These results suggest that a parallel post‐PKS modification mechanism is involved in FD‐891 biosynthesis.     

A Unique Amino Transfer Mechanism for Constructing the β‐Amino Fatty Acid Starter Unit in the Biosynthesis of the Macrolactam Antibiotic Cremimycin

Cremimycin is a 19‐membered macrolactam glycoside antibiotic based on three distinctive substructures: 1) a β‐amino fatty acid starter moiety, 2) a bicyclic macrolactam ring, and 3) a cymarose unit. To elucidate the biosynthetic machineries responsible for these three structures, the cremimycin biosynthetic gene cluster was identified. The cmi gene cluster consists of 33 open reading frames encoding eight polyketide synthases, six deoxysugar biosynthetic enzymes, and a characteristic group of five β‐amino‐acid‐transfer enzymes. Involvement of the gene cluster in cremimycin production was confirmed by a gene knockout experiment. Further, a feeding experiment demonstrated that 3‐aminononanoate is a direct precursor of cremimycin. Two characteristic enzymes of the cremimycin‐type biosynthesis were functionally characterized in vitro. The results showed that a putative thioesterase homologue, CmiS1, catalyzes the Michael addition of glycine to the β‐position of a non‐2‐enoic acid thioester, followed by hydrolysis of the thioester to give N‐carboxymethyl‐3‐aminononanoate. Subsequently, the resultant amino acid was oxidized by a putative FAD‐dependent glycine oxidase homologue, CmiS2, to produce 3‐aminononanoate and glyoxylate. This represents a unique amino transfer mechanism for β‐amino acid biosynthesis.     

Biosynthesis of the Peptide Antibiotic Feglymycin by a Linear Nonribosomal Peptide Synthetase Mechanism

Feglymycin, a peptide antibiotic produced by Streptomyces sp. DSM 11171, consists mostly of nonproteinogenic phenylglycine‐type amino acids. It possesses antibacterial activity against methicillin‐resistant Staphylococcus aureus strains and antiviral activity against HIV. Inhibition of the early steps of bacterial peptidoglycan synthesis indicated a mode of action different from those of other peptide antibiotics. Here we describe the identification and assignment of the feglymycin (feg) biosynthesis gene cluster, which codes for a 13‐module nonribosomal peptide synthetase (NRPS) system. Inactivation of an NRPS gene and supplementation of a hydroxymandelate oxidase mutant with the amino acid l ‐Hpg proved the identity of the feg cluster. Feeding of Hpg‐related unnatural amino acids was not successful. This characterization of the feg cluster is an important step to understanding the biosynthesis of this potent antibacterial peptide.     

Analysis of the tetronomycin gene cluster: insights into the biosynthesis of a polyether tetronate antibiotic

The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open-reading frames. Based on sequence comparison to related biosynthetic gene clusters, the majority of these can be assigned a plausible role in TMN biosynthesis. The identity of the cluster, and the requirement for a number of individual genes, especially those hypothesised to contribute a glycerate unit to the formation of the tetronate ring, were confirmed by specific gene disruption. However, two large genes that are predicted to encode together a multifunctional PKS of a highly unusual type seem not to be involved in this pathway since deletion of one of them did not alter tetronomycin production. Unlike previously characterised polyether PKS systems, oxidative cyclisation appears to take place on the modular PKS rather than after transfer to a separate carrier protein, while tetronate ring formation and concomitant chain release share common mechanistic features with spirotetronate biosynthesis.     

Biosynthesis and identification of volatiles released by the myxobacterium Stigmatella aurantiaca

The volatiles released by agar plate cultures of two strains of the myxobacterium Stigmatella aurantiaca (strains Sg a15 and DW4/3-1) were collected in a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Large numbers of substances from different compound classes (ketones, esters, lactones, terpenes, and sulfur and nitrogen compounds) were identified; several of them are reported from natural sources for the first time. The volatiles 2-methyltridecan-4-one (17), its isomer 3-methyltridecan-4-one (20), and the higher homologue 2-methyltetradecan-4-one (18) were identified in the extracts of both strains and were synthesized. In addition, strain Sg a15 produced 2,12-dimethyltridecan-4-one (19), 2-methyltridec-2-en-4-one (23), and a series of phenyl ketones, among them 1-phenyldecan-1-one (14) and 9-methyl-1-phenyldecan-1-one (16), whereas strain DW4/3-1 emitted traces of 10-methylundecan-2-one (21). The biosynthesis of 14 and 16 was examined in feeding experiments with deuterated precursors carried out on agar plate cultures. The leucine-derived starter unit isovalerate was shown to be incorporated into 16, as was phenylalanine-derived benzoic acid into both 14 and 16. The results point to formation both of the phenyl ketones and of the structurally related aliphatic ketones through an unusual head-to-head coupling between a starter unit such as benzoyl-CoA and a fatty acyl-CoA, followed by decarboxylation.     

