Polyether Cyclization Cascade Alterations in Response to Monensin Polyketide Synthase Mutations

The targeted manipulation of polyketide synthases has in recent years led to numerous new-to-nature polyketides. For type I polyketide synthases the response of post-polyketide synthases (PKS) processing enzymes onto the most frequently polyketide backbone manipulations is so far insufficiently studied. In particular, complex processes such as the polyether cyclisation in the biosynthesis of ionophores such as monensin pose interesting objects of research. We present here a study of the substrate promiscuity of the polyether cyclisation cascade enzymes in monensin biosynthesis in the conversion of redox derivatives of the nascent polyketide chain. LC-HRMS/MS²-based studies revealed a remarkable flexibility of the post-PKS enzymes. They acted on derivatized polyketide backbones based on the three possible polyketide redox states within two different modules and gave rise to an altered polyether structure. One of these monensin derivatives was isolated and characterized by 2D-NMR spectroscopy, crystallography, and bioactivity studies.     

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP‐free intermediate towards the final metabolite. However, the timing of ACP off‐loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone‐2‐carboxylic acid‐N‐acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)‐DNPA, and the product of ActIV reaction was characterized as an ACP‐free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC‐TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.     

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.     

Exploring the Promiscuous Enzymatic Activation of Unnatural Polyketide Extender Units in Vitro and in Vivo for Monensin Biosynthesis

The incorporation of new-to-nature extender units into polyketide synthesis is an important source for diversity yet is restricted by limited availability of suitably activated building blocks in vivo. We here describe a straightforward workflow for the biogenic activation of commercially available new-to-nature extender units. Firstly, the substrate scope of a highly flexible malonyl co-enzyme A synthetase from Streptomyces cinnamonensis was characterized. The results were matched by in vivo experiments in which the said extender units were accepted by both the polyketide synthase and the accessory enzymes of the monensin biosynthetic pathway. The experiments gave rise to a series of predictable monensin derivatives by the exploitation of the innate substrate promiscuity of an acyltransferase and downstream enzyme functions.     

Predicted Incorporation of Non‐native Substrates by a Polyketide Synthase Yields Bioactive Natural Product Derivatives

The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl‐ or methyl‐malonyl‐CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5_mon) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5_mon, insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non‐native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl‐binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules.     

Covalent linkage mediates communication between ACP and TE domains in modular polyketide synthases

Polyketide natural products such as erythromycin A and epothilone are assembled on multienzyme polyketide synthases (PKSs), which consist of modular sets of protein domains. Within these type I systems, the fidelity of biosynthesis depends on the programmed interaction among the multiple domains within each module, centered around the acyl carrier protein (ACP). A detailed understanding of interdomain communication will therefore be vital for attempts to reprogram these pathways by genetic engineering. We report here that the interaction between a representative ACP domain and its downstream thioesterase (TE) is mediated largely by covalent tethering through a short "linker" region, with only a minor energetic contribution from protein-protein molecular recognition. This finding helps explain in part the empirical observation that TE domains can function out of their normal context in engineered assembly lines, and supports the view that overall PKS architecture may dictate at least a subset of interdomain interactions.     

The Ketosynthase Domain Controls Chain Length in Mushroom Oligocyclic Polyketide Synthases

The nonreducing iterative type I polyketide synthases (NR-PKSs) CoPKS1 and CoPKS4 of the webcap mushroom Cortinarius odorifer share 88 % identical amino acids. CoPKS1 almost exclusively produces a tricyclic octaketide product, atrochrysone carboxylic acid, whereas CoPKS4 shows simultaneous hepta- and octaketide synthase activity and also produces the bicyclic heptaketide 6-hydroxymusizin. To identify the region(s) controlling chain length, four chimeric enzyme variants were constructed and assayed for activity in Aspergillus niger as heterologous expression platform. We provide evidence that the β-ketoacyl synthase (KS) domain determines chain length in these mushroom NR-PKSs, even though their KS domains differ in only ten amino acids. A unique proline-rich linker connecting the acyl carrier protein with the thioesterase domain varies most between these two enzymes but is not involved in chain length control.     

