An enzymatic Alder-ene reaction.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites.

α-Pyrone Derivatives from Calcarisporium arbuscula Discovered by Genome Mining.

A highly reducing polyketide synthase (HRPKS) gene cluster from the genome of Calcarisporium arbuscula was identified through genome mining. Heterologous expression of this cluster led to the production of four new α-pyrone compounds, calcapyrones A (1) and B (2), along with their biosynthetic intermediates calcapyrones C (3) and D (4). The structures of these compounds were elucidated on the basis of extensive spectroscopic experiments, and the absolute configurations of the 7,8-diol moieties in 1 and 2 were assigned using Snatzke's method. The biosynthetic pathway of 1 and 2 was established through in vivo and in vitro experiments.

SAM-dependent enzyme-catalysed pericyclic reactions in natural product biosynthesis.

Pericyclic reactions-which proceed in a concerted fashion through a cyclic transition state-are among the most powerful synthetic transformations used to make multiple regioselective and stereoselective carbon-carbon bonds. They have been widely applied to the synthesis of biologically active complex natural products containing contiguous stereogenic carbon centres. Despite the prominence of pericyclic reactions in total synthesis, only three naturally existing enzymatic examples (the intramolecular Diels-Alder reaction, and the Cope and the Claisen rearrangements) have been characterized. Here we report a versatile S-adenosyl-l-methionine (SAM)-dependent enzyme, LepI, that can catalyse stereoselective dehydration followed by three pericyclic transformations: intramolecular Diels-Alder and hetero-Diels-Alder reactions via a single ambimodal transition state, and a retro-Claisen rearrangement. Together, these transformations lead to the formation of the dihydropyran core of the fungal natural product, leporin. Combined in vitro enzymatic characterization and computational studies provide insight into how LepI regulates these bifurcating biosynthetic reaction pathways by using SAM as the cofactor. These pathways converge to the desired biosynthetic end product via the (SAM-dependent) retro-Claisen rearrangement catalysed by LepI. We expect that more pericyclic biosynthetic enzymatic transformations remain to be discovered in naturally occurring enzyme 'toolboxes'. The new role of the versatile cofactor SAM is likely to be found in other examples of enzyme catalysis.

Biosynthesis of para-Cyclophane-Containing Hirsutellone Family of Fungal Natural Products.

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.

Genome-Directed Discovery of Tetrahydroisoquinolines from Deep-Sea Derived Streptomyces niveus SCSIO 3406.

A genome-directed discovery strategy to identify new tetrahydroisoquinolines (THIQs) was applied to deep-sea derived Streptomyces niveus SCSIO 3406; 11 THIQs were found representing three THIQ classes. Known aclidinomycins A (1) and B (2) were isolated along with nine new compounds, aclidinomycins C-K (3-11). The structures were elucidated using extensive spectroscopic analyses and single-crystal X-ray diffraction methods. The core skeleton of compounds 6-9 contains a fused tetrahydropyran (THP) as an integral part of a distinct type of 6/6/6/6/5/5 polycyclic motif. This is the first report of such a system. Beyond their discovery, we also report here a proposed biosynthetic route to these interesting natural products as well as a preliminary survey of their antimicrobial activities.

Extracellularly oxidative activation and inactivation of matured prodrug for cryptic self-resistance in naphthyridinomycin biosynthesis.

Understanding how antibiotic-producing bacteria deal with highly reactive chemicals will ultimately guide therapeutic strategies to combat the increasing clinical resistance crisis. Here, we uncovered a distinctive self-defense strategy featured by a secreted oxidoreductase NapU to perform extracellularly oxidative activation and conditionally overoxidative inactivation of a matured prodrug in naphthyridinomycin (NDM) biosynthesis from Streptomyces lusitanus NRRL 8034. It was suggested that formation of NDM first involves a nonribosomal peptide synthetase assembly line to generate a prodrug. After exclusion and prodrug maturation, we identified a pharmacophore-inactivated intermediate, which required reactivation by NapU via oxidative C-H bond functionalization extracellularly to afford NDM. Beyond that, NapU could further oxidatively inactivate the NDM pharmacophore to avoid self-cytotoxicity if they coexist longer than necessary. This discovery represents an amalgamation of sophisticatedly temporal and spatial shielding mode conferring self-resistance in antibiotic biosynthesis from Gram-positive bacteria.

Fungal Highly Reducing Polyketide Synthases Biosynthesize Salicylaldehydes That Are Precursors to Epoxycyclohexenol Natural Products.

Fungal highly reducing polyketide synthases (HRPKSs) are highly programmed multidomain enzymes that synthesize reduced polyketide structures. Recent reports indicated salicylaldehydes are synthesized by HRPKS biosynthetic gene clusters, which are unexpected based on known enzymology of HRPKSs. Using genome mining of a Trichoderma virens HRPKS gene cluster that encodes a number of redox enzymes, we uncover the strategy used by HRPKS pathways in the biosynthesis of aromatic products such as salicylaldehyde 4, which can be oxidatively modified to the epoxycyclohexanol natural product trichoxide 1. We show selective ß-hydroxyl groups in the linear HRPKS product are individually reoxidized to ß-ketones by short-chain dehydrogenase/reductase enzymes, which enabled intramolecular aldol condensation and aromatization. Our work expands the chemical space of natural products accessible through HRPKS pathways.

