A polylinker approach to reductive loop swaps in modular polyketide synthases.

Multiple versions of the DEBS 1-TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin-producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor "reductive loops," which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1-TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE-catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C-2 and C-3, revealed that the 3-hydroxy configuration (but not the 2-methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination.

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.

Evidence for the role of the monB genes in polyether ring formation during monensin biosynthesis.

Ionophoric polyethers are produced by the exquisitely stereoselective oxidative cyclization of a linear polyketide, probably via a triepoxide intermediate. We report here that deletion of either or both of the monBI and monBII genes from the monensin biosynthetic gene cluster gave strains that produced, in place of monensins A and B, a mixture of C-3-demethylmonensins and a number of minor components, including C-9-epi-monensin A. All the minor components were efficiently converted into monensins by subsequent acid treatment. These data strongly suggest that epoxide ring opening and concomitant polyether ring formation are catalyzed by the MonB enzymes, rather than by the enzyme MonCII as previously thought. Consistent with this, homology modeling shows that the structure of MonB-type enzymes closely resembles the recently determined structure of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis.

Molecular basis of Celmer's rules: stereochemistry of catalysis by isolated ketoreductase domains from modular polyketide synthases.

A system is reported for the recombinant expression of individual ketoreductase (KR) domains from modular polyketide synthases (PKSs) and scrutiny of their intrinsic specificity and stereospecificity toward surrogate diketide substrates. The eryKR(1) and the tylKR(1) domains, derived from the first extension module of the erythromycin PKS and the tylosin PKS, respectively, both catalyzed reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester, with complete stereoselectivity and stereospecificity, even though the substrate is not tethered to an acyl carrier protein or an intact PKS multienzyme. In contrast, and to varying degrees, the isolated enzymes eryKR(2), eryKR(5), and eryKR(6) exercised poorer control over substrate selection and the stereochemical course of ketoreduction. These data, together with modeling of diketide binding to KR(1) and KR(2), demonstrate the fine energetic balance between alternative modes of presentation of ketoacylthioester substrates to KR active sites.

Chain initiation on type I modular polyketide synthases revealed by limited proteolysis and ion-trap mass spectrometry.

Limited proteolysis in combination with liquid chromatography-ion trap mass spectrometry (LC-MS) was used to analyze engineered or natural proteins derived from a type I modular polyketide synthase (PKS), the 6-deoxyerythronolide B synthase (DEBS), and comprising either the first two extension modules linked to the chain-terminating thioesterase (TE) (DEBS1-TE); or the last two extension modules (DEBS3) or the first extension module linked to TE (diketide synthase, DKS). Functional domains were released by controlled proteolysis, and the exact boundaries of released domains were obtained through mass spectrometry and N-terminal sequencing analysis. The acyltransferase-acyl carrier protein required for chain initiation (AT(L)-ACP(L)), was released as a didomain from both DEBS1-TE and DKS, as well as the off-loading TE as a didomain with the adjacent ACP. Mass spectrometry was used successfully to monitor in detail both the release of individual domains, and the patterns of acylation of both intact and digested DKS when either propionyl-CoA or n-butyryl-CoA were used as initiation substrates. In particular, both loading domains and the ketosynthase domain of the first extension module (KS1) were directly observed to be simultaneously primed. The widely available and simple MS methodology used here offers a convenient approach to the proteolytic mapping of PKS multienzymes and to the direct monitoring of enzyme-bound intermediates.

Skipping in a hybrid polyketide synthase. Evidence for ACP-to-ACP chain transfer.

A tetraketide synthase containing a loading module (LM), the extension modules erythromycin module 1, rapamycin module 2, and erythromycin module 2 (LM-Ery1-Rap2-Ery2-TE), when expressed in Saccharopolyspora erythraea strain JC2, produced as previously reported a mixture of tetraketide lactones (minor products) and triketide lactones (major products). Several alternative plausible mechanisms by which this "skipping" phenomenon might occur may be proposed. Site-directed mutagenesis of the ketosynthase (KS) and acylcarrier protein (ACP) domains in the interpolated module has shown that skipping within the hybrid PKS involves passage of the growing polyketide through the interpolated module, by direct ACP-to-ACP transfer of the polyketide chain.

Structural elucidation studies on 14- and 16-membered macrolide aglycones by accurate-mass electrospray sequential mass spectrometry.

Collision induced dissociation sequential mass spectrometry was used to investigate the fragmentation of the heptaketide macrolide aglycones, 6-deoxyerythronolide B (6-dEB), erythronolide B (EB), and acetate-starter EB (Ac-EB). The fragmentations of two previously reported octaketide analogs produced by "stuttering" of the erythromycin polyketide synthase, stuttered-6-dEB and acetate-starter stuttered-6-dEB were also studied. The accuracy with which the mass of each fragment was measured allowed it to be attributed to an unambiguous formula. Most of the experiments were repeated using samples dissolved in deuterated solvents. These data were then used to deduce plausible fragmentation pathways of the five compounds which were shown to have a high degree of similarity. Preliminary fragmentation analysis of a novel octaketide analog was performed and the structure was predicted as stuttered EB. Subsequent scale-up of the bacterial fermentations, followed by isolation and characterization by nuclear magnetic resonance spectroscopy confirmed this prediction. Further fragmentation experiments were then performed on this compound, which provided further evidence of the similarity of the fragmentation schemes. These results demonstrate the utility of collision induced dissociation sequential mass spectrometry analysis in the preliminary screening of bacterial fermentations for new polyketides. These studies were performed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Fragmentation studies on tetronasin by accurate-mass electrospray tandem mass spectrometry.

