Parallel evolution of methyltransferases leads to vobasine biosynthesis in Tabernaemontana elegans and Catharanthus roseus.

Monoterpenoid indole alkaloids (MIA) are one of the largest and most complex alkaloid class in nature, boasting many clinically significant drugs such as anticancer vinblastine and antiarrhythmic ajmaline. Many MIAs undergo nitrogen N-methylation, altering their reactivity and affinity to the biological targets through a straightforward reaction. Remarkably, all known MIA N-methyltransferases (NMT) originate from the neofunctionalization of ancestral γ-tocopherol C-methyltransferases (γTMTs), a phenomenon seemingly unique to the Apocynaceae family. In this study, we unveil and characterize a new γTMT-like enzyme from the plant Tabernaemontana elegans (toad tree): perivine Nß-methyltransferase (TePeNMT). TePeNMT and other homologs form a distinct clade in our phylogenetic study, setting them apart from other γTMTs and γTMT-like NMTs discovered to date. Enzyme kinetic experiments and enzyme homology modeling studies reveal the significant differences in enzyme active sites between TePeNMT and CrPeNMT, a previously characterized perivine Nß-methyltransferase from Catharanthus roseus (Madagascar periwinkle). Collectively, our findings suggest that parallel evolution of ancestral γTMTs may be responsible for the occurrence of perivine N-methylation in T. elegans and C. roseus.

Biosynthetic Strategies of Berberine Bridge Enzyme-like Flavoprotein Oxidases toward Structural Diversification in Natural Product Biosynthesis.

Berberine bridge enzyme-like oxidases are often involved in natural product biosynthesis and are seen as essential enzymes for the generation of intricate pharmacophores. These oxidases have the ability to transfer a hydride atom to the FAD cofactor, which enables complex substrate modifications and rearrangements including (intramolecular) cyclizations, carbon-carbon bond formations, and nucleophilic additions. Despite the diverse range of activities, the mechanistic details of these reactions often remain incompletely understood. In this Review, we delve into the complexity that BBE-like oxidases from bacteria, fungal, and plant origins exhibit by providing an overview of the shared catalytic features and emphasizing the different reactivities. We propose four generalized modes of action by which BBE-like oxidases enable the synthesis of natural products, ranging from the classic alcohol oxidation reactions to less common amine and amide oxidation reactions. Exploring the mechanisms utilized by nature to produce its vast array of natural products is a subject of considerable interest and can lead to the discovery of unique biochemical activities.

Deciphering the SAM- and metal-dependent mechanism of O-methyltransferases in cystargolide and belactosin biosynthesis: A structure-activity relationship study.

Cystargolides and belactosins are natural products with a distinct dipeptide structure and an electrophilic ß-lactone warhead. They are known to inhibit proteases such as the proteasome or caseinolytic protease P, highlighting their potential in treating cancers and neurodegenerative diseases. Recent genetic analyses have shown homology between the biosynthetic pathways of the two inhibitors. Here, we characterize the O-methyltransferases BelI and CysG, which catalyze the initial step of ß-lactone formation. Employing techniques such as crystallography, computational analysis, mutagenesis, and activity assays, we identified a His-His-Asp (HHD) motif in the active sites of the two enzymes, which is crucial for binding a catalytically active calcium ion. Our findings thus elucidate a conserved divalent metal-dependent mechanism in both biosynthetic pathways that distinguish BelI and CysG from previously characterized O-methyltransferases.

Isofunctional but Structurally Different Methyltransferases for Dithiolopyrrolone Diversification.

