Whole-Cell Biotransformation for the Preparation of Chromopyrrolic Acid and 5,5'-dichloro-Chromopyrrolic Acid in Escherichia coli.

Chromopyrrolic acid (CPA) and its congeners are important intermediates for the biosynthesis and synthesis of various dimeric tryptophan natural products. We have constructed two E. coli strains (CPA001/CPA002) harboring a single plasmid carrying genes coding for a combination of two enzymes (LaStaO/LzrO and VioB) that are able to convert L-tryptophan (L-Trp)/5-chloro-L-tryotophan (5-Cl-L-Trp) to chromopyrrolic acid (CPA)/5,5'-dichloro-chromopyrrolic acid (5,5'-diCl-CPA). Effect on the production of CPA were evaluated by varying the parameters of strain cultivation and biotransformation process. Under the optimized conditions, up to 325 mg/L of CPA and 275 mg/L of 5,5'-diCl-CPA could be obtained by supplementing L-Trp and 5-Cl-L-Trp, respectively, to a working culture of CPA001, or to a phosphate buffer-resuspended culture of CPA002. The practicability of this whole-cell biotransformation system could also be served as a potential platform for the preparation of CPA congeners.

Cofactor-independent C-C bond cleavage reactions catalyzed by the AlpJ family of oxygenases in atypical angucycline biosynthesis.

Biosynthesis of atypical angucyclines involves unique oxidative B-ring cleavage and rearrangement reactions, which are catalyzed by AlpJ-family oxygenases, including AlpJ, JadG, and GilOII. Prior investigations established the essential requirement for FADH2/FMNH2 as cofactors when utilizing the quinone intermediate dehydrorabelomycin as a substrate. In this study, we unveil a previously unrecognized facet of these enzymes as cofactor-independent oxygenases when employing the hydroquinone intermediate CR1 as a substrate. The enzymes autonomously drive oxidative ring cleavage and rearrangement reactions of CR1, yielding products identical to those observed in cofactor-dependent reactions of AlpJ-family oxygenases. Furthermore, the AlpJ- and JadG-catalyzed reactions of CR1 could be quenched by superoxide dismutase, supporting a catalytic mechanism wherein the substrate CR1 reductively activates molecular oxygen, generating a substrate radical and the superoxide anion O2 •-. Our findings illuminate a substrate-controlled catalytic mechanism of AlpJ-family oxygenases, expanding the realm of cofactor-independent oxygenases. Notably, AlpJ-family oxygenases stand as a pioneering example of enzymes capable of catalyzing oxidative reactions in either an FADH2/FMNH2-dependent or cofactor-independent manner.

Confirmation of the stereochemistry of spiroviolene.

We confirm the previously revised stereochemistry of spiroviolene by X-ray crystallographically characterizing a hydrazone derivative of 9-oxospiroviolane, which is synthesized by hydroboration/oxidation of spiroviolene followed by oxidation of the resultant hydroxy group. An unexpected thermal boron migration occurred during the hydroboration process of spiroviolene that resulted in the production of a mixture of 1α-hydroxyspiroviolane, 9α- and 9ß-hydroxyspiroviolane after oxidation. The assertion of the cis-orientation of the 19- and 20-methyl groups provided further support for the revised cyclization mechanism of spiroviolene.

Biosynthesis of Phomactin Platelet Activating Factor Antagonist Requires a Two-Enzyme Cascade.

Phomactin diterpenoids possess a unique bicyclo[9.3.1]pentadecane skeleton with multiple oxidative modifications, and are good platelet-activating factor (PAF) antagonists that can inhibit PAF-induced platelet aggregation. In this study, we identified the gene cluster (phm) responsible for the biosynthesis of phomactins from a marine fungus, Phoma sp. ATCC 74077. Despite the complexity of their structures, phomactin biosynthesis only requires two enzymes: a type I diterpene cyclase PhmA and a P450 monooxygenase PhmC. PhmA was found to catalyze the formation of the phomactatriene, while PhmC sequentially catalyzes the oxidation of multiple sites, leading to the generation of structurally diverse phomactins. The rearrangement mechanism of the diterpene scaffold was investigated through isotope labeling experiments. Additionally, we obtained the crystal complex of PhmA with its substrate analogue FGGPP and elucidated the novel metal-ion-binding mode and enzymatic mechanism of PhmA through site-directed mutagenesis. This study provides the first insight into the biosynthesis of phomactins, laying the foundation for the efficient production of phomactin natural products using synthetic biology approaches.

Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products.

A unified strategy for the biomimetic total synthesis of the spiroindimicin family of natural products was reported. Key transformations include a one-pot two-enzyme-catalyzed oxidative dimerization of L-tryptophan/5-chloro-L-tryptophan to afford the bis-indole precursors chromopyrrolic acid/5',5''-dichloro-chromopyrrolic acid, and regioselective C3'-C2'' and C3'-C4'' bond formation converting a common bis-indole skeleton to two skeletally different natural products, including (±)-spiroindimicins D and G with a [5,5] spiro-ring skeleton, and (±)-spiroindimicins A and H with a [5,6] spiro-ring skeleton, respectively.

Crystal Structure Based Mutagenesis of Cattleyene Synthase Leads to the Generation of Rearranged Polycyclic Diterpenes.

The crystal structures of cattleyene synthase (apo-CyS), and CyS complexed with geranylgeranyl pyrophosphate (GGPP) were solved. The CySC59A variant exhibited an increased production of cattleyene and other diterpenes with diverse skeletons. Its structure showed a widened active site cavity explaining the relaxed selectivity. Isotopic labeling experiments revealed a remarkable cyclization mechanism involving several skeletal rearrangements for one of the novel diterpenes.

Characterization of Enzymes Catalyzing the Formation of the Nonproteinogenic Amino Acid l-Dap in Capreomycin Biosynthesis.

Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including l-2,3-diaminopropionic acid (l-Dap), ß-ureidodehydroalanine, l-capreomycidine, and ß-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of l-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from O-phospho-l-Ser (OPS) and l-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD+-dependent oxidative hydrolysis by CmnK to generate l-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD+-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate α-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce l-Dap. These findings modify the original proposed pathway and provide insights into l-Dap formation in the biosynthesis of other related natural products.

Discovery of the leinamycin family of natural products by mining actinobacterial genomes.

Nature's ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF-SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF-SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature's rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature's biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

Metamorphosis of cycloalkenes for the divergent total synthesis of polycyclic indole alkaloids.

From tryptamine and secologanine, nature generates monoterpene indole alkaloids with an unprecedented level of skeletal diversity through a 'couple-divert' sequence. Intrigued by this biosynthetic machinery, new strategies and tactics to access skeletally distinct natural products are continuously emerging. In this Tutorial Review, we'll present a simplified view of nature's logic for biosynthesis and the representative strategies and tactics developed for the divergent total synthesis of monoterpene indole alkaloids. Our group has been developing a 'couple-divert' approach with the strategic use of cycloalkene as a pluripotent motif in the synthetic design and has developed an integrated oxidation/reduction/cyclization (iORC) sequence for transforming functionalized cycloalkenes, easily available by a variety of cross-coupling reactions, to skeletally diverse natural products. The integration of controlled regio-, chemo- and stereo-selective cyclization and heteroannulation reactions into these domino sequences will be highlighted.

Discovery and Characterization of 1-Aminocyclopropane-1-carboxylic Acid Synthase of Bacterial Origin.

The guangnanmycins (GNMs) belong to a small group of natural products featuring a 1-aminocyclopropane-1-carboxylic acid (ACC) moiety. While extensively studied in plants, ACC biosynthesis in bacteria remains poorly understood. Here we report inactivation of gnmY in vivo and biochemical characterization of GnmY in vitro, assigning GnmY as the first bacterial free ACC synthase that catalyzes the synthesis of ACC from S-adenosyl methionine. ACC is activated by GnmS and subsequently incorporated into the GNM scaffold by the GNM hybrid nonribosomal peptide synthetase-polyketide synthase system in GNM biosynthesis. GnmS exhibits relaxed substrate specificity, exploitation of which allowed the incorporation of 1-aminocyclobutane-1-carboxylic acid (ACBC) into the GNM scaffold to produce a GNM analogue with a cyclobutane ring at C-17. This study provides new insights into ACC biosynthesis in bacteria. GnmY and GnmS might be portable to engineer other ACC/ACBC-containing natural products.

Herbicidins from Streptomyces sp. CB01388 Showing Anti- Cryptosporidium Activity.

