Formation and Loading of a (2S)-2-Ethylmalonamyl Starter Unit in the Assembly Line of Polyketide-Nonribosomal Peptide Hybrid Sanglifehrin A

Sanglifehrin A (SFA) is a spirolactam-conjugated, 22-membered macrolide with remarkable immunosuppressive and antiviral activities. This macrolide is a result of a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line that utilizes (2S)-2-ethylmalonamyl as a starter unit. Here, we report that the formation and loading of this starter unit in the SFA assembly line involve two unusual enzymatic reactions that occur on a discrete acyl carrier protein (ACP), SfaO. An amide synthetase, SfaP, catalyzes the amidation of (2S)-2-ethylmalonyl in a SfaO-dependent manner. Then, a β-ketoacyl-ACP synthase III-like protein, SfaN, transfers resultant (2S)-2-ethylmalonamyl from SfaO onto the loading ACP domain of the hybrid PKS-NRPS assembly line to prime SFA biosynthesis. Both SfaP and SfaN display promiscuous activities. This study furthers the appreciation of assembly line chemistry, as a new paradigm for unusual building block formation and incorporation is provided.     

A Unique Pathway for the 3-Aminobutyrate Starter Unit from l-Glutamate through β-Glutamate during Biosynthesis of the 24-Membered Macrolactam Antibiotic, Incednine

Incednine is a 24-membered macrolactam antibiotic produced by Streptomyces sp. ML694-90F3. A previous study demonstrated that its unique nitrogen-containing starter unit was derived from l-glutamate. To elucidate the missing link between l-glutamate and the starter unit, deuterium labeled amino acid feeding experiments were conducted. These experiments revealed that 3-[3-(2)H]aminobutyrate and β-[2,2,4,4-(2)H(4)]glutamate were incorporated into the starter moiety. The results indicate that a novel decarboxylation of β-glutamate to give 3-aminobutyrate is involved in incednine biosynthesis.     

Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase.

BACKGROUND: A single modular polyketide synthase (PKS) gene cluster is responsible for production of both the 14-membered macrolide antibiotic picromycin and the 12-membered macrolide antibiotic methymycin in Streptomyces venezuelae. Building on the success of the heterologous expression system engineered using the erythromycin PKS, we have constructed an analogous system for the picromycin/methymycin PKS. Through heterologous expression and construction of a hybrid PKS, we have examined the contributions that the PKS, its internal thioesterase domain (pikTE) and the Pik TEII thioesterase domain make in termination and cyclization of the two polyketide intermediates. RESULTS: The picromycin/methymycin PKS genes were functionally expressed in the heterologous host Streptomyces lividans, resulting in production of both narbonolide and 10-deoxymethynolide (the precursors of picromycin and methymycin, respectively). Co-expression with the Pik TEII thioesterase led to increased production levels, but did not change the ratio of the two compounds produced, leaving the function of this protein largely unknown. Fusion of the PKS thioesterase domain (pikTE) to 6-deoxyerythronolide B synthase (DEBS) resulted in formation of only 14-membered macrolactones. CONCLUSIONS: These experiments demonstrate that the PKS alone is capable of catalyzing the synthesis of both 14- and 12-membered macrolactones and favor a model by which different macrolactone rings result from a combination of the arrangement between the module 5 and module 6 subunits in the picromycin PKS complex and the selectivity of the pikTE domain.     

Functional expression of genes involved in the biosynthesis of the novel polyketide chain extension unit, methoxymalonyl-acyl carrier protein, and engineered biosynthesis of 2-desmethyl-2-methoxy-6-deoxyerythronolide B

A subcluster of five genes, asm13-17, from the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum was coexpressed in Streptomyces lividans with the genes encoding the 6-deoxyerythronolide B (6-DEB) synthase from Saccharopolyspora erythraea, in which the methylmalonate-specifying AT6 domain had been replaced by the methoxymalonate-specifying AT8 domain from the FK520 cluster of Streptomyces hygroscopicus. The engineered strain produced the predicted product, 2-desmethyl-2-methoxy-DEB, instead of 6-DEB and 2-desmethyl-6-DEB, which were formed in the absence of the asm13-17 cassette, indicating that asm13-17 are sufficient for synthesis of this unusual chain extension unit. Deletion of asm17, encoding a methyltransferase, from the cassette gave 6-DEB instead of its hydroxy analogue, indicating that methylation of the extender unit is required for its incorporation.     

Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase

BACKGROUND: Modular polyketide synthases catalyse the biosynthesis of medically useful natural products by stepwise chain assembly, with each module of enzyme activities catalysing a separate cycle of polyketide chain extension. Domain swapping between polyketide synthases leads to hybrid multienzymes that yield novel polyketides in a more or less predictable way. No experiments have so far been reported which attempt to enlarge a polyketide synthase by interpolating additional modules. RESULTS: We describe here the construction of tetraketide synthases in which an entire extension module from the rapamycin-producing polyketide synthase is covalently spliced between the first two extension modules of the erythromycin-producing polyketide synthase (DEBS). The extended polyketide synthases thus formed are found to catalyse the synthesis of specific tetraketide products containing an appropriate extra ketide unit. Co-expression in Saccharopolyspora erythraea of the extended DEBS multienzyme with multienzymes DEBS 2 and DEBS 3 leads to the formation, as expected, of novel octaketide macrolactones. In each case the predicted products are accompanied by significant amounts of unextended products, corresponding to those of the unaltered DEBS PKS. We refer to this newly observed phenomenon as 'skipping'. CONCLUSIONS: The strategy exemplified here shows far-reaching possibilities for combinatorial engineering of polyketide natural products, as well as revealing the ability of modular polyketide synthases to 'skip' extension modules. The results also provide additional insight into the three-dimensional arrangement of modules within these giant synthases.     

Precursor-directed biosynthesis of 16-membered macrolides by the erythromycin polyketide synthase.

Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1 degrees ), a mutant form of 6-deoxyerythronolide B synthase that is blocked in the formation of 6-deoxyerythronolide B (1, 6-dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain elongation step of 6-dEB biosynthesis. Administration of (2E,4S,5R)-2,4-dimethyl-5-hydroxy-2-heptenoic acid, N-acetylcysteamine thioester (6) an unsaturated triketide analogue of the natural triketide chain elongation intermediate to cultures of S. coelicolor CH999/pJRJ2 results in formation of a 16-membered macrolactone, which is isolated in the hemiketal form 33. The formation of the octaketide 33 indicates that the triketide substrate has been processed by DEBS module 2 as if it were a diketide analogue. The substrate specificity of this novel reaction has been explored by the incubation of three additional analogues of the unsaturated triketide 6, compounds 18, 31, and 32, with S. coelicolor CH999/pJRJ2, resulting in the formation of the corresponding macrolactones 34, 35, and 36. By contrast, the unsaturated triketide 10, lacking a methyl group at C-2, did not give rise to any detectable macrolactone product when incubated with S. coelicolor CH999/pJRJ2.     

Generation of multiple bioactive macrolides by hybrid modular polyketide synthases in Streptomyces venezuelae

The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase, leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products.     

