Quantitative atom probe analysis of nanostructure containing clusters and precipitates with multiple length scales.

A model Al-3 Cu-(0.05 Sn) (wt%) alloy containing a bimodal distribution of relatively shear-resistant θ' precipitates and shearable GP zones is considered in this study. It has recently been shown that the addition of the GP zones to such microstructures can lead to significant increases in strength without a decrease in the uniform elongation. In this study, atom probe tomography (APT) has been used to quantitatively characterise the evolution of the GP zones and the solute distribution in the bimodal microstructure as a function of applied plastic strain. Recent nuclear magnetic resonance (NMR) analysis has clearly shown strain-induced dissolution of the GP zones, which is supported by the current APT data with additional spatial information. There is significant repartitioning of Cu from the GP zones into the solid solution during deformation. A new approach for cluster finding in APT data has been used to quantitatively characterise the evolution of the sizes and shapes of the Cu containing features in the solid solution solute as a function of applied strain.

Engineered biosynthesis of geldanamycin analogs for Hsp90 inhibition.

Geldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the "inert" groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.

Approaches to stabilization of inter-domain recombination in polyketide synthase gene expression plasmids.

Regions of extremely high sequence identity are recurrent in modular polyketide synthase (PKS) genes. Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites. We present two different solutions for circumventing intra-plasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor. In one example, a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence. The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions. Both methods resulted in PKS complexes capable of 6-deoxyerythronolide B analogue biosynthesis in S. coelicolor CH999, permitting reproducible scale-up to at least 5-l stirred-tank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs.

A comparison of EDS microanalysis in FIB-prepared and electropolished TEM thin foils.

This paper reports the results of a fine-probe EDS microanalytical study of cellular precipitation in a Cu-Ti binary alloy. Compositional profiles across the solute depleted Cu-rich FCC lamellae and the Cu4Ti lamellae within isothermally formed cellular colonies were measured in a FEG-TEM from thin-foil specimens prepared by conventional electropolishing and by a technique using a Ga+ focused ion-beam (FIB). The Cliff-Lorimer ratio method, with an absorption correction, was employed to quantify the compositions. Two FIB samples were prepared with different orientations of the lamellae with respect to the ion-milling direction. The compositional profiles across the Cu-rich FCC lamellae and the Cu4Ti compound lamellae in both the FIB-prepared samples and the electropolished sample were, within experimental error, numerically equivalent. The composition of the Cu4Ti compound phase lamellae was very close to the ideal stoichiometric composition of 20 at % Ti. It is concluded that for this system, and for the specimen preparation procedures used in this study, the Ga+ ion-milling process has had no detectable effect on the chemistry changes across the interlamellar interface at the scale studied. These results indicate that the possible sources of chemical artifacts which include redeposition, preferential sputtering and ion-induced atomic migration can be minimized if several precautions are taken during milling in the FIB. Consistent with previous investigators, it was also found that the ion-milling process does introduce significant structural artifacts (e.g., dislocations) into the softer FCC Cu-rich phase compared with a specimen produced by conventional electropolishing.

Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.

Production of aromatic minimal polyketides by the daunorubicin polyketide synthase genes reveals the incompatibility of the heterologous DpsY and JadI cyclases.

Our investigations into whether the biosynthesis of a linearly fused ring system of an aromatic polyketide (jadomycin) could be modified to produce an angularly fused system (daunorubicin) and vice versa showed that introduction of the respective cyclases did not have the desired effect. Genes from the daunorubicin pathway produced a novel 21-carbon polyketide.

Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans.

BACKGROUND: The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis. RESULTS: A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose. CONCLUSIONS: The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.

Conversion of cyclic nonaketides to lovastatin and compactin by a lovC deficient mutant of Aspergillus terreus.

Investigation of the post-PKS biosynthetic steps to the cholesterol-lowering agent lovastatin (1) using an Aspergillus terreus strain with a disrupted lovC gene, which is essential for formation of 4a,5-dihydromonacolin L (3), shows that 7 and 3 are precursors to 1, and demonstrates that lovastatin diketide synthase (lovF protein) does not require lovC.

Combinatorial biosynthesis in microorganisms as a route to new antimicrobial, antitumor and neuroregenerative drugs.

