Analysis of the Valgamicin Biosynthetic Pathway Reveals a General Mechanism for Cyclopropanol Formation across Diverse Natural Product Scaffolds.

Cyclopropanol rings are highly reactive and may function as molecular "warheads" that affect natural product bioactivity. Yet, knowledge on their biosynthesis is limited. Using gene cluster analyses, isotope labeling, and in vitro enzyme assays, we shed first light on the biosynthesis of the cyclopropanol-substituted amino acid cleonine, a residue in the antimicrobial depsipeptide valgamicin C and the cytotoxic glycopeptide cleomycin A2. We decipher the biosynthetic origin of valgamicin C and show that the cleonine cyclopropanol ring is derived from dimethylsulfoniopropionate (DMSP). Furthermore, we demonstrate that part of the biosynthesis is analogous to the formation of malleicyprol polyketides in pathogenic bacteria. By genome mining and metabolic profiling, we identify the potential to produce cyclopropanol rings in other bacterial species. Our results reveal a general mechanism for cyclopropyl alcohol biosynthesis across diverse natural products that may be harnessed for bioengineering and drug discovery.

Controlling Substrate- and Stereospecificity of Condensation Domains in Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are sophisticated molecular machines that biosynthesize peptide drugs. In attempts to generate new bioactive compounds, some parts of NRPSs have been successfully manipulated, but especially the influence of condensation (C-)domains on substrate specificity remains enigmatic and poorly controlled. To understand the influence of C-domains on substrate preference, we extensively evaluated the peptide formation of C-domain mutants in a bimodular NRPS system. Thus, we identified three key mutations that govern the preference for stereoconfiguration and side-chain identity. These mutations show similar effects in three different C-domains (GrsB1, TycB1, and SrfAC) when di- or pentapeptides are synthesized in vitro or in vivo. Strikingly, mutation E386L allows the stereopreference to be switched from d- to l-configured donor substrates. Our findings provide valuable insights into how cryptic specificity filters in C-domains can be re-engineered to clear roadblocks for NRPS engineering and enable the production of novel bioactive compounds.

Novel Biocatalysts from Specialized Metabolism.

Enzymes are increasingly recognized as valuable (bio)catalysts that complement existing synthetic methods. However, the range of biotransformations used in the laboratory is limited. Here we give an overview on the biosynthesis-inspired discovery of novel biocatalysts that address various synthetic challenges. Prominent examples from this dynamic field highlight remarkable enzymes for protecting-group-free amide formation and modification, control of pericyclic reactions, stereoselective hetero- and polycyclizations, atroposelective aryl couplings, site-selective C-H activations, introduction of ring strain, and N-N bond formation. We also explore unusual functions of cytochrome P450 monooxygenases, radical SAM-dependent enzymes, flavoproteins, and enzymes recruited from primary metabolism, which offer opportunities for synthetic biology, enzyme engineering, directed evolution, and catalyst design.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Biosynthetic incorporation of fluorinated amino acids into the nonribosomal peptide gramicidin S.

Fluorine is a key element in medicinal chemistry, as it can significantly enhance the pharmacological properties of drugs. In this study, we aimed to biosynthetically produce fluorinated analogues of the antimicrobial cyclic decapeptide gramicidin S (GS). However, our results show that the A-domain of the NRPS module GrsA rejects 4-fluorinated analogues of its native substrate Phe due to an interrupted T-shaped aromatic interaction in the binding pocket. We demonstrate that GrsA mutant W239S improves the incorporation of 4-fluorinated Phe into GS both in vitro and in vivo. Our findings provide new insights into the behavior of NRPSs towards fluorinated amino acids and strategies for the engineered biosynthesis of fluorinated peptides.

Biomimetic engineering of nonribosomal peptide synthesis.

Nonribosomal peptides (NRPs) have gained attention due to their diverse biological activities and potential applications in medicine and agriculture. The natural diversity of NRPs is a result of evolutionary processes that have occurred over millions of years. Recent studies have shed light on the mechanisms by which nonribosomal peptide synthetases (NRPSs) evolve, including gene duplication, recombination, and horizontal transfer. Mimicking natural evolution could be a useful strategy for engineering NRPSs to produce novel compounds with desired properties. Furthermore, the emergence of antibiotic-resistant bacteria has highlighted the urgent need for new drugs, and NRPs represent a promising avenue for drug discovery. This review discusses the engineering potential of NRPSs in light of their evolutionary history.

Directed Evolution of Piperazic Acid Incorporation by a Nonribosomal Peptide Synthetase.

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N-N bond. This success was achieved by UPLC-MS/MS-based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analogue. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

Macrophage-targeting oligopeptides from Mortierella alpina.

