Combinatorial Biosynthesis Research Articles

Intrinsic and Extrinsic Programming of Product Chain Length and Release Mode in Fungal Collaborating Iterative Polyketide Synthases.

Combinatorial biosynthesis with fungal polyketide synthases (PKSs) promises to produce unprecedented bioactive "unnatural" natural products (uNPs) for drug discovery. Genome mining of the dothideomycete Rhytidhysteron rufulum uncovered a collaborating highly reducing PKS (hrPKS)-nonreducing PKS (nrPKS) pair. These enzymes produce trace amounts of rare S-type benzenediol macrolactone congeners with a phenylacetate core in a heterologous host. However, subunit shuffling and domain swaps with voucher enzymes demonstrated that all PKS domains are highly productive. This contradiction led us to reveal novel programming layers exerted by the starter unit acyltransferase (SAT) and the thioesterase (TE) domains on the PKS system. First, macrocyclic vs linear product formation is dictated by the intrinsic biosynthetic program of the TE domain. Next, the chain length of the hrPKS product is strongly influenced in trans by the off-loading preferences of the nrPKS SAT domain. Last, TE domains are size-selective filters that facilitate or obstruct product formation from certain priming units. Thus, the intrinsic programs of the SAT and TE domains are both part of the extrinsic program of the hrPKS subunit and modulate the observable metaprogram of the whole PKS system. Reconstruction of SAT and TE phylogenies suggests that these domains travel different evolutionary trajectories, with the resulting divergence creating potential conflicts in the PKS metaprogram. Such conflicts often emerge in chimeric PKSs created by combinatorial biosynthesis, reducing biosynthetic efficiency or even incapacitating the system. Understanding the points of failure for such engineered biocatalysts is pivotal to advance the biosynthetic production of uNPs.

The antitumor antibiotic mithramycin: new advanced approaches in modification and production.

The aureolic acid-type polyketide mithramycin (MTM) has a remarkable cytotoxicity against a variety of human tumors and has been used for the treatment of several types of cancer, including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia, and Paget's disease. However, its clinical use is quite limited due to its toxicity. Recently, interest in MTM has been renewed after its identification as a top candidate for the inhibition of the aberrant fusion transcription factor EWS-FLI1, associated with malignant transformation and progression of Ewing sarcoma tumor family. The mechanism of MTM inhibition involves its reversible non-intercalative interaction with GC-rich DNA regions. As a result of this binding, MTM blocks binding of transcription factors (such as Sp1) to their GC-rich promoters and inhibits transcription of several proto-oncogenes and thus suppresses various types of cancer. Knowledge of the biosynthesis of MTM and its gene cluster has enabled genetic modifications of the gene cluster and combinatorial biosynthesis to produce new modified MTM molecules ("mithralogues") with improved efficacy and lower toxicity, which has also renewed interest in the clinical development of MTM. However, production yields of MTM and its analogues are low in the natural production strains. Recent developments in genetic engineering approaches have made it possible to increase MTM production through more rational strategies based on genetic manipulations and heterologous expression in optimized chassis. Recent construction of various genetically modified strains of Streptomyces lividans has shown their use for efficient heterologous production of various biologically active secondary metabolites including MTM. KEY POINTS: • Discovery a novel bifunctional glycosyl hydrolase from uncultured microorganism. • Heterologous production of MTM in engineered S. lividans strains is efficient.

Coevolution-based prediction of protein-protein interactions in polyketide biosynthetic assembly lines.

Polyketide synthases (PKSs) are enzymes that generate diverse molecules of great pharmaceutical importance, including a range of clinically used antimicrobials and antitumor agents. Many polyketides are synthesized by cis-AT modular PKSs, which are organized in assembly lines, in which multiple enzymes line up in a specific order. This order is defined by specific protein-protein interactions (PPIs). The unique modular structure and catalyzing mechanism of these assembly lines makes their products predictable and also spurred combinatorial biosynthesis studies to produce novel polyketides using synthetic biology. However, predicting the interactions of PKSs, and thereby inferring the order of their assembly line, is still challenging, especially for cases in which this order is not reflected by the ordering of the PKS-encoding genes in the genome. Here, we introduce PKSpop, which uses a coevolution-based PPI algorithm to infer protein order in PKS assembly lines. Our method accurately predicts protein orders (93% accuracy). Additionally, we identify new residue pairs that are key in determining interaction specificity, and show that coevolution of N- and C-terminal docking domains of PKSs is significantly more predictive for PPIs than coevolution between ketosynthase and acyl carrier protein domains. The code is available on http://www.bif.wur.nl/ (under 'Software'). Supplementary data are available at Bioinformatics online.