Macrolactones built from the bis-3,4(indol-1-yl)maleimide scaffold

15, 16, and 17-Membered lactones based on the bis-3,4(indol-1-yl)maleimide framework were obtained using intramolecular esterification reaction starting from 3-(1-ω-carboxyalkyl-2,3-dihydroindol-1-yl)-4-(1-ω-hydroxyalkyl-2,3-dihydroindol-1-yl)-maleimides. 3,4-Dibromo-maleimide, ω-(2,3-dihydroindol-3-yl)alkanoic acids, and ω-(2,3-dihydroindol-3-yl)alkanoles were used as starting compounds. Substitution of Br for the substituted indolines followed by the intramolecular cyclization of O-silylated hydroxyl acids derivatives led to macrolactones that incorporated 4-(dihydroindol-1-yl)-3-(indol-1-yl)maleimide moieties. Indoline nuclei in these compounds were dehydrogenated by DDQ in refluxing toluene to give 15, 16 or 17-membered lactones 3-[(ω-3-carboxyalkylindol-1-yl)-4-(ω-hydroxyalkylindol-1-yl)maleimides. Quantum chemical calculations showed that the formation of macrolactones of smaller size (13-membered) corresponds to the higher Gibbs energy ΔG^# and correlates with the absence of the target reaction product.     

Iterative chain elongation by a pikromycin monomodular polyketide synthase

The unique ability of the pikromycin polyketide synthase (Pik PKS) to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes of polyketide synthesis, specifically, the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII and PikAIV and demonstrated the ability of these proteins to generate triketide lactone products using (14)C-methylmalonyl-CoA as the sole substrate. Monomodular PikAIII generates TKL (1) when reacted alone, and synthesizes TKL (2) upon reaction in combination with PikAIV. Product formation remains dependent on the enzymatic decarboxylation of methylmalonyl-CoA and transfer of the acyl chain within the enzyme rather than acylation by propionyl-CoA from spontaneous decarboxylation. We propose that synthesis of TKL (1) by PikAIII involves iterative assembly of the triketide chain within a PikAIII homodimer analogous to the nonmodular type I PKS systems.     

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.     

Biosynthesis of structurally unique fungal metabolite GKK1032A(2): indication of novel carbocyclic formation mechanism in polyketide biosynthesis

The biosynthesis of the antitumor agent GKK1032A(2) (1) has been investigated by administration of isotopically labeled ((13)C and (2)H) precursors to Penicillium sp. GKK1032. These studies showed that the backbone of 1 is constructed from l-tyrosine and a nonaketide chain flanked with five methyl groups probably by a polyketide synthase and a nonribosomal peptide synthetase hybrid. On the basis of the oxidation level of the starter unit and unusual 13-membered macroether formation between the tyrosine hydroxy group and the polyketide chain, novel cyclization mechanisms on the formation of a tricarbocyclic system and a macroether have been proposed. Involvement of a similar type of cyclization in the biosynthesis of structurally related metabolites is discussed.     

Amide N-glycosylation by Asm25, an N-glycosyltransferase of ansamitocins

Ansamitocins are potent antitumor maytansinoids produced by Actinosynnema pretiosum. Their biosynthesis involves the initial assembly of a macrolactam polyketide, followed by a series of postpolyketide synthase (PKS) modifications. Three ansamitocin glycosides were isolated from A. pretiosum and fully characterized structurally as novel ansamitocin derivatives, carrying a beta-D-glucosyl group attached to the macrolactam amide nitrogen in place of the N-methyl group. By gene inactivation and complementation, asm25 was identified as the N-glycosyltransferase gene responsible for the macrolactam amide N-glycosylation of ansamitocins. Soluble, enzymatically active Asm25 protein was obtained from asm25-expressing E. coli by solubilization from inclusion bodies. Its optimal reaction conditions, including temperature, pH, metal ion requirement, and Km/Kcat, were determined. Asm25 also showed broad substrate specificity toward other ansamycins and synthetic indolin-2-ones. To the best of our knowledge, this represents the first in vitro characterization of a purified antibiotic N-glycosyltransferase.     

