Functional Genome Mining Reveals a Class V Lanthipeptide Containing a d-Amino Acid Introduced by an F420H2-Dependent Reductase

Lantibiotics are a type of ribosomally synthesized and post-translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N-terminal (N,N)-dimethyl phenylalanine, C-terminal (2-aminovinyl)-3-methyl-cysteine, and d -Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F₄₂₀H₂-dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d -Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.     

Functional Genome Mining Reveals a Class V Lanthipeptide Containing a d‐Amino Acid Introduced by an F420H2‐Dependent Reductase

Lantibiotics are a type of ribosomally synthesized and post‐translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N‐terminal (N,N)‐dimethyl phenylalanine, C‐terminal (2‐aminovinyl)‐3‐methyl‐cysteine, and d ‐Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F₄₂₀H₂‐dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d ‐Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.     

The Biosynthesis of Norsesquiterpene Aculenes Requires Three Cytochrome P450 Enzymes to Catalyze a Stepwise Demethylation Process

Aculenes are a unique class of norsequiterpenes (C₁₄) that are produced by Aspergillus aculeatus. The nordaucane skeleton in aculenes A–D may be derived from an ent‐daucane precursor through demethylation, however, the enzymes involved remain unexplored. We identified the biosynthetic gene cluster and characterized the biosynthetic pathway based on gene inactivation, feeding experiments, and heterologous reconstitution in Saccharomyces cerevisiae and Aspergillus oryzae. We discovered that three cytochrome P450 monoxygenases are required to catalyze the stepwise demethylation process. AneF converts the 12‐methyl group into a carboxylic acid and AneD installs the 10‐hydroxy group for later tautomerization and stabilization. Finally, AneG installs an electron‐withdrawing carbonyl group at the C‐2 position, which triggers C‐12 decarboxylation to yield the nordaucane skeleton. Additionally, a terpene cyclase (AneC) was found that forms a new product (dauca‐4,7‐diene).     

Staurosporine and rebeccamycin aglycones are assembled by the oxidative action of StaP, StaC, and RebC on chromopyrrolic acid

In the biosynthesis of the antitumor indolocarbazoles rebeccamycin and staurosporine by streptomycetes, assembly of the aglycones involves a complex set of oxidative condensations. Overall formation of aglycones K252c and arcyriaflavin A from their biosynthetic precursor chromopyrrolic acid involves four- and eight-electron oxidations, respectively. This process is catalyzed by the remarkable enzyme StaP, with StaC and RebC acting to direct the level of oxidation in the newly formed five-membered ring. An aryl-aryl coupling reaction is integral to this transformation as well as oxidative decarboxylation of the dicarboxypyrrole moiety of chromopyrrolic acid. Herein we describe the heterologous expression of staP, staC, and rebC in Escherichia coli and the activity of the corresponding enzymes in constructing the two distinct six-ring scaffolds. StaP is a cytochrome P450 enzyme, requiring dioxygen, ferredoxin, flavodoxin NADP(+)-reductase, and NAD(P)H for activity. StaP on its own converts chromopyrrolic acid into three aglycone products, K252c, arcyriaflavin A, and 7-hydroxy-K252c; in the presence of StaC, K252c is the predominant product, while the presence of RebC directs formation of arcyriaflavin A. (18)O-Labeling studies indicate that the oxygen(s) of the pyrrolinone and maleimide functionalities of the aglycones formed are all derived from dioxygen. This work allowed for the in vitro reconstitution of the full biosynthetic pathway from l-tryptophan to the staurosporine and rebeccamycin aglycones, K252c and 1,11-dichloroarcyriaflavin A.     