Rational Design of Flavonoid Production Routes Using Combinatorial and Precursor-Directed Biosynthesis.

Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multienzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bioretrosynthetic approaches, leading to the design of alternative and more convenient routes to produce relevant molecules. In this perspective, plant-derived polyketides are extremely adaptable to those synthetic biological applications. Here, we present a combination of an in vitro CoA ligase activity assay coupled with a bacterial multigene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. When the vast knowledge from protein databases is exploited, the herein presented procedure can be easily repeated with additional plant-derived polyketides. Lastly, we report an efficient in vivo expression system that can be further exploited to heterologously express pathways not necessarily related to plant polyketide synthases.

An aurora of natural products-based drug discovery is coming

Natural products (NPs), a nature's reservoir possessing enormous structural and functional diversity far beyond the current ability of chemical synthesis, are now proving themselves as most wonderful gifts from mother nature for human beings. Many of them have been used successfully as medicines, as well as the most important sources of drug leads, food additives, and many industry relevant products for millennia. Most notably, more than half of the antibiotics and anti-cancer drugs currently in use are, or derived from, natural products. However, the speed and outputs of NP-based drug discovery has been slowing down dramatically after the fruitful harvest of the “low-hanging fruit” during the golden age of 1950s-1960s. With recent scientific advances combining metabolic sciences and technology, multi-omics, big data, combinatorial biosynthesis, synthetic biology, genome editing technology (such as CRISPR), artificial intelligence (AI), and 3D printing, the “high-hanging fruit” is becoming more and more accessible with reduced costs. We are now more and more confident that a new age of natural products discovery is dawning.

Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin

BackgroundMithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear.ResultsExpression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps.ConclusionsWe established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7–C12), followed by formation of the second and third rings (C5–C14; C3–C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1–C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation.

Production of the bioactive plant-derived triterpenoid morolic acid in engineered Saccharomyces cerevisiae.

Morolic acid is a plant-derived triterpenoid that possesses pharmacological properties such as cytotoxicity, as well as anti-HIV, anti-HSV, anti-inflammatory, and antidiabetic effects. The significant therapeutic properties of morolic acid are desirable in the context of pharmacological and drug development research, but the low accessibility of morolic acid from natural resources limits its applications. In the present study, we developed a microbial system for the production of morolic acid. Using a combinatorial biosynthesis approach, a novel production pathway was constructed in Saccharomycescerevisiae by coexpressing BfOSC2 (germanicol synthase) from Bauhinia forficata and CYP716A49 (triterpene C-28 oxidase) from Beta vulgaris. Moreover, we reconstructed the cellular galactose regulatory network by introducing a chimeric transcriptional activator (fusion of Gal4dbd.ER.VP16) to overdrive the genes under the control of the galactose promoter. We also overexpressed truncated HMG1, encoding feedback-inhibition-resistant form of 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 and sterol-regulating transcription factor upc2-1, to increase the isoprenoid precursors in the mevalonate pathway. Using this yeast system, we achieved morolic acid production up to 20.7 ± 1.8 mg/L in batch culture. To our knowledge, this is the highest morolic acid titer reported from a heterologous host, indicating a promising approach for obtaining rare natural triterpenoids.

Discovery of Cryptic Largimycins in Streptomyces Reveals Novel Biosynthetic Avenues Enriching the Structural Diversity of the Leinamycin Family.

Largimycins are hybrid nonribosomal peptide-polyketides that constitute a new group of metabolites in the leinamycin family of natural products displaying unique structural features such as containing an oxazole instead of a thiazole ring or being oxime ester macrocycles, unprecedented in nature, rather than macrolactams. Their discovery in Streptomyces argillaceus and Streptomyces canus has relied on the activation of two homologous silent gene clusters by overexpressing a transcriptional activator and cultivating in specific media. The proposed biosynthesis of largimycins includes the key action of the oxidoreductase LrgO, responsible for the formation of the oxime group involved in macrocyclization, and two putative cryptic biosynthetic steps consisting of chlorination of l-Thr by the NRPS loading module and incorporation of an olefinic exomethylene group by LrgJ PKS. The discovery of largimycins uncovers novel biosynthetic avenues employed in nature to enrich the structural diversity of leinamycins and provides tools for combinatorial biosynthesis.