Dithiolopyrrolone (DTP) natural products are produced by several different bacteria and have potent antibacterial, antifungal and anticancer activities. While the amide of their DTP core can be methylated to fine-tune bioactivity, the enzyme responsible for the amide N-methylation has remained elusive in most taxa. Here, we identified the amide methyltransferase XrdM that is responsible for xenorhabdin (XRD) methylation in Xenorhabdus doucetiae but encoded outside of the XRD gene cluster. XrdM turned out to be isofunctional with the recently reported methyltransferase DtpM, that is involved in the biosynthesis of the DTP thiolutin, although its X-ray structure is unrelated to that of DtpM. To investigate the structural basis for ligand binding in both enzymes, we used X-ray crystallography, modeling, site-directed mutagenesis, and kinetic activity assays. Our study expands the limited knowledge of post-non-ribosomal peptide synthetase (NRPS) amide methylation in DTP biosynthesis and reveals an example of convergent evolution of two structurally completely different enzymes for the same reaction in different organisms.

Metabolic engineering of Saccharomyces cerevisiae for chelerythrine biosynthesis.

BACKGROUND: Chelerythrine is an important alkaloid used in agriculture and medicine. However, its structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from plants. Here, we reconstructed and optimized the complete biosynthesis pathway for chelerythrine from (S)-reticuline in Saccharomyces cerevisiae using genetic reprogramming. RESULTS: The first-generation strain Z4 capable of producing chelerythrine was obtained via heterologous expression of seven plant-derived enzymes (McoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, and PsCPR) in S. cerevisiae W303-1 A. When this strain was cultured in the synthetic complete (SC) medium supplemented with 100 µM of (S)-reticuline for 10 days, it produced up to 0.34 µg/L chelerythrine. Furthermore, efficient metabolic engineering was performed by integrating multiple-copy rate-limiting genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, INO2, and AtATR1), tailoring the heme and NADPH engineering, and engineering product trafficking by heterologous expression of MtABCG10 to enhance the metabolic flux of chelerythrine biosynthesis, leading to a nearly 900-fold increase in chelerythrine production. Combined with the cultivation process, chelerythrine was obtained at a titer of 12.61 mg per liter in a 0.5 L bioreactor, which is over 37,000-fold higher than that of the first-generation recombinant strain. CONCLUSIONS: This is the first heterologous reconstruction of the plant-derived pathway to produce chelerythrine in a yeast cell factory. Applying a combinatorial engineering strategy has significantly improved the chelerythrine yield in yeast and is a promising approach for synthesizing functional products using a microbial cell factory. This achievement underscores the potential of metabolic engineering and synthetic biology in revolutionizing natural product biosynthesis.

The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain.

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual ß-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for ß-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the ß-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized ß-lactam-forming biosynthetic gene clusters by genome mining.

Structural diversity of decalin forming Diels-Alderase.

The Diels-Alder (DA) reaction, specifically referring to the [4 + 2] cycloaddition reaction in pericyclic reactions, is a process that forms two carbon-carbon covalent bonds in a single step via an electron ring transition state. Among the secondary metabolites produced by microorganisms, numerous compounds are biosynthesized through DA reactions, most of which are enzymatic. Our research group has discovered an enzyme named Diels-Alderase (DAase) that catalyzes the DA reaction in filamentous fungi, and we have been investigating its catalytic mechanism. This review describes the reported microbial DAase enzymes, with a particular focus on those involved in the construction of the decalin ring.

Multinuclear non-heme iron dependent oxidative enzymes (MNIOs) involved in unusual peptide modifications.

Multinuclear non-heme iron dependent oxidative enzymes (MNIOs), formerly known as domain of unknown function 692 (DUF692), are involved in the post-translational modification of peptides during the biosynthesis of peptide-based natural products. These enzymes catalyze highly unusual and diverse chemical modifications. Several class-defining features of this large family (>14 000 members) are beginning to emerge. Structurally, the enzymes are characterized by a TIM-barrel fold and a set of conserved residues for a di- or tri-iron binding site. They use molecular oxygen to modify peptide substrates, often in a four-electron oxidation taking place at a cysteine residue. This review summarizes the current understanding of MNIOs. Four modifications are discussed in detail: oxazolone-thioamide formation, ß-carbon excision, hydantoin-macrocycle formation, and 5-thiooxazole formation. Briefly discussed are two other reactions that do not take place on Cys residues.