An Unusual Thioesterase Promotes Isochromanone Ring Formation in Ajudazol Biosynthesis

The ajudazols are antifungal secondary metabolites produced by a hybrid polyketide synthase (PKS)‐nonribosomal peptide synthetase (NRPS) multienzyme “assembly line” in the myxobacterium Chondromyces crocatus Cm c5. The most striking structural feature of these compounds is an isochromanone ring system; such an aromatic moiety is only known from two other complex polyketides, the electron transport inhibitor stigmatellin and the polyether lasalocid. The cyclization and aromatization reactions in the stigmatellin pathway are presumed to be catalyzed by a cyclase domain located at the end of the PKS, while the origin of the lasalocid benzenoid ring remains obscure. Notably, the ajudazol biosynthetic machinery does not incorporate a terminal cyclase, but instead a variant thioesterase (TE) domain. Here we present detailed phylogenetic and sequence analysis, coupled with experiments both in vitro and in vivo, that suggest that this TE promotes formation of the isochromanone ring, a novel reaction for this type of domain. As the ajudazol TE has homologues in several other secondary‐metabolite pathways, these results are likely to be generalizable.     

Specifically immobilised aldo/keto reductase AKR1A1 shows a dramatic increase in activity relative to the randomly immobilised enzyme

The difference between site-specific and random immobilisation of the aldo/keto reductase AKR1A1 was explored. AKR1A1 was recombinantly expressed as a thioester by the intein strategy. The thioester was selectively modified with a biotin label by the expressed protein ligation method, and subsequent immobilisation on streptavidin templates was performed. Adsorption of wild-type AKR1A1 to streptavidin templates and of biotinylated AKR1A1 to uncoated templates was used to study randomly immobilised enzymes. Investigation of the kinetic parameters revealed remarkably improved activity for the site-specifically immobilised enzyme, which was comparable to that of the wild-type enzyme in solution and 60-300-fold greater than that of the randomly immobilized enzymes. Furthermore, the enzyme was surprisingly stable. No loss of activity was observed for over a week, and even after 50 days more than 35% of activity was maintained.     

Establishing a new methodology for genome mining and biosynthesis of polyketides and peptides through yeast molecular genetics

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.     

A Stable Chemical SUMO1–Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2–E3 Ligase Complex

Ubiquitin and ubiquitin‐like (Ubl) modifiers such as SUMO are conjugated to substrate proteins by E1, E2, and E3 enzymes. In the presence of an E3 ligase, the E2～Ubl thioester intermediate becomes highly activated and is prone to chemical decomposition, thus making biochemical and structural studies difficult. Here we explored a stable chemical conjugate of the E2 enzyme from the SUMO pathway, Ubc9, with its modifier SUMO1 as a structural analogue of the Ubc9～SUMO1 thioester intermediate, by introducing a triazole linkage by biorthogonal click chemistry. The chemical conjugate proved stable against proteolytic cleavage, in contrast to a Ubc9–SUMO1 isopeptide analogue obtained by auto‐SUMOylation. Triazole‐linked Ubc9–SUMO1 bound specifically to the preassembled E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9, thus suggesting that it is a suitable thioester mimic. We anticipate interesting prospects for its use as a research tool to study protein complexes involving E2 and E3 enzymes.     

Discovery and Elucidation of the Biosynthesis of Aspernidgulenes: Novel Polyenes from Aspergillus Nidulans by Using Serial Promoter Replacement

Through serial promoter exchanges, we isolated several novel polyenes, the aspernidgulenes, from Aspergillus nidulans and uncovered their succinct biosynthetic pathway involving only four enzymes. An enoyl reductase (ER)-less highly reducing polyketide synthase (HR-PKS) putatively produces a 5,6-dihydro-α-pyrone polyene, which undergoes bisepoxidation, epoxide ring opening, cyclization, and hydrolytic cleavage by three tailoring enzymes to generate aspernidgulene A1 and A2. Our findings demonstrate the prowess of fungal-tailoring enzymes to transform a polyketide scaffold concisely and efficiently into complex structures. Moreover, comparison with citreoviridin and aurovertin biosynthesis suggests that methylation of the α-pyrone hydroxy group by methyltransferase (CtvB or AurB) is the branching point at which the biosynthesis of these two classes of compounds diverge. Therefore, scanning for the presence or absence of the gatekeeping α-pyrone methyltransferase gene in homologous clusters might be a potential way to classify the product bioinformatically as belonging to methylated α-pyrone polyenes or polyenes containing rings derived from the cyclization of the unmethylated 5,6-dihydro-α-pyrone, such as 2,3-dimethyl-γ-lactone and oxabicyclo[2.2.1]heptane.     

