Genome Mining of a Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthetase Pathway to Synthesize a Phytotoxic N-Acyl Amino Acid.

Guided by the retrobiosynthesis hypothesis, we characterized a fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid megasynthetase pathway to generate 2-trans-4-trans-2-methylsorbyl-d-leucine (1), a polyketide amino acid conjugate that inhibits Arabidopsis root growth. The biosynthesis of 1 includes a PKS-NRPS enzyme to assemble an N-acyl amino alcohol intermediate, which is further oxidized to an N-acyl amino acid (NAAA), demonstrating a new biosynthetic logic for synthesizing NAAAs and expanding the chemical space of products encoded by fungal PKS-NRPS clusters.

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.

Discovery and biosynthesis of macrophasetins from the plant pathogen fungus Macrophomina phaseolina.

3-Decalinoyltetramic acids (DTAs) are a class of natural products with chemical diversity and potent bioactivities. In fungal species there is a general biosynthetic route to synthesize this type of compounds, which usually features a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) and a lipocalin-like Diels-Alderase (LLDAse). Using a synthetic biology approach, combining the bioinformatics analysis prediction and heterologous expression, we mined a PKS-NRPS and LLDAse encoding gene cluster from the plant pathogenic fungus Macrophomina phaseolina and characterized the cluster to be responsible for the biosynthesis of novel DTAs, macrophasetins. In addition, we investigated the biosynthesis of these compounds and validated the accuracy of the phylogeny-guided bioinformatics analysis prediction. Our results provided a proof of concept example to this approach, which may facilitate the discovery of novel DTAs from the fungal kingdom.

Flavoprotein StnP2 Catalyzes the ß-Carboline Formation during the Streptonigrin Biosynthesis.

ß-Carboline (ßC) alkaloids constitute a large family of indole alkaloids that exhibit diverse pharmacological properties, such as antitumor, antiviral, antiparasitic, and antimicrobial activities. Here, we report that a flavoprotein StnP2 catalyzes the dehydrogenation at C1-N2 of a tetrahydro-ß-carboline (THßC) generating a 3,4-dihydro-ß-carboline (DHßC), and the DHßC subsequently undergoes a spontaneous dehydrogenation to ßC formation involved in the biosynthesis of the antitumor agent streptonigrin. Biochemical characterization showed that StnP2 catalyzed the highly regio- and stereo-selective dehydrogenation, and StnP2 exhibits promiscuity toward different THßCs. This study provides an alternative kind of enzyme catalyzing the biosynthesis of ßC alkaloids and enhances the importance of flavoproteins.

Genome Mining Discovery of a New Benzazepine Alkaloid Pseudofisnin A from the Marine Fungus Neosartorya pseudofischeri F27-1.

l-Kynurenine (Kyn) is an intermediate in the kynurenine pathway and is also found to be a building block or biosynthetic precursor to bioactive natural products. Recent studies revealed that l-Kyn can be incorporated via nonribosomal peptide synthetase (NRPS) biosynthetic routes to generate 1-benzazepine-containing compounds, while 1-benzazepine is a pharmaceutically important scaffold that is rarely found in natural products. Using a core biosynthetic enzyme-guided genome-mining approach, we discovered a biosynthetic gene cluster from Neosartorya pseudofischeri and identified that it encodes for the biosynthesis of pseudofisnins, novel 1-benzazepine-containing compounds. The biosynthetic pathway of pseudofisnins was elucidated through in vivo and in vitro experiments. The methyltransferase PseC from the pathway was biochemically characterized to be an iterative methyltransferase that catalyzes off-NRPS line di-methylation on an amine group.

Combinatorial Biosynthesis of Terpenoids through Mixing-and-Matching Sesquiterpene Cyclase and Cytochrome P450 Pairs.

Terpenoids are an important class of natural products with diverse structures and bioactivities. Their hydrocarbon scaffolds are mainly derived from the terpenes produced by terpene cyclases (TCs). Otherwise, new hydrocarbon scaffolds can be achieved through oxidative rearrangement catalyzed by oxygenases such as P450s. Herein, we report the functional characterization of α/ß-trans-bergamotene-producing TCs and their multifunctional P450 partners mined from different fungal species. In addition, novel sesquiterpenoids with hydrocarbon scaffolds different from bergamotenes were generated by combinatorial biosynthesis through mixing-and-matching these TC and P450 pairs. Our results provide a successful example of expanding the chemical diversity of terpenoids by combining genome mining and synthetic biology.

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics.

Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.

TerC Is a Multifunctional and Promiscuous Flavoprotein Monooxygenase That Catalyzes Bimodal Oxidative Transformations.

The flavoprotein monooxygenase (FPMO) TerC is encoded by all known cyclopentene biosynthetic gene clusters. It can catalyze oxidative dearomatization toward a series of 6-HM analogues and further induces different skeletal distortions to form either benzoquinone or pyrone by bimodal reaction cascades, which is only governed by the C7 substitutions. Beyond our study demonstrated bimodal reaction cascades and advanced the biosynthetic knowledge of fungal cyclopentenes, this work also sets the stage for the bioengineering of 6-HM polyketides.

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.

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.

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.

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.

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.

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.

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.

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.

Late-Stage Terpene Cyclization by an Integral Membrane Cyclase in the Biosynthesis of Isoprenoid Epoxycyclohexenone Natural Products.

Macrophorins are representative examples of isoprenoid epoxycyclohexenones containing cyclized drimane moieties. We located and characterized the biosynthetic gene cluster of macrophorin from Penicillium terrestris. MacJ encoded by this cluster was characterized to be the first example of a membrane-bound type-II terpene cyclase catalyzing the cyclization of meroterpenoids via direct protonation of the terminal olefinic bond in acyclic yanuthones. The late-stage functionalization and substrate promiscuity of MacJ make it a potential biocatalyst for the synthesis of macrophorin analogues.

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.