The realm of natural products of early diverging fungi such as Mortierella species is largely unexplored. Herein, the nonribosomal peptide synthetase (NRPS) MalA catalysing the biosynthesis of the surface-active biosurfactants, malpinins, has been identified and biochemically characterised. The investigation of the substrate specificity of respective adenylation (A) domains indicated a substrate-tolerant enzyme with an unusual, inactive C-terminal NRPS module. Specificity-based precursor-directed biosynthesis yielded 20 new congeners produced by a single enzyme. Moreover, MalA incorporates artificial, click-functionalised amino acids which allowed postbiosynthetic coupling to a fluorophore. The fluorescent malpinin conjugate penetrates mammalian cell membranes via an phagocytosis-mediated mechanism, suggesting Mortierella oligopeptides as carrier peptides for directed cell targeting. The current study demonstrates substrate-specificity testing as a powerful tool to identify flexible NRPS modules and highlights basal fungi as reservoir for chemically tractable compounds in pharmaceutical applications.

Proof-Reading Thioesterase Boosts Activity of Engineered Nonribosomal Peptide Synthetase.

Nonribosomal peptide synthetases (NRPSs) are a vast source of valuable natural products, and re-engineering them is an attractive path toward structurally diversified active compounds. NRPS engineering often requires heterologous expression, which is hindered by the enormous size of NRPS proteins. Protein splitting and docking domain insertion have been proposed as a strategy to overcome this limitation. Here, we have applied the splitting strategy to the gramicidin S NRPS: Despite better production of the split proteins, gramicidin S production almost ceased. However, the addition of type II thioesterase GrsT boosted production. GrsT is an enzyme encoded in the gramicidin S biosynthetic gene cluster that we have produced and characterized for this purpose. We attribute the activity enhancement to the removal of a stalled intermediate from the split NRPS that is formed due to misinitiation. These results highlight type II thioesterases as useful tools for NRPS engineering.

Pathogenic bacteria remodel central metabolic enzyme to build a cyclopropanol warhead.

Bacteria of the Burkholderia pseudomallei (BP) group pose a global health threat, causing the infectious diseases melioidosis, a common cause of pneumonia and sepsis, and glanders, a contagious zoonosis. A trait of BP bacteria is a conserved gene cluster coding for the biosynthesis of polyketides (malleicyprols) with a reactive cyclopropanol unit that is critical for virulence. Enzymes building this warhead represent ideal targets for antivirulence strategies but the biochemical basis of cyclopropanol formation is unknown. Here we describe the formation of the malleicyprol warhead. We show that BurG, an unusual NAD+-dependent member of the ketol-acid reductoisomerase family, constructs the strained cyclopropanol ring. Biochemical assays and a suite of eight crystal structures of native and mutated BurG with bound analogues and inhibitors provide snapshots of each step of the complex reaction mechanism, involving a concealed oxidoreduction and a C-S bond cleavage. Our findings illustrate a remarkable case of neofunctionalisation, where a biocatalyst from central metabolism has been evolutionarily repurposed for warhead production in pathogens.

Total Synthesis and Functional Evaluation of IORs, Sulfonolipid-based Inhibitors of Cell Differentiation in Salpingoeca rosetta.

The choanoflagellate Salpingoeca rosetta is an important model system to study the evolution of multicellularity. In this study we developed a new, modular, and scalable synthesis of sulfonolipid IOR-1A (six steps, 27 % overall yield), which acts as bacterial inhibitor of rosette formation in S. rosetta. The synthesis features a decarboxylative cross-coupling reaction of a sulfonic acid-containing tartaric acid derivative with alkyl zinc reagents. Synthesis of 15 modified IOR-1A derivatives, including fluorescent and photoaffinity-based probes, allowed quantification of IOR-1A, localization studies within S. rosetta cells, and evaluation of structure-activity relations. In a proof of concept study, an inhibitory bifunctional probe was employed in proteomic profiling studies, which allowed to deduce binding partners in bacteria and S. rosetta. These results showcase the power of synthetic chemistry to decipher the biochemical basis of cell differentiation processes within S. rosetta.

Engineering DNA-Templated Nonribosomal Peptide Synthesis.

Diffusive escape of intermediates limits the rate enhancement that nanocontainers or macromolecular scaffolds can provide for artificial biocatalytic cascades. Nonribosomal peptide synthetases (NRPSs) naturally form gigantic assembly lines and prevent escape by covalently tethering intermediates. Here, we have built DNA-templated NRPS (DT-NRPS) by adding zinc-finger tags to split NRPS modules. The zinc fingers direct the NRPS modules to 9-bp binding sites on a DNA strand, where they form a catalytically active enzyme cascade. Geometric constraints of the DT-NRPSs were investigated using the template DNA as a molecular ruler. Up to four DT-NRPS modules were assembled on DNA to synthesize peptides. DT-NRPSs outperform previously reported DNA-templated enzyme cascades in terms of DNA acceleration, which demonstrates that covalent intermediate channeling is possible along the DNA template. Attachment of assembly line enzymes to a DNA scaffold is a promising catalytic strategy for the sequence-controlled biosynthesis of nonribosomal peptides and other polymers.

Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina.

Fungi are traditionally considered a reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early-diverging fungi such as Mortierella Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC 32222. The molecular structures of malpicyclins were elucidated by high-resolution tandem mass spectrometry (HR-MS/MS), Marfey's method, and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative quantitative real-time PCR (qRT-PCR) expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPSs), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicated a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as-yet-unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as a novel and underrated resource of natural products.IMPORTANCE Fungal natural compounds are industrially produced, with application in antibiotic treatment, cancer medications, and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but reidentification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early-diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, designated malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much more closely related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella Both enzymes were biochemically characterized and are involved in as-yet-unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early-diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor.

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.

Emergence of a Negative Activation Heat Capacity during Evolution of a Designed Enzyme.

Temperature influences the reaction kinetics and evolvability of all enzymes. To understand how evolution shapes the thermodynamic drivers of catalysis, we optimized the modest activity of a computationally designed enzyme for an elementary proton-transfer reaction by nearly 4 orders of magnitude over 9 rounds of mutagenesis and screening. As theorized for primordial enzymes, the catalytic effects of the original design were almost entirely enthalpic in origin, as were the rate enhancements achieved by laboratory evolution. However, the large reductions in ΔH⧧ were partially offset by a decrease in TΔS⧧ and unexpectedly accompanied by a negative activation heat capacity, signaling strong adaptation to the operating temperature. These findings echo reports of temperature-dependent activation parameters for highly evolved natural enzymes and are relevant to explanations of enzymatic catalysis and adaptation to changing thermal environments.

Adenylation Domains in Nonribosomal Peptide Engineering.

Nonribosomal peptides are a prolific source of bioactive molecules biosynthesized on large, modular assembly line synthetases. Synthetic biologists seek to obtain tailored peptides with tuned or novel bioactivities by engineering modules and domains of these nonribosomal peptide synthetases. The activation step catalyzed by adenylation domains primarily selects which amino acids are incorporated into nonribosomal peptides. Here, we review experimental protocols for probing the adenylation reaction that are applicable in natural product discovery and engineering. Several alternatives to the established pyrophosphate exchange assay will be compared and potential pitfalls pointed out. Binding pocket mutagenesis of adenylation domains has been successfully conducted to adjust substrate preferences. Novel screening methods relying on yeast surface display, for instance, search a larger sequence space for improved mutants and thus allow more substantial changes in peptide structure.

Biocatalytic Strategies towards [4+2] Cycloadditions.

Long sought after [4+2] cyclases have sprouted up in numerous biosynthetic pathways in recent years, raising hopes for biocatalytic solutions to cycloaddition catalysis, an important problem in chemical synthesis. In a few cases, detailed pictures of the inner workings of these catalysts have emerged, but intense efforts to gain deeper understanding are underway by means of crystallography and computational modelling. This Minireview aims to shed light on the catalytic strategies that this highly diverse family of enzymes employs to accelerate and direct the course of [4+2] cycloadditions with reference to small-molecule catalysts and designer enzymes. These catalytic strategies include oxidative or reductive triggers and lid-like movements of enzyme domains. A precise understanding of natural cycloaddition catalysts will be instrumental for customizing them for various synthetic applications.

Structure elucidation of the syringafactin lipopeptides provides insight in the evolution of nonribosomal peptide synthetases.

Modular biosynthetic machineries such as polyketide synthases (PKSs) or nonribosomal peptide synthetases (NRPSs) give rise to a vast structural diversity of bioactive metabolites indispensable in the treatment of cancer or infectious diseases. Here, we provide evidence for different evolutionary processes leading to the diversification of modular NRPSs and thus, their respective products. Discovery of a novel lipo-octapeptide family from Pseudomonas, the virginiafactins, and detailed structure elucidation of closely related peptides, the cichofactins and syringafactins, allowed retracing recombinational diversification of the respective NRPS genes. Bioinformatics analyses allowed us to spot an evolutionary snapshot of these processes, where recombination occurred both within the same and between different biosynthetic gene clusters. Our systems feature a recent diversification process, which may represent a typical paradigm to variations in modular biosynthetic machineries.

HAMA: a multiplexed LC-MS/MS assay for specificity profiling of adenylate-forming enzymes.

Adenylation enzymes selecting substrates for ribosomal and nonribosomal protein and peptide biosynthesis have been popular targets of enzyme engineering. Previous standard assays for adenylation specificity have been cumbersome and failed to reflect the competition conditions inside a cell because they measure substrates one at a time. We have developed an adenylation assay based on hydroxamate quenching and LC-MS/MS detection of hydroxamate products testing dozens of competing amino acid substrates in parallel. Streamlined specificity profiling of adenylation enzymes will facilitate engineering and directed evolution of ribosomal and nonribosomal peptide synthesis.