Genome-Mined Diels-Alderase Catalyzes Formation of the cis-Octahydrodecalins of Varicidin A and B.

Pericyclases are an emerging family of enzymes catalyzing pericyclic reactions. A class of lipocalin-like enzymes recently characterized as Diels-Alderases (DAses) catalyze decalin formation through intramolecular Diels-Alder (IMDA) reactions between electron-rich dienes and electron-deficient dienophiles. Using this class of enzyme as a beacon for genome mining, we discovered a biosynthetic gene cluster from Penicillium variabile and identified that it encodes for the biosynthesis of varicidin A (1), a new antifungal natural product containing a cis-octahydrodecalin core. Biochemical analysis reveals a carboxylative deactivation strategy used in varicidin biosynthesis to suppress the nonenzymatic IMDA reaction of an early acyclic intermediate that favors trans-decalin formation. A P450 oxidizes the reactive intermediate to yield a relatively unreactive combination of an electron-deficient diene and an electron-deficient dienophile. The DAse PvhB catalyzes the final stage IMDA on the carboxylated intermediate to form the cis-decalin that is important for the antifungal activity.

Thioesterase-Catalyzed Aminoacylation and Thiolation of Polyketides in Fungi.

Fungal highly reducing polyketide synthases (HRPKSs) biosynthesize polyketides using a single set of domains iteratively. Product release is a critical step in HRPKS function to ensure timely termination and enzyme turnover. Nearly all of the HRPKSs characterized to date employ a separate thioesterase (TE) or acyltransferase enzyme for product release. In this study, we characterized two fungal HRPKSs that have fused C-terminal TE domains, a new domain architecture for fungal HRPKSs. We showed that both HRPKS-TEs synthesize aminoacylated polyketides in an ATP-independent fashion. The KU42 TE domain selects cysteine and homocysteine and catalyzes transthioesterification using the side-chain thiol group as the nucleophile. In contrast, the KU43 TE domain selects leucine methyl ester and performs a direct amidation of the polyketide, a reaction typically catalyzed by nonribosomal peptide synthetase (NRPS) domains. The characterization of these HRPKS-TE enzymes showcases the functional diversity of HRPKS enzymes and provides potential TE domains as biocatalytic tools to diversify HRPKS structures.

Oxidative Cyclization in Natural Product Biosynthesis.

Oxidative cyclizations are important transformations that occur widely during natural product biosynthesis. The transformations from acyclic precursors to cyclized products can afford morphed scaffolds, structural rigidity, and biological activities. Some of the most dramatic structural alterations in natural product biosynthesis occur through oxidative cyclization. In this Review, we examine the different strategies used by nature to create new intra(inter)molecular bonds via redox chemistry. This Review will cover both oxidation- and reduction-enabled cyclization mechanisms, with an emphasis on the former. Radical cyclizations catalyzed by P450, nonheme iron, α-KG-dependent oxygenases, and radical SAM enzymes are discussed to illustrate the use of molecular oxygen and S-adenosylmethionine to forge new bonds at unactivated sites via one-electron manifolds. Nonradical cyclizations catalyzed by flavin-dependent monooxygenases and NAD(P)H-dependent reductases are covered to show the use of two-electron manifolds in initiating cyclization reactions. The oxidative installations of epoxides and halogens into acyclic scaffolds to drive subsequent cyclizations are separately discussed as examples of "disappearing" reactive handles. Last, oxidative rearrangement of rings systems, including contractions and expansions, will be covered.

Genome Mining and Assembly-Line Biosynthesis of the UCS1025A Pyrrolizidinone Family of Fungal Alkaloids.

UCS1025A is a fungal polyketide/alkaloid that displays strong inhibition of telomerase. The structures of UCS1025A and related natural products are featured by a tricyclic furopyrrolizidine connected to a trans-decalin fragment. We mined the genome of a thermophilic fungus and activated the ucs gene cluster to produce UCS1025A at a high titer. Genetic and biochemical analysis revealed a PKS-NRPS assembly line that activates 2S,3S-methylproline derived from l-isoleucine, followed by Knoevenagel condensation to construct the pyrrolizidine moiety. Oxidation of the 3S-methyl group to a carboxylate leads to an oxa-Michael cyclization and furnishes the furopyrrolizidine. Our work reveals a new strategy used by nature to construct heterocyclic alkaloid-like ring systems using assembly line logic.

Collaborative Biosynthesis of Maleimide- and Succinimide-Containing Natural Products by Fungal Polyketide Megasynthases.

Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precisely positioned olefin from the unnatural amino acid is proposed to facilitate a scaffold rearrangement of the PKS-NRPS product to forge the maleimide and succinimide cores of oxaleimides.

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,ß-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

Catalysis of Extracellular Deamination by a FAD-Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin†A.

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process, at the end of which the matured compounds are exported outside the cells. The biosynthesis of saframycin A (SFM-A), an antitumor antibiotic, requires a cryptic fatty acyl chain to guide the construction of a pentacyclic tetrahydroisoquinoline scaffold; however, the follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, a membrane-bound peptidase, hydrolyzes the fatty acyl chain to release the amino group; and SfmCy2, a secreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly. These results not only fill in the missing steps of SFM-A biosynthesis, but also reveal that a FAD-binding oxidoreductase catalyzes an unexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain.

Fungal polyketides have significant biological activities, yet the biosynthesis by highly reducing polyketide synthases (HRPKSs) remains enigmatic. An uncharacterized group of HRPKSs was found to contain a C-terminal domain with significant homology to carnitine O-acyltransferase (cAT). Characterization of one such HRPKS (Tv6-931) from Trichoderma virens showed that the cAT domain is capable of esterifying the polyketide product with polyalcohol nucleophiles. This process is readily reversible, as confirmed through the holo ACP-dependent transesterification of the released product. The methyltransferase (MT) domain of Tv6-931 can perform two consecutive α-methylation steps on the last ß-keto intermediate to yield an α,α-gem-dimethyl product, a new programing feature among HRPKSs. Recapturing of the released product by cAT domain is suggested to facilitate complete gem-dimethylation by the MT.

Biosynthesis of Complex Indole Alkaloids: Elucidation of the Concise Pathway of Okaramines.

The okaramines are a class of complex indole alkaloids isolated from Penicillium and Aspergillus species. Their potent insecticidal activity arises from selectively activating glutamate-gated chloride channels (GluCls) in invertebrates, not affecting human ligand-gated anion channels. Okaramines B (1) and D (2) contain a polycyclic skeleton, including an azocine ring and an unprecedented 2-dimethyl-3-methyl-azetidine ring. Owing to their complex scaffold, okaramines have inspired many total synthesis efforts, but the enzymology of the okaramine biosynthetic pathway remains unexplored. Here, we identified and characterized the biosynthetic gene cluster (oka) of 1 and 2, then elucidated the pathway with target gene inactivation, heterologous reconstitution, and biochemical characterization. Notably, we characterized an α-ketoglutarate-dependent non-heme FeII dioxygenase that forged the azetidine ring on the okaramine skeleton.

Biosynthesis of Strained Piperazine Alkaloids: Uncovering the Concise Pathway of Herquline A.

Nature synthesizes many strained natural products that have diverse biological activities. Uncovering these biosynthetic pathways may lead to biomimetic strategies for organic synthesis of such compounds. In this work, we elucidated the concise biosynthetic pathway of herquline A, a highly strained and reduced fungal piperazine alkaloid. The pathway builds on a nonribosomal peptide synthetase derived dityrosine piperazine intermediate. Following enzymatic reduction of the P450-cross-linked dicyclohexadienone, N-methylation of the piperazine serves as a trigger that leads to a cascade of stereoselective and nonenzymatic transformations. Computational analysis of key steps in the pathway rationalizes the observed reactivities.

Biochemical Characterization of a Eukaryotic Decalin-Forming Diels-Alderase.

The trans-decalin structure formed by intramolecular Diels-Alder cycloaddition is widely present among bioactive natural products isolated from fungi. We elucidated the concise three-enzyme biosynthetic pathway of the cytotoxic myceliothermophin and biochemically characterized the Diels-Alderase that catalyzes the formation of trans-decalin from an acyclic substrate. Computational studies of the reaction mechanism rationalize both the substrate and stereoselectivity of the enzyme.

Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast.

The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here, using Saccharomyces cerevisiae as a heterologous host and a phylogenetically guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologues identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavinine and anominine and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.

Efficient Biosynthesis of Fungal Polyketides Containing the Dioxabicyclo-octane Ring System.

Aurovertins are fungal polyketides that exhibit potent inhibition of adenosine triphosphate synthase. Aurovertins contain a 2,6-dioxabicyclo[3.2.1]octane ring that is proposed to be derived from a polyene precursor through regioselective oxidations and epoxide openings. In this study, we identified only four enzymes required to produce aurovertin E. The core polyketide synthase produces a polyene α-pyrone. Following pyrone O-methylation by a methyltransferase, a flavin-dependent mono-oxygenase and an epoxide hydrolase can iteratively transform the terminal triene portion of the precursor into the dioxabicyclo[3.2.1]octane scaffold. We demonstrate that a tetrahydrofuranyl polyene is the first stable intermediate in the transformation, which can undergo epoxidation and anti-Baldwin 6-endo-tet ring opening to yield the cyclic ether product. Our results further demonstrate the highly concise and efficient ways in which fungal biosynthetic pathways can generate complex natural product scaffolds.