Substrate Recognition by the Class II Lanthipeptide Synthetase HalM2.

Class II lanthipeptides belong to a diverse group of natural products known as ribosomally synthesized and post-translationally modified peptides (RiPPs). Most RiPP precursor peptides contain an N-terminal recognition sequence known as the leader peptide, which is typically recognized by biosynthetic enzymes that catalyze modifications on the C-terminal core peptide. For class II lanthipeptides, these are carried out by a bifunctional lanthipeptide synthetase (LanM) that catalyzes dehydration and cyclization reactions on peptidic substrates to generate thioether-containing, macrocyclic molecules. Some lanthipeptide synthetases are extraordinarily substrate tolerant, making them promising candidates for biotechnological applications such as combinatorial biosynthesis and cyclic peptide library construction. In this study, we characterized the mode of leader peptide recognition by HalM2, the lanthipeptide synthetase responsible for the production of the antimicrobial peptide haloduracin β. Using NMR spectroscopic techniques, in vitro binding assays, and enzyme activity assays, we identified substrate residues that are important for binding to HalM2 and for post-translational modification of the peptide substrates. Additionally, we provide evidence of the binding site on the enzyme using binding assays with truncated enzyme variants, hydrogen-deuterium exchange mass spectrometry, and photoaffinity labeling. Understanding the mechanism by which lanthipeptide synthetases recognize their substrate will facilitate their use in biotechnology, as well as further our general understanding of how RiPP enzymes recognize their substrates.

Synthetic biology based construction of biological activity-related library of fungal decalin-containing diterpenoid pyrones

A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host, Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.

Expanding the Diversity of Plant Monoterpenoid Indole Alkaloids Employing Human Cytochrome P450 3A4.

Human drug‐metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R,20R)‐epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.

Constructing Combinatorial Synthases Using Acyl Carrier Protein Chimeras

Synthases encoded by biosynthetic gene clusters in microorganisms are capable of producing structurally complex molecules of diverse function. The ability to mix‐and‐match enzymes from different synthases to create “unnatural natural products” could enable the environmentally‐responsible construction of new pharmaceutically relevant molecules. Acyl carrier proteins (ACPs) are central hubs within biosynthetic pathways as they mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural product. Past combinatorial biosynthesis efforts have failed because of incompatible ACP‐enzyme interactions. To overcome this obstacle, chimeric ACPs capable of cross‐talk between different biosynthetic pathways were designed to serve as a lynchpin in functional hybrid synthases. Chimeric ACPs were constructed by combining features of the actinorhodin polyketide synthase ACP (ACT ACP) and of E. coli fatty acid synthase ACP (AcpP). Given the premise that AcpP interacts with its cognate ketosynthase, FabF, whereas ACT ACP does not, the ability of chimeric ACPs to form a mechanistically‐relevant crosslink with FabF was evaluated. A colorimetric assay was used to assess ACP interactions and revealed the role of Helix II of AcpP in driving ACP‐FabF binding. Results suggest that the residues guiding ACP‐FabF compatibility may reside in Helix II. Future directions will include using site directed mutagenesis to investigate the specific residues responsible for ACP‐FabF recognition.Support or Funding InformationNational Institutes of Health (R15GM120704 to L.K.C)National Science Foundation CAREER Award (CHE‐1652424 to L.K.C.)Cottrell Scholars Award (24350 to L.K.C.)

CanE, an Iron/2-Oxoglutarate-Dependent Lasso Peptide Hydroxylase from Streptomyces canus.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that feature a unique lariat-knot topology. Canucin A, a post-translationally hydroxylated lasso peptide, was recently discovered via activation of its otherwise silent biosynthetic gene cluster in Streptomyces canus. The biosynthesis of canucin A, notably the introduction of a hydroxyl group at the β-carbon of the terminal aspartate residue, is the topic of the current report. We combine genetic and biochemical experiments to show that an iron/2-oxoglutarate-dependent enzyme, CanE, installs the hydroxyl group onto the precursor peptide in vivo and in vitro. Moreover, we show that hydroxylation occurs prior to macrocyclization and that the RiPP recognition element (RRE), encoded within the gene cluster to facilitate the initial proteolytic reaction, also increases the yield of hydroxylation, hinting at a dual role for the RRE. Our results have implications for the combinatorial biosynthesis of lasso peptides.