Combinatorial biosynthesis utilizes the genes of biosynthetic pathways that produce microbial products to create novel chemical structures. The engineering of mondular polyketide synthase (PKS) genes has been the major focus of this effort and has led to the production of analogs of macrolide antibiotics like the erythromycins and their derived ketolides, and of the immunosuppressive macrolide FK-520 (Fujisawa Pharmaceutical Co Ltd). Approaches to making analogs of the promising antitumor compounds known as epothilones are also being explored. Lead compounds for further study have resulted and routes to analogs of other pharmacologically important compounds have been established. To facilitate this work, many new tools for manipulating and studying the multifunctional PKSs have been developed including the development of Escherichia coli as a PKS expression last. These developments have resulted in faster ways of engineering PKS to produce new compounds for the development of chemotherapeutic agents from natural products.

Triple hydroxylation of tetracenomycin A2 to tetracenomycin C involving two molecules of O(2) and one molecule of H(2)O.

The TcmG or ElmG oxygenase-catalyzed triple hydroxylation of tetracenomycin (Tcm) A2 to Tcm C proceeds via a novel monooxygenase-dioxygenase mechanism, deriving the 4- and 12a-OH groups of Tcm C from two molecules of O(2) and the 4a-OH group of Tcm C from a molecule of H(2)O. These results suggest a mechanistic analogy among TcmG, ElmG, and the bacterial and fungal hydroquinone epoxidizing dioxygenases, as well as the mammalian vitamin K-dependent gamma-glutamyl carboxylase.

Thioesterases and the premature termination of polyketide chain elongation in rifamycin B biosynthesis by Amycolatopsis mediterranei S699.

The role of two thioesterase genes in the premature release of polyketide synthase intermediates during rifamycin biosynthesis in the Amycolatopsis mediterranei S699 strain was investigated. Creation of an in-frame deletion in the rifR gene led to a 30 approximately 60% decrease in the production of both rifamycin B by the S699 strain or a series of tetra- to decaketide shunt products of polyketide chain assembly by the rifF strain. Since a similar percentage decrease was seen in both genetic backgrounds, we conclude that the RifR thioesterase 2 is not involved in premature release of the carbon chain assembly intermediates. Similarly, fusion of the Saccharopolyspora erythraea DEBS3 thioesterase I domain to the C-terminus of the RifE PKS subunit did not result in a noticeable increase in the amount of the undecaketide intermediate formed nor in the amounts of the tetra- to decaketide shunt products. Hence, premature release of the carbon chain assembly intermediates is an unusual property of the Rif PKS itself.

An acyl-coenzyme A carboxylase encoding gene associated with jadomycin biosynthesis in Streptomyces venezuelae ISP5230.

Analysis of a region of chromosomal DNA lying between jadR1 and jadI in the gene cluster for jadomycin biosynthesis in Streptomyces venezuelae ISP5230 detected an ORF encoding 584 amino acids similar in sequence to the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) components of acyl-coenzyme A carboxylases. Multiple sequence alignments of the deduced Jad protein with acyl-coenzyme A carboxylases from various sources located the BC and BCCP components in the N- and C-terminal regions, respectively, of the deduced polypeptides. The organization and amino acid sequence of the deduced polypeptide most closely resembled those in other Gram-positive bacteria broadly classified as actinomycetes. Disrupting the gene, designated jadJ, severely reduced but did not eliminate jadomycin production. The disruption had no effect on growth or morphology of the organism, implying that the product of jadJ is not essential for fatty acid biosynthesis. It is concluded that jadJ supplies malonyl-coenzyme A for biosynthesis of the polyketide intermediate that is eventually processed to form the antibiotic jadomycin B.

Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases.

Lovastatin biosynthesis in Aspergillus terreus involves two unusual type I multifunctional polyketide syntheses (PKSs). Lovastatin nonaketide synthase (LNKS), the product of the lovB gene, is an iterative PKS that interacts with LovC, a putative enoyl reductase, to catalyze the 35 separate reactions in the biosynthesis of dihydromonacolin L, a lovastatin precursor. LNKS also displays Diels-Alderase activity in vitro. Lovastatin diketide synthase (LDKS) made by lovF, in contrast, acts non-iteratively like the bacterial modular PKSs to make (2R)-2-methylbutyric acid. Then, like LNKS, LDKS interacts closely with another protein, the LovD transesterase enzyme that catalyzes attachment of the 2-methylbutyric acid to monacolin J in the final step of the lovastatin pathway. Key features of the genes for these four enzymes and others, plus the regulatory and self-resistance factors involved in lovastatin production, are also described.