A high-content imaging assay was used to screen the fraction collection of the Natural Product Library at The Scripps Research Institute for inhibitors of Cryptosporidium parvum. A chemical investigation of one strain, Streptomyces sp. CB01388, resulted in the isolation of six herbicidins (1-6), one of which is new (herbicidin L, 1). Five of the six herbicidins (1-3, 5, 6) showed moderate inhibitory activity against C. parvum, with 1 and 6 comparable to the FDA-approved drug nitazoxanide, and 2-6 showed no toxicity to the host HCT-8 cells and human HEK293T and HepG2 cells. These findings highlight the herbicidin scaffold for anti- Cryptosporidium drug development.

A Long-Range Acting Dehydratase Domain as the Missing Link for C17-Dehydration in Iso-Migrastatin Biosynthesis.

The dehydratase domains (DHs) of the iso-migrastatin (iso-MGS) polyketide synthase (PKS) were investigated by systematic inactivation of the DHs in module-6, -9, -10 of MgsF (i.e., DH6, DH9, DH10) and module-11 of MgsG (i.e., DH11) in vivo, followed by structural characterization of the metabolites accumulated by the mutants, and biochemical characterization of DH10 in vitro, using polyketide substrate mimics with varying chain lengths. These studies allowed us to assign the functions for all four DHs, identifying DH10 as the dedicated dehydratase that catalyzes the dehydration of the C17 hydroxy group during iso-MGS biosynthesis. In contrast to canonical DHs that catalyze dehydration of the ß-hydroxy groups of the nascent polyketide intermediates, DH10 acts in a long-range manner that is unprecedented for type I PKSs, a novel dehydration mechanism that could be exploited for polyketide structural diversity by combinatorial biosynthesis and synthetic biology.

Enantioselective Total Syntheses of (-)-Rhazinilam, (-)-Leucomidine†B, and (+)-Leuconodine†F.

A divergent total synthesis of three structurally distinct natural products from imine 9 was accomplished through an approach featuring: 1) a Pd-catalyzed decarboxylative cross-coupling, and 2) heteroannulation of 9 with bromoacetaldehyde and oxalyl chloride to give tetrahydroindolizine 6 and dioxopyrrole 7, respectively. The former was converted into (-)-rhazinilam, while the latter was converted into (-)-leucomidine B and (+)-leuconodine F. A substrate-directed highly diastereoselective reduction of a sterically unbiased double bond by using a homogeneous palladium catalyst was developed. A self-induced diastereomeric anisochronism (SIDA) phenomenon was observed for leucomidine B.

Total Syntheses of (-)-Mersicarpine, (-)-Scholarisine G, (+)-Melodinine E, (-)-Leuconoxine, (-)-Leuconolam, (-)-Leuconodine A, (+)-Leuconodine F, and (-)-Leuconodine C: Self-Induced Diastereomeric Anisochronism (SIDA) Phenomenon for Scholarisine G and Leuconodines A and C.

Enantioselective total syntheses of title natural products from a common cyclohexenone derivative (S)-18 were reported. Ozonolysis of (S)-18 afforded a stable diketo ester (R)-17 that was subsequently converted to two skeletally different natural products, i.e., (-)-mersicarpine (8) with a [6.5.6.7] fused tetracyclic ring system and (-)-scholarisine G (9) with a [6.5.6.6.5] fused pentacyclic skeleton, respectively. The postcyclization diversification was realized by taking advantage of the facile conversion of (+)-melodinine E (6) to N-acyliminium ion 7, from which a hydroxy group was selectively introduced to the C6, C7, C10 and the central C21 position of diazafenestrane system, leading to (-)-leuconodine A (11), (+)-leuconodine F (12), (-)-scholarisine G (9), (-)-leuconodine C (13), and skeletally different (-)-leuconolam (5). Furthermore, an unprecedented non-natural oxabridged oxadiazafenestrane 68 was formed by oxidation of (+)-melodinine E (6). During the course of this study, a strong self-induced diastereomeric anisochronism (SIDA) phenomenon was observed for scholarisine G (9), leuconodines A (11) and C (13). X-ray structures of both the racemic and the enantiopure natural products 9, 11, and 13 were obtained. The different crystal packing of these two forms nicely explained the chemical shift differences observed in the (1)H NMR spectra of the racemic and the enantio-enriched compounds in an achiral environment.

An Enantioselective Total Synthesis of (-)-Isoschizogamine.

A concise enantioselective total synthesis of (-)-isoschizogamine, a complex bridged polycyclic monoterpene indole alkaloid, was accomplished. N-Alkylation of an enantio-enriched imine with an alkyl iodide afforded an iminium salt, which, upon heating by microwave irradiation in the presence of pivalic acid, was converted into the hexacyclic structure of natural product by a complex but ordered domino sequence. The one-pot process leading to the formation of one C-C bond and three C-N bonds created three rings and three contiguous stereogenic centers with complete control of both the relative and absolute stereochemistry.