Biosynthesis of Amino Acid Derived α-Pyrones by an NRPS-NRPKS Hybrid Megasynthetase in Fungi.

A nonribosomal peptide synthetase (NRPS)-nonreducing polyketide synthase (NRPKS) hybrid enzyme (AnATPKS) from Aspergillus niger was shown to produce amino acid derived α-pyrone natural products (pyrophen and campyrone B). Biochemical characterization of the NRPS module in vitro reveals that the adenylation domain is promiscuous toward a variety of substituted phenylalanine analogues. Using precursor feeding and heterologous expression of AnATPKS and an associated O-methyltransferase (AnOMT), we were able to access a library of substituted pyrophen analogues. Our study paves the way for future combinatorial biosynthesis of diverse α-pyrone natural products using NRPS-NRPKS hybrids.

Revised Structure of Anthelvencin A and Characterization of the Anthelvencin Biosynthetic Gene Cluster.

Anthelvencins A and B are pyrrolamide metabolites produced by Streptomyces venezuelae ATCC 14583 and 14585. Isolated in 1965, they were reported to exhibit anthelmintic and moderate antibacterial activities. In this study, we revise the structure of anthelvencin A and identify a third anthelvencin metabolite, bearing two N-methylated pyrrole groups, which we named anthelvencin C. We sequenced the genome of S. venezuelae ATCC 14583 and identified a gene cluster predicted to direct the biosynthesis of anthelvencins. Functional analysis of this gene cluster confirmed its involvement in anthelvencin biosynthesis and allowed us to propose a biosynthetic pathway for anthelvencins. In addition to a nonribosomal peptide synthetase (NRPS), the assembly of anthelvencins involves an enzyme from the ATP-grasp ligase family, Ant23. We propose that Ant23 uses a PCP-loaded 4-aminopyrrole-2-carboxylate as substrate. As observed for the biosynthesis of the other pyrrolamides congocidine (produced by Streptomyces ambofaciens ATCC 25877) and distamycin (produced by Streptomyces netropsis DSM 40846), the NRPS assembling anthelvencins is composed of stand-alone domains only. Such NRPSs, sometimes called type II NRPSs, are less studied than the classical multimodular NRPSs. Yet, they constitute an interesting model to study protein-protein interactions in NRPSs and are good candidates for combinatorial biosynthesis approaches.

Engineered Biosynthesis of 5/5/6 Type Polycyclic Tetramate Macrolactams in an Ikarugamycin (5/6/5 Type)-Producing Chassis.

Combinatorial biosynthesis of 5/5/6 type polycyclic tetramate macrolactams (PoTeMs) was achieved in an engineered ikarugamycin (5/6/5 type) producer by introducing a set of 5/5/6 type PoTeM biosynthetic genes from Streptomyces griseus. This study supports the idea that PoTeMs share a common polyene tetramate precursor generated by the hybrid polyketide synthase/nonribosomal peptide synthetase enzymes. The X-ray crystal structure of pactamide G (7) sets an example for resolving the absolute configuration of 5/5/6 type PoTeMs.

New Sipanmycin Analogues Generated by Combinatorial Biosynthesis and Mutasynthesis Approaches Relying on the Substrate Flexibility of Key Enzymes in the Biosynthetic Pathway.