We report here the first full fragmentation study of tetronasin 1. Fragmentation was carried out by high-resolution ESI-CID-MS(n). The formulae of the fragment ions were determined by accurate mass measurements. It is demonstrated that the fragmentation routes observed derive essentially from a first loss of water via two different mechanisms. One minor route consists of a charge remote neutral loss and the second major route occurs via the formation of a carbocation. The fragments obtained from this carbocation were produced by subsequent complex neutral eliminations and the structures were inferred, in some cases, by carbocation stability.

Novel gas-phase ion-molecule aromatic nucleophilic substitution in beta-carbolines.

We report a novel gas-phase ion-molecule aromatic-nucleophilic substitution reaction between beta-carbolines and water vapour, that accounts for the observation of ions with higher masses than the precursor ion in the MS/MS spectra.

Identification using LC-MSn of co-metabolites in the biosynthesis of the polyketide toxin mycolactone by a clinical isolate of Mycobacterium ulcerans.

LC-MSn analysis of mycolactone toxin from extracts of Mycobacterium ulcerans has shown that minor co-metabolites, including two previously unreported, differ structurally from mycolactone only in a small portion of the polyketide side-chain.

Engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents.

Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea, in which the beta-D-forosamine moiety in glycosidic linkage to the hydroxy group at C17 is replaced by alpha-L-mycarose.

A novel erythromycin, 6-desmethyl erythromycin D, made by substituting an acyltransferase domain of the erythromycin polyketide synthase.

The acyltransferase (AT) domain in module 4 of the erythromycin polyketide synthase (PKS) was substituted with an AT domain from the rapamycin PKS module 2 in order to alter the substrate specificity from methylmalonyl-CoA to malonyl-CoA. The resulting strain produced 6-desmethyl erythromycin D as the predominant product. This AT domain swap completes the library of malonyl-CoA AT swaps on the erythromycin PKS and reinforces PKS engineering as a robust and generic tool.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of dextran and dextrin derivatives.

Application of matrix-assisted laser desorption/ionization (MALDI) to the analysis of dextran and dextrin derivatives, specifically glucose saccharides, by time-of-flight (TOF) mass spectrometry has been reported. MALDI-TOF analysis was carried out on alpha-, beta- and gamma-cyclodextrin, two O-methylated-beta-cyclodextrins of differing degrees of substitution (DS) and dextrans (a linear glucose saccharide), as pure and doped solutions and as mixtures of two or more of these. Doping was carried out with trace amounts of inorganic salts. The purpose of the analysis of the cyclodextrins was to determine whether they would form inclusion complexes with the various cations added, or whether less specific cation addition/exchange was occurring either prior to desorption or in the gas phase.

The Thioesterase of the Erythromycin-Producing Polyketide Synthase: Influence of Acyl Chain Structure on the Mode of Release of Substrate Analogues from the Acyl Enzyme Intermediates.

The production of genetically engineered polyketides depends critically on thioesterase activity for product release. In vitro studies with the thioesterase from the erythromycin polyketide synthase (PKS) have demonstrated that the ability of this enzyme to act as a universal decoupler is limited, but stereochemical variation is readily tolerated. Synthetic analogues with all four stereochemical configurations of the natural substrate's 2-methyl-3-hydroxy substitution pattern (1-4; X=p-nitrophenoxy) were substrates for the enzyme.

The changing patterns of covalent active site occupancy during catalysis on a modular polyketide synthase multienzyme revealed by ion-trap mass spectrometry.

A catalytically competent, homodimeric diketide synthase comprising the first extension module of the erythromycin polyketide synthase was analysed using MS, after limited proteolysis to release functional domains, to determine the pattern of covalent attachment of substrates and intermediates to active sites during catalysis. Using the natural substrates, the acyltransferase and acylcarrier protein of the loading module were found to be heavily loaded with propionyl starter groups, while the ketosynthase was fully propionylated. The acylcarrier protein of the extension module was partly occupied by the product diketide, and the adjacent chain-releasing thioesterase domain was vacant, implying that the rate-limiting step is transfer of the diketide from the acylcarrier protein to the thioesterase domain. The data suggest an attractive model for preventing iterative chain extension by efficient repriming of the ketosynthase domain after condensation. Use of the alternative starter unit valeryl-CoA produced an altered pattern, in which a significant proportion of the extension acylcarrier protein was loaded with methylmalonate, not diketide, consistent with the condensation step having become an additional slow step. Strikingly, when NADPH was omitted, the extension acylcarrier protein contained methylmalonate and none of the expected keto diketide, in contrast to results obtained previously by mixing individual recombinant domains, showing the importance of also studying intact modules. The detailed patterns of loading of the extension acylcarrier protein (of which there are two in the homodimer) also provided the first evidence for simultaneous loading of both acylcarrier proteins and for the coordination of timing between the two active centres for chain extension.