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.

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.

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.

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.

Precision is essential for efficient catalysis in an evolved Kemp eliminase.

Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities, have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination--a well-studied model system for proton transfer from carbon--we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 10(8)-fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about the prospects of designing more sophisticated catalysts.

Inverted stereocontrol of iridoid synthase in snapdragon.

The natural product class of iridoids, found in various species of flowering plants, harbors astonishing chemical complexity. The discovery of iridoid biosynthetic genes in the medicinal plant Catharanthus roseus has provided insight into the biosynthetic origins of this class of natural product. However, not all iridoids share the exact five- to six-bicyclic ring scaffold of the Catharanthus iridoids. For instance, iridoids in the ornamental flower snapdragon (Antirrhinum majus, Plantaginaceae family) are derived from the C7 epimer of this scaffold. Here we have cloned and characterized the iridoid synthase enzyme from A. majus (AmISY), the enzyme that is responsible for converting 8-oxogeranial into the bicyclic iridoid scaffold in a two-step reduction-cyclization sequence. Chiral analysis of the reaction products reveals that AmISY reduces C7 to generate the opposite stereoconfiguration in comparison with the Catharanthus homologue CrISY. The catalytic activity of AmISY thus explains the biosynthesis of 7-epi-iridoids in Antirrhinum and related genera. However, although the stereoselectivity of the reduction step catalyzed by AmISY is clear, in both AmISY and CrISY, the cyclization step produces a diastereomeric mixture. Although the reduction of 8-oxogeranial is clearly enzymatically catalyzed, the cyclization step appears to be subject to less stringent enzyme control.

Structural determinants of reductive terpene cyclization in iridoid biosynthesis.

The carbon skeleton of ecologically and pharmacologically important iridoid monoterpenes is formed in a reductive cyclization reaction unrelated to canonical terpene cyclization. Here we report the crystal structure of the recently discovered iridoid cyclase (from Catharanthus roseus) bound to a mechanism-inspired inhibitor that illuminates substrate binding and catalytic function of the enzyme. Key features that distinguish iridoid synthase from its close homolog progesterone 5ß-reductase are highlighted.

Identification and Characterization of the Iridoid Synthase Involved in Oleuropein Biosynthesis in Olive (Olea europaea) Fruits.

The secoiridoids are the main class of specialized metabolites present in olive (Olea europaea L.) fruit. In particular, the secoiridoid oleuropein strongly influences olive oil quality because of its bitterness, which is a desirable trait. In addition, oleuropein possesses a wide range of pharmacological properties, including antioxidant, anti-inflammatory, and anti-cancer activities. In accordance, obtaining high oleuropein varieties is a main goal of molecular breeding programs. Here we use a transcriptomic approach to identify candidate genes belonging to the secoiridoid pathway in olive. From these candidates, we have functionally characterized the olive homologue of iridoid synthase (OeISY), an unusual terpene cyclase that couples an NAD (P)H-dependent 1,4-reduction step with a subsequent cyclization, and we provide evidence that OeISY likely generates the monoterpene scaffold of oleuropein in olive fruits. OeISY, the first pathway gene characterized for this type of secoiridoid, is a potential target for breeding programs in a high value secoiridoid-accumulating species.

Biosynthetic engineering of nonribosomal peptide synthetases.

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Reprogramming nonribosomal peptide synthetases for "clickable" amino acids.

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan-to-serine mutation in phenylalanine-specific NRPS adenylation domains enables the efficient activation of non-natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 10(5)-fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O-propargyl-L-tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non-natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides.

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.

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.

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.