The appearance of new infectious diseases, the increase in multidrug-resistant bacteria, and the need for more effective chemotherapeutic agents have oriented the interests of researchers toward the search for metabolites with novel or improved bioactivities. Sipanmycins are disaccharyl glycosylated macrolactams that exert antibiotic and cytotoxic activities. By applying combinatorial biosynthesis and mutasynthesis approaches, we have generated eight new members of the sipanmycin family. The introduction of plasmids harboring genes responsible for the biosynthesis of several deoxysugars into sipanmycin-producing Streptomyces sp. strain CS149 led to the production of six derivatives with altered glycosylation patterns. After structural elucidation of these new metabolites, we conclude that some of these sugars are the result of the combination of the enzymatic machinery encoded by the introduced plasmids and the native enzymes of the d-sipanose biosynthetic pathway of the wild-type CS149 strain. In addition, two analogues of the parental compounds with a modified polyketide backbone were generated by a mutasynthesis approach, feeding cultures of a mutant strain defective in sipanmycin biosynthesis with 3-aminopentanoic acid. The generation of new sipanmycin analogues shown in this work relied on the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, particularly the glycosyltransferase pair SipS9/SipS14 and enzymes SipL3, SipL1, SipL7, and SipL2, which are involved in the incorporation of the polyketide synthase starting unit.IMPORTANCE Combinatorial biosynthesis has proved its usefulness in generating derivatives of already known compounds with novel or improved pharmacological properties. Sipanmycins are a family of glycosylated macrolactams produced by Streptomyces sp. strain CS149, whose antiproliferative activity is dependent on the sugar moieties attached to the aglycone. In this work, we report the generation of several sipanmycin analogues with different deoxysugars, showing the high degree of flexibility exerted by the glycosyltransferase machinery with respect to the recognition of diverse nucleotide-activated sugars. In addition, modifications in the macrolactam ring were introduced by mutasynthesis approaches, indicating that the enzymes involved in incorporating the starter unit have a moderate ability to introduce different types of β-amino acids. In conclusion, we have proved the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, especially the glycosyltransferases, which can be exploited in future experiments.

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.

Heterologous and Engineered Biosynthesis of Nematocidal Polyketide-Nonribosomal Peptide Hybrid Macrolactone from Extreme Thermophilic Fungi.

Fungal polyketide-nonribosomal peptide (PK-NRP) hybrid macrolactones are a growing family of natural products with biomedical and agricultural activities. One of the most important families is the thermolides, which are produced by extreme thermophilic fungi and exhibit strong nematocidal activity. We show here that the genes ThmABCE from Talaromyces thermophilus NRRL 2155 are critical for thermolide synthesis. Two separate single-module hrPKS (ThmA) and NRPS (ThmB) enzymes collaborate to synthesize the core macrolactone backbone (6 or 7), and the NRPS ThmB-CT domain catalyzes the key macrocyclization step in PK-NRP intermediate release via ester bond formation, representing a novel function of fungal NRPS C domains. We also show that heterologous and engineered expression of the Thm genes in the type strains of Aspergillus nidulans and Escherichia coli not only dramatically enhances the yields of thermolides but also affords different esterified analogues, such as butyryl- (thermolides J and K, 15 and 16), hexanoyl-, and octanyl- derivatives or mixed thermolides. Thermolides L and M (18 and 19), discovered via genome mining-based combinatorial biosynthesis, represent the first l-phenylalanine-based thermolides. Our work shows a unique biosynthetic mechanism of PK-NRP hybrid macrolactones from extremophiles, which led to the discovery of novel compounds and furthers our biosynthetic knowledge.

Expanded toolbox for directing the biosynthesis of macrocyclic peptides in bacterial cells.

The macrocyclization of recombinant polypeptides by means of genetically encodable non-canonical amino acids has recently provided an attractive strategy for the screening and discovery of macrocyclic peptide inhibitors of protein-protein interactions. Here, we report the development of an expanded suite of electrophilic unnatural amino acids (eUAAs) useful for directing the biosynthesis of genetically encoded thioether-bridged macrocyclic peptides in bacterial cells (E. coli). These reagents are shown to provide efficient access to a broad range of macrocyclic peptide scaffolds spanning from 2 to 20 amino acid residues, with the different eUAAs offering complementary reactivity profiles toward mediating short- vs. long-range macrocyclizations. Swapping of the eUAA cyclization module in a cyclopeptide inhibitor of streptavidin and Keap1 led to compounds with markedly distinct binding affinity toward the respective target proteins, highlighting the effectiveness of this strategy toward tuning the structural and functional properties of bioactive macrocyclic peptides. The peptide cyclization strategies reported here expand opportunities for the combinatorial biosynthesis of natural product-like peptide macrocycles in bacterial cells or in combination with display platforms toward the discovery of selective agents capable of targeting proteins and protein-mediated interactions.