Aqueous Titanium Trichloride Promoted Reductive Cyclization of o-Nitrostyrenes to Indoles: Development and Application to the Synthesis of Rizatriptan and Aspidospermidine.

Treatment of o-nitrostyrenes with aqueous TiCl3 solution at room temperature afforded indoles through a formal reductive C(sp(2) )-H amination process. A range of functions such as halides (Cl, Br), carbonyl (ester, carbamate), cyano, hydroxy, and amino groups were tolerated. From ß,ß-disubstituted o-nitrostyrenes, 2,3-disubstituted indoles were formed by a domino reduction/cyclization/migration process. Mild conditions, simple experimental procedure, ready accessibility of the starting materials and good to excellent yields characterize the present transformation. The methodology was used as a key step in a concise synthesis of rizatriptan and a formal total synthesis of aspidospermidine.

Unified strategy to monoterpene indole alkaloids: total syntheses of (±)-goniomitine, (±)-1,2-dehydroaspidospermidine, (±)-aspidospermidine, (±)-vincadifformine, and (±)-kopsihainanine A.

Total syntheses of (±)-goniomitine, (±)-1,2-dehydroaspidospermidine, (±)-aspidospermidine, (±)-vincadifformine, and (±)-kopsihainanine A were achieved featuring two common key steps: (1) a palladium-catalyzed decarboxylative vinylation that provides quick access to cyclopentene intermediates containing all of the carbons present in the natural products and (2) an integrated oxidation/reduction/cyclization (iORC) sequence for skeletal reorganization that converts the cyclopentenes to the pentacyclic structures of the natural products. By incorporation of a geometric constraint to iORC substrates, both the chemoselectivity (C7 vs N1 cyclization) and the stereoselectivity (trans- vs cis-fused ring system) of the cyclization process can be controlled.

Enantioselective total syntheses of leuconolam-leuconoxine-mersicarpine group monoterpene indole alkaloids.

A unified strategy allowing enantioselective total syntheses of (-)-mersicarpine, (-)-scholarisine G, (+)-melodinine E, (-)-leuconoxine, and (-)-leuconolam from a common cyclohexenone derivative was reported. The Suzuki-Miyaura reaction was used to couple two simple fragments incorporating the key elements for total synthesis, and unprecedented oxidation/reduction/cyclization processes were developed that converted the substituted cyclohexenone to either a mersicarpine or leuconoxine skeleton. In a reverse biomimetic synthesis fashion, (+)-melodinine E was converted to (-)-leuconolam under acidic conditions.

Biallelic Variants in Lanosterol Synthase (LSS) Cause Palmoplantar Keratoderma-Congenital Alopecia Syndrome Type 2.

Palmoplantar keratoderma-congenital alopecia syndrome type 2 is an autosomal recessive disorder with an unknown genetic basis. In this study, we identified biallelic variants in the LSS gene in two unrelated palmoplantar keratoderma-congenital alopecia syndrome type 2 cases (c.3G>A, p.Met1? and c.1025T>G, p.Ile342Ser in patient 1; c.1522G>T, p.Gly508Trp and c.428+42T>A in patient 2) presenting with additional clinical features, including early-onset cataracts, pseudoainhum, and agenesis of the corpus callosum. LSS encodes lanosterol synthase (LSS), which functions in the cholesterol biosynthesis pathway by converting (S)-2,3-oxidosqualene to lanosterol. The c.3G>A variant resulted in an alternative translation initiation at residue Met81, producing an N-terminal truncated protein (LSS-ΔN80), as shown by immunoblotting. The c.428+42T>A variant introduced a potential splicing site, leading to a premature stop codon. Ex vivo studies revealed downregulation of LSS in both patients. Remarkably decreased lanosterol levels were found in vitro in three LSS variants, LSS-ΔN80, p.Ile342Ser, and p.Gly508Trp, suggesting a loss of enzymatic activity. Transmission electron microscopy and immunofluorescence showed abnormal cornified envelope formation in the stratum corneum of the patients. Taken together, our findings indicate LSS as a causative gene for palmoplantar keratoderma-congenital alopecia syndrome type 2, which emphasizes the importance of the cholesterol synthesis pathway in human skin cornification.

Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.