Harnessing regulatory networks in Actinobacteria for natural product discovery.

Microbes typically live in complex habitats where they need to rapidly adapt to continuously changing growth conditions. To do so, they produce an astonishing array of natural products with diverse structures and functions. Actinobacteria stand out for their prolific production of bioactive molecules, including antibiotics, anticancer agents, antifungals, and immunosuppressants. Attention has been directed especially towards the identification of the compounds they produce and the mining of the large diversity of biosynthetic gene clusters (BGCs) in their genomes. However, the current return on investment in random screening for bioactive compounds is low, while it is hard to predict which of the millions of BGCs should be prioritized. Moreover, many of the BGCs for yet undiscovered natural products are silent or cryptic under laboratory growth conditions. To identify ways to prioritize and activate these BGCs, knowledge regarding the way their expression is controlled is crucial. Intricate regulatory networks control global gene expression in Actinobacteria, governed by a staggering number of up to 1000 transcription factors per strain. This review highlights recent advances in experimental and computational methods for characterizing and predicting transcription factor binding sites and their applications to guide natural product discovery. We propose that regulation-guided genome mining approaches will open new avenues toward eliciting the expression of BGCs, as well as prioritizing subsets of BGCs for expression using synthetic biology approaches. ONE-SENTENCE SUMMARY: This review provides insights into advances in experimental and computational methods aimed at predicting transcription factor binding sites and their applications to guide natural product discovery.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Programming Dynamic Division of Labor Using Horizontal Gene Transfer.

The metabolic engineering of microbes has broad applications, including biomanufacturing, bioprocessing, and environmental remediation. The introduction of a complex, multistep pathway often imposes a substantial metabolic burden on the host cell, restraining the accumulation of productive biomass and limiting pathway efficiency. One strategy to alleviate metabolic burden is the division of labor (DOL) in which different subpopulations carry out different parts of the pathway and work together to convert a substrate into a final product. However, the maintenance of different engineered subpopulations is challenging due to competition and convoluted interstrain population dynamics. Through modeling, we show that dynamic division of labor (DDOL), which we define as the DOL between indiscrete populations capable of dynamic and reversible interchange, can overcome these limitations and enable the robust maintenance of burdensome, multistep pathways. We propose that DDOL can be mediated by horizontal gene transfer (HGT) and use plasmid genomics to uncover evidence that DDOL is a strategy utilized by natural microbial communities. Our work suggests that bioengineers can harness HGT to stabilize synthetic metabolic pathways in microbial communities, enabling the development of robust engineered systems for deployment in a variety of contexts.

Functional Plasticity of a Viral Terpene Synthase, OILTS, that Shows Non-Specific Metal Cofactor Binding and Metal-Dependent Biosynthesis.

OILTS is a viral class I terpene synthase found from the giant virus Orpheovirus IHUMI-LCC2. It exhibits a unique structure and demonstrates high plasticity to metal cofactors, allowing it to biosynthesize different cyclic terpene frameworks. Notably, while OILTS produces only (+)-germacrene D-4-ol with the most common cofactor, Mg2+, it also biosynthesizes a different cyclic terpene, (+)-cubebol, with Mn2+, Co2+, or Ni2+, presenting a rare instance of cofactor-dependent enzyme catalysis. This is the first report of (+)-cubebol biosynthesis, to our knowledge. In addition, OILTS can uptake Zn2+ as a cofactor, which is uncommon among ordinary terpene synthases. These findings suggest that OILTS's functional plasticity may benefit the virus in diverse host environments, highlighting potential evolutionary implications.

Unusual Enzymatic C-C Bond Formation and Cleavage Reactions during Natural Product Biosynthesis.