A multiplasmid approach to preparing large libraries of polyketides.

A three-plasmid system for heterologous expression of 6-deoxyerythronolide B synthase (DEBS) has been developed to facilitate combinatorial biosynthesis of polyketides made by type I modular polyketide synthases (PKSs). The eryA PKS genes encoding the three DEBS subunits were individually cloned into three compatible Streptomyces vectors carrying mutually selectable antibiotic resistance markers. A strain of Streptomyces lividans transformed with all three plasmids produced 6-deoxyerythronolide B at a level similar to that of a strain transformed with a single plasmid containing all three genes. The utility of this system in combinatorial biosynthesis was demonstrated through production of a library of modified polyketide macrolactones by using versions of each plasmid constructed to contain defined mutations. Combinations of these vector sets were introduced into S. lividans, resulting in strains producing a wide range of 6-deoxyerythronolide B analogs. This method can be extended to any modular PKS and has the potential to produce thousands of novel natural products, including ones derived from further modification of the PKS products by tailoring enzymes.

Genetic engineering of doxorubicin production in Streptomyces peucetius: a review.

The genetics and biochemistry of daunorubicin and doxorubicin production by Streptomyces peucetius is reviewed, with a focus on how such information can be used for the genetic engineering of strains having improved titers of these two antitumor antibiotics.

Purification and properties of the Streptomyces peucetius DpsC beta-ketoacyl:acyl carrier protein synthase III that specifies the propionate-starter unit for type II polyketide biosynthesis.

Biosynthesis of the polyketide-derived carbon skeleton of daunorubicin (DNR) begins with propionate rather than acetate, which is the starter unit for most other aromatic polyketides. The dpsCgene has been implicated in specifying the unique propionate-starter unit, and it encodes a protein that is very similar to the Escherichia coli beta-ketoacyl:acyl carrier protein (ACP) synthase III (FabH or KS III) enzyme of fatty acid biosynthesis. Purified DpsC was found to use propionyl-coenzyme A as substrate and to be acylated by propionate at the Ser-118 residue. DpsC exhibits KS III activity in catalyzing the condensation of propionyl-CoA and malonyl-ACP, and also functions as an acyltransferase in the transfer of propionate to an ACP. The DpsC enzyme has a high-substrate specificity, utilizing only propionyl-CoA, and not malonyl-CoA, 2-methylmalonyl-CoA or acetyl-CoA, as the starter unit of DNR biosynthesis.

The Streptomyces peucetius dpsC gene determines the choice of starter unit in biosynthesis of the daunorubicin polyketide.

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of Streptomyces peucetius, directly downstream of the genes encoding the beta-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC and dpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.

Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis.

Polyketides, the ubiquitous products of secondary metabolism in microorganisms, are made by a process resembling fatty acid biosynthesis that allows the suppression of reduction or dehydration reactions at specific biosynthetic steps, giving rise to a wide range of often medically useful products. The lovastatin biosynthesis cluster contains two type I polyketide synthase genes. Synthesis of the main nonaketide-derived skeleton was found to require the previously known iterative lovastatin nonaketide synthase (LNKS), plus at least one additional protein (LovC) that interacts with LNKS and is necessary for the correct processing of the growing polyketide chain and production of dihydromonacolin L. The noniterative lovastatin diketide synthase (LDKS) enzyme specifies formation of 2-methylbutyrate and interacts closely with an additional transesterase (LovD) responsible for assembling lovastatin from this polyketide and monacolin J.

DnrD cyclase involved in the biosynthesis of doxorubicin: purification and characterization of the recombinant enzyme.

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester (AAME) to aklaviketone (AK), an intermediate in the biosynthetic pathway to daunorubicin (DNR) and doxorubicin. To investigate the role of DnrD in this transformation, its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyze the conversion of AAME to AK presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin (TCM) C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the AAME precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters that are important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.