Catalytic promiscuity in the alpha/beta-hydrolase superfamily: hydroxamic acid formation, C--C bond formation, ester and thioester hydrolysis in the C--C hydrolase family

The haloperoxidase family of alpha/beta-hydrolases contains enzymes of several different catalytic activities, including esterases, C--C hydrolases and cofactor-independent haloperoxidases (perhydrolases), but the molecular basis of this catalytic promiscuity is not fully understood. The C--C hydrolase enzyme MhpC from E. coli is shown to possess esterase and thioesterase activity, and the ability to activate hydroxylamine as a nucleophile to form hydroxamic acid products. The ratio of these activities was examined for nine site-directed mutant enzymes that contained mutations at nonessential residues in the enzyme active site. Higher levels of esterase and thioesterase activity were found in mutants Phe173Gly and Trp264Gly; this might be due to increased amounts of space in the active site. Higher levels of hydroxamic acid formation activity were found in mutant Asn109His-a mutation found in many haloperoxidase enzymes. Wild-type and mutant MhpC enzymes were also capable of C--C bond formation in organic solvents, and the highest activity was observed in nonpolar solvents. The results provide experimental support for the catalytic promiscuity shown in this family of enzymes, and indicate that differences in catalytic function can be introduced by point mutations.     

Biosynthetic Code for Divergolide Assembly in a Bacterial Mangrove Endophyte

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3‐amino‐5‐hydroxybenzoate and the rare extender units ethylmalonyl‐CoA and isobutylmalonyl‐CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer–Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.     

The Thioesterase Bhp is Involved in the Formation of β‐Hydroxytyrosine during Balhimycin Biosynthesis in Amycolatopsis balhimycina

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel‐chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with “haloperoxidase”/perhydrolase activity, it did not show any enzymatic activity with standard “haloperoxidase”/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p‐nitrophenyl acetate, p‐nitrophenyl phosphate and S‐thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S‐β‐hydroxytyrosyl‐N‐acetyl cysteamine thioester (β‐OH‐Tyr‐SNAC) with 15 times the efficiency of S‐L ‐tyrosyl‐N‐acetyl cysteamine thioester (L ‐Tyr‐SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β‐OH‐Tyr‐S‐PCP (PCP=peptidyl carrier protein) to free β‐hydroxytyrosine (β‐OH‐Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP‐bound β‐OH‐Tyr and not a “haloperoxidase”/perhydrolase or nonspecific esterase.     

Probing enzyme promiscuity of SGNH hydrolases

Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstA(N)-EstP(C) and EstP(N)-EstA(C) were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.     

Ketoreduction in mycolactone biosynthesis: insight into substrate specificity and stereocontrol from studies of discrete ketoreductase domains in vitro

Mycolactone, a polyketide toxin responsible for the extensive tissue destruction seen in Buruli ulcer, is assembled on a modular polyketide synthase (PKS). Despite operating on structurally different intermediates during synthesis, many of the ketoreductase (KR) domains of the mycolactone (MLS) PKS have identical sequences. This suggests that these enzymes might exhibit an unusually high level of substrate promiscuity. However, we show here that when recombinant mycolactone KR domains are tested with a range of surrogate substrates, their specificity closely matches that of KR domains derived from other PKS systems. In addition, our findings reinforce the role of substrate tethering for achieving stereochemical control in modular PKSs by affecting the delicate energetics of ketoreduction.     

Rapid Biocatalytic Synthesis of Aromatic Acid CoA Thioesters by Using Microbial Aromatic Acid CoA Ligases

Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants. As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.     

Fluorescent Mechanism‐Based Probe for Aerobic Flavin‐Dependent Enzyme Activity

Diversity in non‐ribosomal peptide and polyketide secondary metabolism is facilitated by interactions between biosynthetic domains with discrete monomer loading and their cognate tailoring enzymes, such as oxidation or halogenation enzymes. The cooperation between peptidyl carrier proteins and flavin‐dependent enzymes offers a specialized strategy for monomer selectivity for oxidization of small molecules from within a complex cellular milieu. In an effort to study this process, we have developed fluorescent probes to selectively label aerobic flavin‐dependent enzymes. Here we report the preparation and implementation of these tools to label oxidase, monooxygenase, and halogenase flavin‐dependent enzymes.     

An Unusual Fatty Acyl:Adenylate Ligase (FAAL)–Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed by a unique polyene cyclisation mechanism, in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from polyene cyclisation and to transfer it to its associated acyl carrier protein (ACP). After oxidative decarboxylation, the intermediate is channelled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation–thiolation didomain with a very unusual substrate selectivity code but has not yet been biochemically studied. On the basis of sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase (FAAL)–acyl carrier protein didomain with unusual substrate specificity. The expected adenylate‐forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalised fatty acids and unsaturated and aromatic carboxylic acids. HPLC‐MS analysis and competition experiments show that AmbG preferentially acylates its ACP with long‐chain hydrophobic acids and tolerates a π system and a branch near the carboxylic acid. AmbG is the first characterised example of a FAAL–ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as partial reconstitution of the ambruticin pathway to elucidate DVC formation.