Natural products from plants and microorganisms provide a valuable reservoir of pharmaceutical compounds. C-C bond formation and cleavage are crucial events during natural product biosynthesis, playing pivotal roles in generating diverse and intricate chemical structures that are essential for biological functions. This review summarizes our recent findings regarding biosynthetic enzymes that catalyze unconventional C-C bond formation and cleavage reactions during natural product biosynthesis.

Evolution and diversification of carboxylesterase-like [4+2] cyclases in aspidosperma and iboga alkaloid biosynthesis.

Monoterpene indole alkaloids (MIAs) are a large and diverse class of plant natural products, and their biosynthetic construction has been a subject of intensive study for many years. The enzymatic basis for the production of aspidosperma and iboga alkaloids, which are produced exclusively by members of the Apocynaceae plant family, has recently been discovered. Three carboxylesterase (CXE)-like enzymes from Catharanthus roseus and Tabernanthe iboga catalyze regio- and enantiodivergent [4+2] cycloaddition reactions to generate the aspidosperma (tabersonine synthase, TS) and iboga (coronaridine synthase, CorS; catharanthine synthase, CS) scaffolds from a common biosynthetic intermediate. Here, we use a combined phylogenetic and biochemical approach to investigate the evolution and functional diversification of these cyclase enzymes. Through ancestral sequence reconstruction, we provide evidence for initial evolution of TS from an ancestral CXE followed by emergence of CorS in two separate lineages, leading in turn to CS exclusively in the Catharanthus genus. This progression from aspidosperma to iboga alkaloid biosynthesis is consistent with the chemotaxonomic distribution of these MIAs. We subsequently generate and test a panel of chimeras based on the ancestral cyclases to probe the molecular basis for differential cyclization activity. Finally, we show through partial heterologous reconstitution of tabersonine biosynthesis using non-pathway enzymes how aspidosperma alkaloids could have first appeared as "underground metabolites" via recruitment of promiscuous enzymes from common protein families. Our results provide insight into the evolution of biosynthetic enzymes and how new secondary metabolic pathways can emerge through small but important sequence changes following co-option of preexisting enzymatic functions.

Recent progress in the reprogramming of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described.

Three-membered ring formation catalyzed by α-ketoglutarate-dependent nonheme iron enzymes.

Epoxides, aziridines, and cyclopropanes are found in various medicinal natural products, including polyketides, terpenes, peptides, and alkaloids. Many classes of biosynthetic enzymes are involved in constructing these ring structures during their biosynthesis. This review summarizes our current knowledge regarding how α-ketoglutarate-dependent nonheme iron enzymes catalyze the formation of epoxides, aziridines, and cyclopropanes in nature, with a focus on enzyme mechanisms.

A dissected non-ribosomal peptide synthetase maintains activity.

Non-ribosomal peptide synthetases (NRPSs) generate chemically complex compounds and their modular architecture suggests that changing their domain organization can predictably alter their products. Ebony, a small three-domain NRPS, catalyzes the formation of ß-alanine containing amides from biogenic amines. To examine the necessity of interdomain interactions, we modeled and docked domains of Ebony to reveal potential interfaces between them. Testing the same domain combinations in vitro showed that 8 % of activity was preserved after Ebony was dissected into a di-domain and a detached C-terminal domain, suggesting that sufficient interaction was maintained after dissection. Our work creates a model to identify domain interfaces necessary for catalysis, an important step toward utilizing Ebony as a combinatorial engineering platform for novel amides.

Peptide mixed phosphonates for covalent complex formation with thioesterases in nonribosomal peptide synthetases.