An Unprecedented Cyclization Mechanism in the Biosynthesis of Carbazole Alkaloids in Streptomyces

Carquinostatin A (CQS), a potent neuroprotective substance, is a unique carbazole alkaloid with both an ortho‐quinone function and an isoprenoid moiety. We identified the entire gene cluster responsible for CQS biosynthesis in Streptomyces exfoliatus through heterologous production of CQS and gene deletion. Biochemical characterization of seven CQS biosynthetic gene products (CqsB1–7) established the total biosynthetic pathway of CQS. Reconstitution of CqsB1 and CqsB2 showed that the synthesis of the carbazole skeleton involves CqsB1‐catalyzed decarboxylative condensation of an α‐hydroxyl‐β‐keto acid intermediate with 3‐hydroxybutyryl‐ACP followed by CqsB2‐catalyzed oxidative cyclization. Based on crystal structures and mutagenesis‐based biochemical assays, a detailed mechanism for the unique deprotonation‐initiated cyclization catalyzed by CqsB2 is proposed. Finally, analysis of the substrate specificity of the biosynthetic enzymes led to the production of novel carbazoles.     

Putative Biosynthesis of Talarodioxadione & Talarooxime from Talaromyces stipitatus

Polyesters containing 2,4-dihydroxy-6-(2-hydroxypropyl)benzoate and 3-hydroxybutyrate moieties have been isolated from many fungal species. Talaromyces stipitatus was previously reported to produce a similar polyester, talapolyester G. The complete genome sequence and the development of bioinformatics tools have enabled the discovery of the biosynthetic potential of this microorganism. Here, a putative biosynthetic gene cluster (BGC) of the polyesters encoding a highly reducing polyketide synthase (HR-PKS) and nonreducing polyketide synthase (NR-PKS), a cytochrome P450 and a regulator, was identified. Although talapolyester G does not require an oxidative step for its biosynthesis, further investigation into the secondary metabolite production of T. stipitatus resulted in isolating two new metabolites called talarodioxadione and talarooxime, in addition to three known compounds, namely 6-hydroxymellein, 15G256α and transtorine that have never been reported from this organism. Interestingly, the biosynthesis of the cyclic polyester 15G256α requires hydroxylation of an inactive methyl group and thus could be a product of the identified gene cluster. The two compounds, talarooxime and transtorine, are probably the catabolic metabolites of tryptophan through the kynurenine pathway. Tryptophan metabolism exists in almost all organisms and has been of interest to many researchers. The biosynthesis of the new oxime is proposed to involve two subsequent N-hydroxylation of 2-aminoacetophenone.     

Biosynthesis of the Structurally Unique Polycyclopropanated Polyketide–Nucleoside Hybrid Jawsamycin (FR‐900848)

The biosynthetic gene cluster of antifungal agent jawsamycin (FR‐900848) has been identified by heterologous expression. A series of gene inactivations and in vitro and in vivo analysis of key enzymes in the biosynthetic pathway established their functions. A novel mechanism involving a radical S‐adenosyl methionine (SAM) cyclopropanase collaborating with an iterative polyketide synthase is proposed for the construction of the unique polycyclopropanated backbone. Our reconstitution system sets the stage for studying the catalytic mechanism of this intriguing contiguous cyclopropanation.     

A Monooxygenase from Boreostereum vibrans Catalyzes Oxidative Decarboxylation in a Divergent Vibralactone Biosynthesis Pathway

The oxidative decarboxylation of prenyl 4‐hydroxybenzoate to prenylhydroquinone has been frequently proposed for the biosynthesis of prenylated (hydro)quinone derivates (sometimes meroterpenoids), yet no corresponding genes or enzymes have so far been reported. A FAD‐binding monooxygenase (VibMO1) was identified that converts prenyl 4‐hydroxybenzoate into prenylhydroquinone and is likely involved in the biosynthesis of vibralactones and other meroterpenoids in the basidiomycete Boreostereum vibrans. Feeding of 3‐allyl‐4‐hydroxybenzylalcohol, an analogue of the vibralactone pathway intermediate 3‐prenyl‐4‐hydroxybenzylalcohol, generated 20 analogues with different scaffolds. This demonstrated divergent pathways to skeletally distinct compounds initiating from a single precursor, thus providing the first insight into a novel biosynthetic pathway for 3‐substituted γ‐butyrolactones from a shikimate origin.     

Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation‐initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two‐step P450‐catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.     