Synthesis and Hydrogen Bonding Capabilities of Biphenyl-Based Amino Acids Designed To Nucleate beta-Sheet Structure

The syntheses of 3'-(aminoethyl)-2-biphenylpropionic acid (1) and 2-amino-3'-biphenylcarboxylic acid (2) are described. These residues were designed to nucleate beta-sheet structure in aqueous solution when incorporated into small, amphiphilic peptides in place of the backbone of the i + 1 and i + 2 residues of the beta-turn. N-Benzyl-3'-(2-(benzylamido)ethyl)-2-biphenylpropamide (3) and N-benzyl-(2-benzylamido)-3'-biphenylamide (4) were synthesized and studied as model compounds to investigate the hydrogen-bonding capabilities of residues 1 and 2, respectively. The X-ray crystal structure of 3 indicates that a 13-membered intramolecular hydrogen-bonded ring is formed, while the remaining amide proton and carbonyl are involved in intermolecular hydrogen bonding. Infrared and variable-temperature NMR experiments indicate that, in solution (CH(2)Cl(2)), 3 exists as an equilibrium mixture of the 13- and the 15-membered intramolecularly hydrogen-bonded conformers with the 15-membered ring conformer being favored. Amide 4 was shown to exist in solution (CH(2)Cl(2)) as an equilibrium mixture of the 11-membered intramolecular hydrogen-bonded ring and a nonbonded conformation. No contribution from the 9-membered hydrogen-bonded ring conformation was observed. The X-ray crystal structure of 4 indicated the absence of intramolecular hydrogen bonding in the solid state.     

Total synthesis of the ristocetin aglycon

The first total synthesis of the ristocetin aglycon is described employing a modular and highly convergent strategy. An effective 12-step (12% overall) synthesis of the ABCD ring system 3 from its amino acid subunits sequentially features an intramolecular aromatic nucleophilic substitution reaction for formation of the diaryl ether and closure of the 16-membered CD ring system (65%), a respectively diastereoselective (3:1, 86%) Suzuki coupling for installation of the AB biaryl linkage on which the atropisomer stereochemistry can be further thermally adjusted, and an effective macrolactamization (51%) for closure of the 12-membered AB ring system. A similarly effective 13-step (14% overall) synthesis of the 14-membered EFG ring system 4 was implemented employing a room-temperature intermolecular S(N)Ar reaction of an o-fluoronitroaromatic for formation of the FG diaryl ether (69%) and a key macrolactamization (92%) with formation of the amide linking residues 1 and 2. The two key fragments 3 and 4 were coupled, and the remaining 16-membered DE ring system was closed via diaryl ether formation to provide the ristocetin tetracyclic ring system (15 steps, 8% overall) enlisting an unusually facile (25 degrees C, 8 h, DMF, >/=95%) and diastereoselective (>/=15:1) aromatic nucleophilic substitution reaction that benefits from substrate preorganization.     

Analysis of amide bond formation with an alpha-hydroxy-beta-amino acid derivative, 3-amino-2-hydroxy-4-phenylbutanoic acid, as an acyl component: byproduction of homobislactone

In the synthesis of peptidomimetics containing alpha-hydroxy-beta-amino acid, the coupling of this N(beta)-protected beta-amino acid with amine components was generally performed without the protection of its alpha-hydroxyl group. However, the formation of dipeptides in low yield was often observed when sterically hindered amine components were used. Boc-Apns-OH [Apns: (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid, allophenylnorstatine] (6), which is one of such beta-amino acid derivatives, is intensively employed as a core structure in the development of HIV-1 protease inhibitors. There have been no precise studies, to date, that have examined amide bond formation with alpha-hydroxy-beta-amino acid derivatives as an acyl component. To determine the cause of this low-yield reaction, we studied the amide bond formation focusing on the activation step of N(beta)-protected alpha-hydroxy-beta-amino acid by using a model coupling reaction between 6 and H-Dmt-OR [Dmt: (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid] (7). A significant amount of homobislactone 9 was formed through the activation of the carboxyl group of 6 to the benzotriazole-type active esters such as OBt and OAt. In addition, this homobislactone formation was markedly increased in the presence of a catalytic amount of a base, which exhibited good correlation with the low yield of the amide bond formation, suggesting that homobislactone formation is one major reason for the low yield of the amide bond formation. Moreover, homobislactones were also formed in other derivatives of the N(beta)-protected alpha-hydroxy-beta-amino acid, suggesting a common feature of this type of amino acids. The use of a strong activation method like EDC--HOAt without base addition enhanced amide bond formation, although a small amount of homobislactone may be formed during the coupling reaction.     