Natural macrocyclic peptides derived from microorganisms are medicinal resources that are important for the development of new therapeutic agents. Most of these molecules are biosynthesized by a nonribosomal peptide synthetase (NRPS). The thioesterase (TE) domain in NRPS is responsible for the macrocyclization of mature linear peptide thioesters in a final biosynthetic step. NRPS-TEs can cyclize synthetic linear peptide analogs and can be utilized as biocatalysts for the preparation of natural product derivatives. Although the structures and enzymatic activities of TEs have been investigated, the substrate recognition and substrate-TE interaction during the macrocyclization step are still unknown. To understand the TE-mediated macrocyclization, here we report the development of a substrate-based analog with mixed phosphonate warheads, which can react irreversibly with the Ser residue at the active site of TE. We have demonstrated that the tyrocidine A linear peptide (TLP) with a p-nitrophenyl phosphonate (PNP) enables efficient complex formation with tyrocidine synthetase C (TycC)-TE containing tyrocidine synthetase.

TetR and OmpR family regulators in natural product biosynthesis and resistance.

This article provides a comprehensive review and sequence-structure analysis of transcription regulator (TR) families, TetR and OmpR/PhoB, involved in specialized secondary metabolite (SSM) biosynthesis and resistance. Transcription regulation is a fundamental process, playing a crucial role in orchestrating gene expression to confer a survival advantage in response to frequent environmental stress conditions. This process, coupled with signal sensing, enables bacteria to respond to a diverse range of intra and extracellular signals. Thus, major bacterial signaling systems use a receptor domain to sense chemical stimuli along with an output domain responsible for transcription regulation through DNA-binding. Sensory and output domains on a single polypeptide chain (one component system, OCS) allow response to stimuli by allostery, that is, DNA-binding affinity modulation upon signal presence/absence. On the other hand, two component systems (TCSs) allow cross-talk between the sensory and output domains as they are disjoint and transmit information by phosphorelay to mount a response. In both cases, however, TRs play a central role. Biosynthesis of SSMs, which includes antibiotics, is heavily regulated by TRs as it diverts the cell's resources towards the production of these expendable compounds, which also have clinical applications. These TRs have evolved to relay information across specific signals and target genes, thus providing a rich source of unique mechanisms to explore towards addressing the rapid escalation in antimicrobial resistance (AMR). Here, we focus on the TetR and OmpR family TRs, which belong to OCS and TCS, respectively. These TR families are well-known examples of regulators in secondary metabolism and are ubiquitous across different bacteria, as they also participate in a myriad of cellular processes apart from SSM biosynthesis and resistance. As a result, these families exhibit higher sequence divergence, which is also evident from our bioinformatic analysis of 158 389 and 77 437 sequences from TetR and OmpR family TRs, respectively. The analysis of both sequence and structure allowed us to identify novel motifs in addition to the known motifs responsible for TR function and its structural integrity. Understanding the diverse mechanisms employed by these TRs is essential for unraveling the biosynthesis of SSMs. This can also help exploit their regulatory role in biosynthesis for significant pharmaceutical, agricultural, and industrial applications.

Crystallographic and thermodynamic evidence of negative cooperativity of flavin and tryptophan binding in the flavin-dependent halogenases AbeH and BorH.

The flavin-dependent halogenase AbeH produces 5-chlorotryptophan in the biosynthetic pathway of the chlorinated bisindole alkaloid BE-54017. We report that in vitro, AbeH (assisted by the flavin reductase AbeF) can chlorinate and brominate tryptophan as well as other indole derivatives and substrates with phenyl and quinoline groups. We solved the X-ray crystal structures of AbeH alone and complexed with FAD, as well as crystal structures of the tryptophan-6-halogenase BorH alone, in complex with 6-chlorotryptophan, and in complex with FAD and tryptophan. Partitioning of FAD and tryptophan into different chains of BorH and failure to incorporate tryptophan into AbeH/FAD crystals suggested that flavin and tryptophan binding are negatively coupled in both proteins. ITC and fluorescence quenching experiments confirmed the ability of both AbeH and BorH to form binary complexes with FAD or tryptophan and the inability of tryptophan to bind to AbeH/FAD or BorH/FAD complexes. FAD could not bind to BorH/tryptophan complexes, but FAD appears to displace tryptophan from AbeH/tryptophan complexes in an endothermic entropically-driven process.