James Staunton

A chain initiation factor common to both modular and aromatic polyketide synthases

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named ‘chain-length factor’, is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases

The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl‐CoA or malonyl‐CoA onto acyl carrier protein (ACP) have been compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl‐CoA or methylmalonyl‐CoA. These differences are sufficiently clear to allow unambiguous assignment of newly‐sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently‐determined structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, though not close enough to make direct contact with bound substrate.

A hybrid modular polyketide synthase obtained by domain swapping

BACKGROUND Modular polyketide synthases govern the synthesis of a number of medically important antibiotics, and there is therefore great interest in understanding how genetic manipulation may be used to produce hybrid synthases that might synthesize novel polyketides. In particular, we aimed to show whether an individual domain can be replaced by a comparable domain from a different polyketide synthase to form a functional hybrid enzyme. To simplify the analysis, we have used our previously-developed model system DEBS1-TE, consisting of the first two chain-extension modules of the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. RESULTS We show here that replacing the entire acyltransferase (AT) domain from module 1 of DEBS1-TE by the AT domain from module 2 of the rapamycin-producing polyketide synthase leads, as predicted, to the synthesis of two novel triketide lactones in good yield, in place of the two lactones produced by DEBS1-TE. Both of the novel products specifically lack a methyl group at C-4 of the lactone ring. CONCLUSIONS Although the AT domain is a core structural domain of a modular polyketide synthase, it has been swapped to generate a truly hybrid multienzyme with a rationally altered specificity of chain extension. Identical manipulations carried out on known polyketide antibiotics might therefore generate families of potentially useful analogues that are inaccessible by chemical synthesis. These results also encourage the belief that other domains may be similarly swapped.

Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units.

BACKGROUND Modular polyketide synthases (PKSs) function as molecular assembly lines in which polyketide chains are assembled by successive addition of chain extension units. At the end of the assembly line, there is usually a covalently linked type I thioesterase domain (TE I), which is responsible for release of the completed acyl chain from its covalent link to the synthase. Additionally, some PKS clusters contain a second thioesterase gene (TE II) for which there is no established role. Disruption of the TE II genes from several PKS clusters has shown that the TE II plays an important role in maintaining normal levels of antibiotic production. It has been suggested that the TE II fulfils this role by removing aberrant intermediates that might otherwise block the PKS complex. RESULTS We show that recombinant tylosin TE II behaves in vitro as a TE towards a variety of N-acetylcysteamine and p-nitrophenyl esters. The trends of hydrolytic activity determined by the kinetic parameter k(cat)/K(M) for the analogues tested indicates that simple fatty acyl chains are effective substrates. Analogues that modelled aberrant forms of putative tylosin biosynthetic intermediates were hydrolysed at low rates. CONCLUSIONS The behaviour of tylosin TE II in vitro is consistent with its proposed role as an editing enzyme. Aberrant decarboxylation of a malonate-derived moiety attached to an acyl carrier protein (ACP) domain may generate an acetate, propionate or butyrate residue on the ACP thiol. Our results suggest that removal of such groups is a significant role of TE II.

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 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.

Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization.

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway‐specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently‐proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin‐linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.

Reconstruction of phase-sensitive two-dimensional NMR spectra by maximum entropy

Abstract The problems of reconstructing phase-sensitive 2D NMR spectra using conventional methods and the advantages of using our previously proposed implementation of the maximum entropy method (MEM) are analyzed. It is shown that when a phase-sensitive 2D spectrum is reconstructed using MEM, a higher resolution can be obtained for a given measuring time. The method should prove to be most useful, however, when applied to the reconstruction of spectra that cannot after conventional data processing be phased such that all peaks have pure absorption phase. MEM is shown to be capable of producing such spectra. For example, when applied to the reconstruction of a phase-sensitive COSY spectrum the cross peaks and diagonal peaks can be separated into subspectra where all peaks have pure absorption phase. In effect this also removes the diagonal from the COSY spectrum. Finally the method is shown to be capable of reducing t1 noise.

Polyketide synthesis in vitro on a modular polyketide synthase

BACKGROUND The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, contains some 30 active sites distributed between three multienzyme polypeptides (designated DEBS1-3). This complexity has hitherto frustrated mechanistic analysis of such enzymes. We previously produced a mutant strain of S. erythraea in which the chain-terminating cyclase domain (TE) is fused to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension in S. erythraea. This mutant strain produces triketide lactone in vivo. We set out to purify the chimaeric enzyme and to determine its activity in vitro. RESULTS The purified DEBS1-TE multienzyme catalyzes synthesis of triketide lactones in vitro. The synthase specifically uses the (2S)-isomer of methylmalonyl-CoA, as previously proposed, but has a more relaxed specificity for the starter unit than in vivo. CONCLUSIONS We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.

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.

Combinatorial biosynthesis of polyketides and nonribosomal peptides.

The engineering of polyketide biosynthesis has begun to provide robust targeted libraries for screening against pharmaceutically relevant targets. New technologies that offer methodology for the rapid generation of more structurally diverse libraries have now been demonstrated.