Understanding thioamitide biosynthesis using pathway engineering and untargeted metabolomics

Thiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very few thioamitide biosynthetic steps have been elucidated, even though the biosynthetic gene clusters (BGCs) of multiple thioamitides have been identified. We hypothesised that engineering the thiostreptamide S4 BGC in a heterologous host could provide insights into its biosynthesis when coupled with untargeted metabolomics and targeted mutations of the precursor peptide. Modified BGCs were constructed, and in-depth metabolomics enabled a detailed understanding of the biosynthetic pathway to thiostreptamide S4, including the identification of a protein critical for amino acid dehydration that has homology to HopA1, an effector protein used by a plant pathogen to aid infection. We use this biosynthetic understanding to bioinformatically identify diverse RiPP-like BGCs, paving the way for future RiPP discovery and engineering.

Heterologous expression, pathway mutations and detailed metabolomic analysis were used to deduce a model for the biosynthesis of thiostreptamide S4, which belongs the thioamitide family of antitumour RiPPs.     

Discovery of a Mosaic‐Like Biosynthetic Assembly Line with a Decarboxylative Off‐Loading Mechanism through a Combination of Genome Mining and Imaging

The biosynthetic gene cluster for the antiplasmodial natural product siphonazole was identified by using a combination of genome mining, imaging, and expression studies in the natural producer Herpetosiphon sp. B060. The siphonazole backbone is assembled from an unusual starter unit from the shikimate pathway that is extended by the action of polyketide synthases and non‐ribosomal peptide synthetases with unusual domain structures, including several split modules and a large number of duplicated domains and domains predicted to be inactive. Product release proceeds through decarboxylation and dehydration independent of the thioesterase SphJ and yields the diene terminus of siphonazole. High variation in terms of codon‐usage within the gene cluster, together with the dislocated domain organization, suggest a recent emergence in evolutionary terms.     

Mechanism of the Pd‐catalyzed Decarboxylative Allylation of α‐Imino Esters: Decarboxylation via Free Carboxylate Ion

The Pd‐catalyzed decarboxylative allylation of α‐(diphenylmethylene)imino esters ( 1 ) or allyl diphenylglycinate imines ( 2 ) is an efficient method to construct new C(sp³)? C(sp³) bonds. The detailed mechanism of this reaction was studied by theoretical calculations [ONIOM(B3LYP/LANL2DZ+p:PM6)] combined with experimental observations. The overall catalytic cycle was found to consist of three steps: oxidative addition, decarboxylation, and reductive allylation. The oxidative addition of 1 to [(dba)Pd(PPh₃)₂] (dba=dibenzylideneacetone) produces an allylpalladium cation and a carboxylate anion with a low activation barrier of +9.1 kcal mol^?1. The following rate‐determining decarboxylation proceeds via a solvent‐exposed α‐imino carboxylate anion rather than an O‐ligated allylpalladium carboxylate with an activation barrier of +22.7 kcal mol^?1. The 2‐azaallyl anion generated by this decarboxylation attacks the face of the allyl ligand opposite to the Pd center in an outer‐sphere process to produce major product 3 , with a lower activation barrier than that of the minor product 4 . A positive linear Hammett correlation [ρ=1.10 for the PPh₃ ligand] with the observed regioselectivity ( 3 versus 4 ) supports an outer‐sphere pathway for the allylation step. When Pd combined with the bis(diphenylphosphino)butane (dppb) ligand is employed as a catalyst, the decarboxylation still proceeds via the free carboxylate anion without direct assistance of the cationic Pd center. Consistent with experimental observations, electron‐withdrawing substituents on 2 were calculated to have lower activation barriers for decarboxylation and, thus, accelerate the overall reaction rates.     

Biosynthesis of the antitumor agent chartreusin involves the oxidative rearrangement of an anthracyclic polyketide

Chartreusin is a potent antitumor agent with a mixed polyketide-carbohydrate structure produced by Streptomyces chartreusis. Three type II polyketide synthase (PKS) gene clusters were identified from an S. chartreusis HKI-249 genomic cosmid library, one of which encodes chartreusin (cha) biosynthesis, as confirmed by heterologous expression of the entire cha gene cluster in Streptomyces albus. Molecular analysis of the approximately 37 kb locus and structure elucidation of a linear pathway intermediate from an engineered mutant reveal that the unusual bis-lactone aglycone chartarin is derived from an anthracycline-type polyketide. A revised biosynthetic model involving an oxidative rearrangement is presented.     

Synthetic Study of Carthamin: Biomimetic Synthesis of Carthamin Model via Peroxidase‐Catalyzed Oxidative Decarboxylation of Its Precursor Model

A chiral carthamin model (3S,3′S)‐1‐[5‐acetyl‐2,6‐diketo‐3‐C‐β‐d ‐glucopyranosylcyclohex‐4‐enylidene]‐1′‐[5′‐acetyl‐3′‐C‐β‐d ‐glucopyranosyl‐2′,3′,4′‐trihydroxy‐6′‐oxocyclohexa‐1′,4′‐dienyl]methane, in which two cinnamoyl groups were replaced by an acetyl group, was synthesized by the dimerization of (S)‐2‐acetyl‐4‐C‐(per‐O‐acetyl‐β‐d ‐glucopyranosyl)cyclohexadienone with glyoxylic acid, followed by peroxidase‐catalyzed oxidative decarboxylation and de‐O‐acetylation, or de‐O‐acetylation and peroxidase‐catalyzed oxidative decarboxylation. The corresponding total yields were 12.5% or 17.1% from 3‐C‐(per‐O‐acetyl‐β‐d ‐glucopyranosyl)phloroacetophenone, and the reaction pathway was identical to the biosynthetic pathway.     

Assessment and acceleration of binding energy calculations for protein–ligand complexes by the fragment molecular orbital method

In the field of drug discovery, it is important to accurately predict the binding affinities between target proteins and drug applicant molecules. Many of the computational methods available for evaluating binding affinities have adopted molecular mechanics‐based force fields, although they cannot fully describe protein–ligand interactions. A noteworthy computational method in development involves large‐scale electronic structure calculations. Fragment molecular orbital (FMO) method, which is one of such large‐scale calculation techniques, is applied in this study for calculating the binding energies between proteins and ligands. By testing the effects of specific FMO calculation conditions (including fragmentation size, basis sets, electron correlation, exchange‐correlation functionals, and solvation effects) on the binding energies of the FK506‐binding protein and 10 ligand complex molecule, we have found that the standard FMO calculation condition, FMO2‐MP2/6‐31G(d), is suitable for evaluating the protein–ligand interactions. The correlation coefficient between the binding energies calculated with this FMO calculation condition and experimental values is determined to be R = 0.77. Based on these results, we also propose a practical scheme for predicting binding affinities by combining the FMO method with the quantitative structure–activity relationship (QSAR) model. The results of this combined method can be directly compared with experimental binding affinities. The FMO and QSAR combined scheme shows a higher correlation with experimental data (R = 0.91). Furthermore, we propose an acceleration scheme for the binding energy calculations using a multilayer FMO method focusing on the protein–ligand interaction distance. Our acceleration scheme, which uses FMO2‐HF/STO‐3G:MP2/6‐31G(d) at R_int = 7.0 Å, reduces computational costs, while maintaining accuracy in the evaluation of binding energy. © 2015 Wiley Periodicals, Inc.     

A Unique Tryptophan C‐Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

Cyanobactins are a rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES‐88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES‐88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C‐3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan‐prenylated cyclic peptides.     

The Biosynthetic Gene Cluster of Mushroom-Derived Antrocin Encodes Two Dual-Functional Haloacid Dehalogenase-like Terpene Cyclases

(−)-Antrocin ( 1 ), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase-like terpene cyclase AncC that biosynthesizes the drimane-type sesquiterpene (+)-albicanol ( 2 ) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2 . Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)-trans-γ-monocyclofarnesol instead of 2 . Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane-type scaffold.     

Discovery of the Azaserine Biosynthetic Pathway Uncovers a Biological Route for α-Diazoester Production

Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.