Evidence that a novel thioesterase is responsible for polyketide chain release during biosynthesis of the polyether ionophore monensin

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.     

In vivo mutational analysis of the mupirocin gene cluster reveals labile points in the biosynthetic pathway: the "leaky hosepipe" mechanism

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.     

Pacidamycin Biosynthesis: Identification and Heterologous Expression of the First Uridyl Peptide Antibiotic Gene Cluster

The pacidamycins are antimicrobial nucleoside antibiotics produced by Streptomyces coeruleorubidus that inhibit translocase I, an essential bacterial enzyme yet to be clinically targeted. The novel pacidamycin scaffold is composed of a pseudopeptide backbone linked by a unique exocyclic enamide to an atypical 3′‐deoxyuridine nucleoside. In addition, the peptidyl chain undergoes a double inversion caused by the incorporation of a diamino acid residue and a rare internal ureido moiety. The pacidamycin gene cluster was identified and sequenced, thereby providing the first example of a biosynthetic cluster for a member of the uridyl peptide family of antibiotics. Analysis of the 22 ORFs provided an insight into the biosynthesis of the unique structural features of the pacidamycins. Heterologous expression in Streptomyces lividans resulted in the production of pacidamycin D and the newly identified pacidamycin S, thus confirming the identity of the pacidamycin biosynthetic gene cluster. Identification of this cluster will enable the generation of new uridyl peptide antibiotics through combinatorial biosynthesis. The concise cluster will provide a useful model system through which to gain a fundamental understanding of the way in which nonribosomal peptide synthetases interact.     

Functional analysis of the aureothin iterative type I polyketide synthase

The modular-type polyketide synthase (PKS) that is involved in aureothin (aur) biosynthesis represents one of the first examples in which a single PKS module (AurA) is used in an iterative fashion. Here we report on the heterologous expression of an engineered AurAB fusion protein that unequivocally proves the iterative nature of AurA. In addition, point mutations reveal that aur PKS module 4 participates in polyketide biosynthesis despite its aberrant acyltransferase domain.     

Understanding the Glycopeptide Antibiotic Crosslinking Cascade: In Vitro Approaches Reveal the Details of a Complex Biosynthesis Pathway

The glycopeptide antibiotics (GPAs) are a fascinating example of complex natural product biosynthesis, with the nonribosomal synthesis of the peptide core coupled to a cytochrome P450-mediated cyclisation cascade that crosslinks aromatic side chains within this peptide. Given that the challenges associated with the synthesis of GPAs stems from their highly crosslinked structure, there is great interest in understanding how biosynthesis accomplishes this challenging set of transformations. In this regard, the use of in vitro experiments has delivered important insights into this process, including the identification of the unique role of the X-domain as a platform for P450 recruitment. In this minireview, we present an analysis of the results of in vitro studies into the GPA cyclisation cascade that have demonstrated both the tolerances and limitations of this process for modified substrates, and in turn developed rules for the future reengineering of this important antibiotic class.     

A biosynthetic pathway to isovaleryl-CoA in myxobacteria: the involvement of the mevalonate pathway

A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched-chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched-chain alpha-keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched-chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl-CoA is derived from 3,3-dimethylacrylyl-CoA (DMA-CoA). Here we show that DMA-CoA is an isomerization product of 3-methylbut-3-enoyl-CoA (3MB-CoA). This compound is directly derived from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate. Incubation of cell-free extracts of a bkd mutant with HMG-CoA gave product(s) with the molecular mass of 3MB-CoA or DMA-CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L-leucine as precursor.