The hidden steps of domain skipping: macrolactone ring size determination in the pikromycin modular polyketide synthase

The pikromycin (Pik) polyketide synthase (PKS) from Streptomyces venezuelae comprises four multifunctional polypeptides (PikAI, PikAII, PikAIII, and PikAIV). This PKS can generate 12- and 14-membered ring macrolactones (10-deoxymethynolide and narbonolide, respectively) through the activity of its terminal modules (PikAIII and PikAIV). We performed a series of experiments involving the functional replacement of PikAIV in mutant strains with homodimeric and heterodimeric PikAIV modules to investigate the details of macrolactone ring size determination. The results suggest a new and surprising mechanism by which the penultimate hexaketide chain elongation intermediate is transferred from PikAIII ACP5 to PikAIV ACP6 before release by the terminal thioesterase domain. Elucidation of this chain transfer mechanism provides important new details about alternative macrolactone ring size formation in modular PKSs and contributes to the potential for rational design of structural diversity by combinatorial biosynthesis.     

Inactivation of the carbamoyltransferase gene refines post-polyketide synthase modification steps in the biosynthesis of the antitumor agent geldanamycin

The post-polyketide synthase modification of geldanamycin (1) biosynthesis is of interest as a means of introducing structural diversity into the compound. From the inactivation of a gene encoding carbamoyltransferase, we demonstrated that the C-17 hydroxylation and the C-21 oxidation precede O-carbamoylation and that the hypothetical progeldanamycin does not possess a double bond at C-4 and C-5. More importantly, our result revealed new intermediates 4,5-dihydro-7-O-descarbamoyl-7-hydroxygeldanamycin (3) and 4,5-dihydrogeldanamycin (5), indicating that O-carbamoylation occurs prior to the C-4,5 cis double bond formation in geldanamycin biosynthesis.     

Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase

The unique ability of the pikromycin (Pik) polyketide synthase to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes underlying polyketide synthesis, specifically the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII (module 5) and PikAIV (module 6) and assessed the ability of these proteins to generate tri- and tetraketide lactone products using N-acetylcysteamine-activated diketides and (14)C-methylmalonyl-CoA as substrates. Comparison of the stereochemical specificities for PikAIII and PikAIV and the reported values for the DEBS modules reveals significant differences between these systems.     

Carbohydrates Containing Nitrogen or Sulfur in the “Hemiacetal” Ring

In 5-amino-5-deoxy-and 5-thioaldoses, a 6-membered hemiacetal ring can be formed by incorporation of a nitrogen or sulfur atom into the ring instead of the oxygen present in pyranoses. In the case of 5-(N-acylamido) -5- deoxyaldoses, the nucleophilic strength of the amide grouping is lowered, with the result that ring closure occurs only when sterically favored, or when a side reaction with formation of furanose is not possible. On the other hand, 5-amino-5-deoxyaldoses containing a free amino group readily from 6-membered “hemiacetal” rings. The resulting piperidinoses possess properties that are unusual in sugars, since they contain the 2-hydroxypiperidine ring system. Piperidinoses are stable to alkalis and labile to acids; they rearrange in the presence of acids to give Amadori compounds, or lose three molecules of water to give derivatives of β-hydroxypyridines. Owing to the high reactivity of the mercapto group, 5-thioaldoses from only sugars with rings containing sulfur, the behavior of which resembles that of the oxygen analogues. 4-Amino-4-deoxy- and 4-thioaldoses can form 5-membered rings containing nitrogen and sulfur.     

Solving the Puzzle of One‐Carbon Loss in Ripostatin Biosynthesis

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis‐ and trans‐acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl‐C2 unit is derived from phenylpyruvate (phenyl‐C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl‐C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl‐S ‐acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin.