Leah B. Bushin

All Publications

• Growth-coupled microbial biosynthesis of the animal pigment xanthommatin NATURE BIOTECHNOLOGY Bushin, L. B., Alter, T. B., Alvan-Vargas, M. V. G., Durr, L., Olson, E. C., Avila, M. J., Volke, D. C., Puiggene, O., Kim, T., Deravi, L. F., Feist, A. M., Nikel, P. I., Moore, B. S. 2025

  Abstract

  Engineering heterologous natural product pathways in bacteria has achieved broad success but most approaches suffer from low initial production levels that require extensive, resource-heavy iterative strain optimization. Xanthommatin is a structurally complex, color-changing animal ommochrome with material and cosmetic applications, yet production in microbial cell factories has been difficult. Here, we introduce a growth-coupled biosynthetic strategy involving a feedback loop where an excised one-carbon (C1) moiety is used as a driver of bacterial growth, simultaneously boosting bioproduction of the target compound. This broadly applicable, plug-and-play strategy is illustrated by enabling xanthommatin biosynthesis in a 5,10-methylenetetrahydrofolate auxotroph of the platform soil bacterium Pseudomonas putida. In this design, formate released during xanthommatin production relieves the C1 deficiency, thereby effectively coupling bacterial growth to pigment synthesis. Adaptive laboratory evolution streamlined xanthommatin's gram-scale bioproduction from glucose, establishing C1 restoration as a general biosynthetic approach to accelerate the engineering of natural product biosynthesis in bacteria.

  View details for DOI 10.1038/s41587-025-02867-7

  View details for Web of Science ID 001606176200001

  View details for PubMedID 41184490

  View details for PubMedCentralID 9400054

• Expanding the Landscape of Noncanonical Amino Acids in RiPP Biosynthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Johnson, B. A., Clark, K. A., Bushin, L. B., Spolar, C. N., Seyedsayamdost, M. R. 2024; 146 (6): 3805-3815

  Abstract

  Advancements in DNA sequencing technologies and bioinformatics have enabled the discovery of new metabolic reactions from overlooked microbial species and metagenomic sequences. Using a bioinformatic co-occurrence strategy, we previously generated a network of ∼600 uncharacterized quorum-sensing-regulated biosynthetic gene clusters that code for ribosomally synthesized and post-translationally modified peptide (RiPP) natural products and are tailored by radical S-adenosylmethionine (RaS) enzymes in streptococci. The most complex of these is the GRC subfamily, named after a conserved motif in the precursor peptide and found exclusively in Streptococcus pneumoniae, the causative agent of bacterial pneumonia. In this study, using both in vivo and in vitro approaches, we have elucidated the modifications installed by the grc biosynthetic enzymes, including a ThiF-like adenylyltransferase/cyclase that generates a C-terminal Glu-to-Cys thiolactone macrocycle, and two RaS enzymes, which selectively epimerize the β-carbon of threonine and desaturate histidine to generate the first instances of l-allo-Thr and didehydrohistidine in RiPP biosynthesis. RaS-RiPPs that have been discovered thus far have stood out for their exotic macrocycles. The product of the grc cluster breaks this trend by generating two noncanonical residues rather than an unusual macrocycle in the peptide substrate. These modifications expand the landscape of nonproteinogenic amino acids in RiPP natural product biosynthesis and motivate downstream biocatalytic applications of the corresponding enzymes.

  View details for DOI 10.1021/jacs.3c10824

  View details for Web of Science ID 001163294800001

  View details for PubMedID 38316431

• Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs NATURE CHEMICAL BIOLOGY Bushin, L. B., Covington, B. C., Clark, K. A., Caruso, A., Seyedsayamdost, M. R. 2022; 18 (10): 1135-+

  Abstract

  Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic β-ether and a heterocyclic sp3-sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.

  View details for DOI 10.1038/s41589-022-01090-8

  View details for Web of Science ID 000839563200004

  View details for PubMedID 35953547

  View details for PubMedCentralID 6686864

• Discovery and Biosynthesis of Streptosactin, a Sactipeptide with an Alternative Topology Encoded by Commensal Bacteria in the Human Microbiome JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bushin, L. B., Covington, B. C., Rued, B. E., Federle, M. J., Seyedsayamdost, M. R. 2020; 142 (38): 16265-16275

  Abstract

  Mammalian microbiomes encode thousands of biosynthetic gene clusters (BGCs) and represent a new frontier in natural product research. We recently found an abundance of quorum sensing-regulated BGCs in mammalian microbiome streptococci that code for ribosomally synthesized and post-translationally modified peptides (RiPPs) and contain one or more radical S-adenosylmethionine (RaS) enzymes, a versatile superfamily known to catalyze some of the most unusual reactions in biology. In the current work, we target a widespread group of streptococcal RiPP BGCs and elucidate both the reaction carried out by its encoded RaS enzyme and identify its peptide natural product, which we name streptosactin. Streptosactin is the first sactipeptide identified from Streptococcus spp.; it contains two sequential four amino acid sactionine macrocycles, an unusual topology for this compound family. Bioactivity assays reveal potent but narrow-spectrum activity against the producing strain and its closest relatives that carry the same BGC, suggesting streptosactin may be a long-suspected fratricidal agent of Streptococcus thermophilus. Our results highlight mammalian streptococci as a rich source of unusual enzymatic chemistries and bioactive natural products.

  View details for DOI 10.1021/jacs.0c05546

  View details for Web of Science ID 000575684100021

  View details for PubMedID 32845143

• Total Synthesis and Stereochemical Assignment of Streptide JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Isley, N. A., Endo, Y., Wu, Z., Covington, B. C., Bushin, L. B., Seyedsayamdost, M. R., Boger, D. L. 2019; 141 (43): 17361-17369

  Abstract

  Streptide (1) is a peptide-derived macrocyclic natural product that has attracted considerable attention since its discovery in 2015. It contains an unprecedented post-translational modification that intramolecularly links the β-carbon (C3) of a residue 2 lysine with the C7 of a residue 6 tryptophan, thereby forming a 20-membered cyclic peptide. Herein, we report the first total synthesis of streptide that confirms the regiochemistry of the lysine-tryptophan cross-link and provides an unambiguous assignment of the stereochemistry (3R vs 3S) of the lysine-2 C3 center. Both the 3R and the originally assigned 3S lysine diastereomers were independently prepared by total synthesis, and it is the former, not the latter, that was found to correlate with the natural product. The approach enlists a powerful Pd(0)-mediated indole annulation for the key macrocyclization of the complex core peptide, utilizes an underdeveloped class of hypervalent iodine(III) aryl substrates in a palladium-catalyzed C-H activation/β-arylation reaction conducted on a lysine derivative, and provides access to material with which the role of streptide and related natural products may be examined.

  View details for DOI 10.1021/jacs.9b09067

  View details for Web of Science ID 000493866300042

  View details for PubMedID 31577142

  View details for PubMedCentralID PMC6821584

• Macrocyclization via an Arginine-Tyrosine Crosslink Broadens the Reaction Scope of Radical <i>S</i>-Adenosylmethionine Enzymes JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Caruso, A., Martinie, R. J., Bushin, L. B., Seyedsayamdost, M. R. 2019; 141 (42): 16610-16614

  Abstract

  Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an ascendant class of natural products with diverse structures and functions. Recently, we identified a wide array of RiPP gene clusters that are regulated by quorum sensing and encode one or more radical S-adenosylmethionine (RaS) enzymes, a diverse protein superfamily capable of catalyzing chemically difficult transformations. In this work, we characterize a novel reaction catalyzed by one such subfamily of RaS enzymes during RiPP biosynthesis: installation of a macrocyclic carbon-carbon bond that links the unactivated δ-carbon of an arginine side chain to the ortho-position of a tyrosine-phenol. Moreover, we show that this transformation is, unusually for RiPP biogenesis, largely insensitive to perturbations of the leader portion of the precursor peptide. This reaction expands the already impressive scope of RaS enzymes and contributes a unique macrocyclization motif to the growing body of RiPP architectures.

  View details for DOI 10.1021/jacs.9b09210

  View details for Web of Science ID 000492800500059

  View details for PubMedID 31596076

• Aliphatic Ether Bond Formation Expands the Scope of Radical SAM Enzymes in Natural Product Biosynthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Clark, K. A., Bushin, L. B., Seyedsayamdost, M. R. 2019; 141 (27): 10610-10615

  Abstract

  The biosynthetic pathways of microbial natural products provide a rich source of novel enzyme-catalyzed transformations. Using a new bioinformatic search strategy, we recently identified an abundance of gene clusters for ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain at least one radical S-adenosylmethionine (RaS) metalloenzyme and are regulated by quorum sensing. In the present study, we characterize a RaS enzyme from one such RiPP gene cluster and find that it installs an aliphatic ether cross-link at an unactivated carbon center, linking the oxygen of a Thr side chain to the α-carbon of a Gln residue. This reaction marks the first ether cross-link installed by a RaS enzyme. Additionally, it leads to a new heterocyclization motif and underlines the utility of our bioinformatics approach in finding new families of RiPP modifications.

  View details for DOI 10.1021/jacs.9b05151

  View details for Web of Science ID 000475533500008

  View details for PubMedID 31246011

• A genetics-free method for high-throughput discovery of cryptic microbial metabolites NATURE CHEMICAL BIOLOGY Xu, F., Wu, Y., Zhang, C., Davis, K. M., Moon, K., Bushin, L. B., Seyedsayamdost, M. R. 2019; 15 (2): 161-+

  Abstract

  Bacteria contain an immense untapped trove of novel secondary metabolites in the form of 'silent' biosynthetic gene clusters (BGCs). These can be identified bioinformatically but are not expressed under normal laboratory growth conditions. Methods to access their products would dramatically expand the pool of bioactive compounds. We report a universal high-throughput method for activating silent BGCs in diverse microorganisms. Our approach relies on elicitor screening to induce the secondary metabolome of a given strain and imaging mass spectrometry to visualize the resulting metabolomes in response to ~500 conditions. Because it does not require challenging genetic, cloning, or culturing procedures, this method can be used with both sequenced and unsequenced bacteria. We demonstrate the power of the approach by applying it to diverse bacteria and report the discovery of nine cryptic metabolites with potentially therapeutic bioactivities, including a new glycopeptide chemotype with potent inhibitory activity against a pathogenic virus.

  View details for DOI 10.1038/s41589-018-0193-2

  View details for Web of Science ID 000456188100015

  View details for PubMedID 30617293

  View details for PubMedCentralID PMC6339573

• Radical Approach to Enzymatic β-Thioether Bond Formation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Caruso, A., Bushin, L. B., Clark, K. A., Martinie, R. J., Seyedsayamdost, M. R. 2019; 141 (2): 990-997

  Abstract

  Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products that harbor diverse chemical functionalities, usually introduced via the action of a small number of tailoring enzymes. We have been interested in RiPP biosynthetic gene clusters that encode unusual metalloenzymes, as these may install as yet unknown alterations. Using a new bioinformatic search strategy, we recently identified an array of unexplored RiPP gene clusters that are quorum sensing-regulated and contain one or more uncharacterized radical S-adenosylmethionine (RaS) metalloenzymes. Herein, we investigate the reaction of one of these RaS enzymes and find that it installs an intramolecular β-thioether bond onto its substrate peptide by connecting a Cys-thiol group to the β-carbon of an upstream Asn residue. The enzyme responsible, NxxcB, accepts several amino acids in place of Asn and introduces unnatural β-thioether linkages at unactivated positions. This new transformation adds to the growing list of Nature's peptide macrocyclization strategies and expands the already impressive catalytic repertoire of the RaS enzyme superfamily.

  View details for DOI 10.1021/jacs.8b11060

  View details for Web of Science ID 000456350300039

  View details for PubMedID 30521328

• Charting an Unexplored Streptococcal Biosynthetic Landscape Reveals a Unique Peptide Cyclization Motif JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bushin, L. B., Clark, K. A., Pelczer, I., Seyedsayamdost, M. R. 2018; 140 (50): 17674-17684

  Abstract

  Peptide natural products are often used as signals or antibiotics and contain unusual structural modifications, thus providing opportunities for expanding our understanding of Nature's therapeutic and biosynthetic repertoires. Herein, we have investigated the under-explored biosynthetic potential of Streptococci, prevalent bacteria in mammalian microbiomes that include mutualistic, commensal, and pathogenic members. Using a new bioinformatic search strategy, in which we linked the versatile radical S-adenosylmethionine (RaS) enzyme superfamily to an emerging class of natural products in the context of quorum sensing control, we identified numerous, uncharted biosynthetic loci. Focusing on one such locus, we identified an unprecedented post-translational modification, consisting of a tetrahydro[5,6]benzindole cyclization motif in which four unactivated positions are linked by two C-C bonds in a regio- and stereospecific manner by a single RaS enzyme. Our results expand the scope of reactions that microbes have at their disposal in concocting complex ribosomal peptides.

  View details for DOI 10.1021/jacs.8b10266

  View details for Web of Science ID 000454383400049

  View details for PubMedID 30398325

• Guidelines for Determining the Structures of Radical SAM Enzyme-Catalyzed Modifications in the Biosynthesis of RiPP Natural Products RADICAL SAM ENZYMES Bushin, L. B., Seyedsayamdost, M. R. edited by Bandarian 2018; 606: 439-460

  Abstract

  Radical S-adenosylmethionine (RaS) enzymes catalyze some of the most fascinating transformations in Nature. With only ~100 of the >300,000 members studied to date, it is safe to assume that a plethora of new reactions and reaction mechanisms remain to be elucidated. It is by now relatively easy to spot RaS enzymes in microbial genomes. However, to determine the reactions that they carry out, detailed structural characterization of the product(s) is necessary, a process that still represents a significant roadblock in the study of RaS enzymes. We have recently combined natural products structural elucidation along with RaS enzymology to provide a proof of concept for how the confluence of these approaches can lead to the discovery of new natural products and RaS enzyme-mediated transformations. Herein, we provide guidelines for expressing, purifying, and reconstituting a subclass of RaS enzymes that contain a so-called SPASM domain, as well as characterizing the reactions that they catalyze using a combination of HR/MSn and NMR investigations. Application of these approaches will aid in expanding the chemical and biosynthetic repertoire of RaS enzymes in the future.

  View details for DOI 10.1016/bs.mie.2018.04.016

  View details for Web of Science ID 000452358500017

  View details for PubMedID 30097102

• Discovery of a Cryptic Antifungal Compound from <i>Streptomyces albus</i> J1074 Using High-Throughput Elicitor Screens JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Xu, F., Nazari, B., Moon, K., Bushin, L. B., Seyedsayamdost, M. R. 2017; 139 (27): 9203-9212

  Abstract

  An important unresolved issue in microbial secondary metabolite production is the abundance of biosynthetic gene clusters that are not expressed under typical laboratory growth conditions. These so-called silent or cryptic gene clusters are sources of new natural products, but how they are silenced, and how they may be rationally activated are areas of ongoing investigation. We recently devised a chemogenetic high-throughput screening approach ("HiTES") to discover small molecule elicitors of silent biosynthetic gene clusters. This method was successfully applied to a Gram-negative bacterium; it has yet to be implemented in the prolific antibiotic-producing streptomycetes. Herein we have developed a high-throughput transcriptional assay format in Streptomyces spp. by leveraging eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, we successfully used HiTES to activate a silent gene cluster in Streptomyces albus J1074. Our results revealed the cytotoxins etoposide and ivermectin as potent inducers, allowing us to isolate and structurally characterize 14 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under standard growth conditions. The successful application of HiTES will allow future interrogations of the biological regulation and chemical output of the countless silent gene clusters in Streptomyces spp.

  View details for DOI 10.1021/jacs.7b02716

  View details for Web of Science ID 000405642400024

  View details for PubMedID 28590725

  View details for PubMedCentralID PMC5617735

• Discovery of <i>scmR</i> as a global regulator of secondary metabolism and virulence in <i>Burkholderia thailandensis</i> E264 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mao, D., Bushin, L. B., Moon, K., Wu, Y., Seyedsayamdost, M. R. 2017; 114 (14): E2920-E2928

  Abstract

  Bacteria produce a diverse array of secondary metabolites that have been invaluable in the clinic and in research. These metabolites are synthesized by dedicated biosynthetic gene clusters (BGCs), which assemble architecturally complex molecules from simple building blocks. The majority of BGCs in a given bacterium are not expressed under normal laboratory growth conditions, and our understanding of how they are silenced is in its infancy. Here, we have addressed this question in the Gram-negative model bacterium Burkholderia thailandensis E264 using genetic, transcriptomic, metabolomic, and chemical approaches. We report that a previously unknown, quorum-sensing-controlled LysR-type transcriptional regulator, which we name ScmR (for secondary metabolite regulator), serves as a global gatekeeper of secondary metabolism and a repressor of numerous BGCs. Transcriptionally, we find that 13 of the 20 BGCs in B. thailandensis are significantly (threefold or more) up- or down-regulated in a scmR deletion mutant (ΔscmR) Metabolically, the ΔscmR strain displays a hyperactive phenotype relative to wild type and overproduces a number of compound families by 18- to 210-fold, including the silent virulence factor malleilactone. Accordingly, the ΔscmR mutant is hypervirulent both in vitro and in a Caenorhabditis elegans model in vivo. Aside from secondary metabolism, ScmR also represses biofilm formation and transcriptionally activates ATP synthesis and stress response. Collectively, our data suggest that ScmR is a pleiotropic regulator of secondary metabolism, virulence, biofilm formation, and other stationary phase processes. A model for how the interplay of ScmR with pathway-specific transcriptional regulators coordinately silences virulence factor production is proposed.

  View details for DOI 10.1073/pnas.1619529114

  View details for Web of Science ID 000398159000016

  View details for PubMedID 28320949

  View details for PubMedCentralID PMC5389298

• Acinetodin and Klebsidin, RNA Polymerase Targeting Lasso Peptides Produced by Human Isolates of <i>Acinetobacter gyllenbergii</i> and <i>Klebsiella pneumoniae</i> ACS CHEMICAL BIOLOGY Metelev, M., Arseniev, A., Bushin, L. B., Kuznedelov, K., Artamonova, T. O., Kondratenko, R., Khodorkovskii, M., Seyedsayamdost, M. R., Severinov, K. 2017; 12 (3): 814-824

  Abstract

  We report the bioinformatic prediction and structural validation of two lasso peptides, acinetodin and klebsidin, encoded by the genomes of several human-associated strains of Acinetobacter and Klebsiella. Computation of the three-dimensional structures of these peptides using NMR NOESY constraints verifies that they contain a lasso motif. Despite the lack of sequence similarity to each other or to microcin J25, a prototypical lasso peptide and transcription inhibitor from Escherichia coli, acinetodin and klebsidin also inhibit transcript elongation by the E. coli RNA polymerase by binding to a common site. Yet, unlike microcin J25, acinetodin and klebsidin are unable to permeate wild type E. coli cells and inhibit their growth. We show that the E. coli cells become sensitive to klebsidin when expressing the outer membrane receptor FhuA homologue from Klebsiella pneumoniae. It thus appears that specificity to a common target, the RNA polymerase secondary channel, can be attained by a surprisingly diverse set of primary sequences folded into a common threaded-lasso fold. In contrast, transport into cells containing sensitive targets appears to be much more specific and must be the major determinant of the narrow range of bioactivity of known lasso peptides.

  View details for DOI 10.1021/acschembio.6b01154

  View details for Web of Science ID 000397077700026

  View details for PubMedID 28106375

• Structure and biosynthesis of a macrocyclic peptide containing an unprecedented lysine-to-tryptophan crosslink NATURE CHEMISTRY Schramma, K. R., Bushin, L. B., Seyedsayamdost, M. R. 2015; 7 (5): 431-437

  Abstract

  Streptococcal bacteria use peptide signals as a means of intraspecies communication. These peptides can contain unusual post-translational modifications, providing opportunities for expanding our understanding of nature's chemical and biosynthetic repertoires. Here, we have combined tools from natural products discovery and mechanistic enzymology to elucidate the structure and biosynthesis of streptide, a streptococcal macrocyclic peptide. We show that streptide bears an unprecedented post-translational modification involving a covalent linkage between two unactivated carbons within the side chains of lysine and tryptophan. The biosynthesis of streptide was addressed by genetic and biochemical studies. The former implicated a new SPASM-domain-containing radical SAM enzyme StrB, while the latter revealed that StrB contains two [4Fe-4S] clusters and installs the unusual lysine-to-tryptophan crosslink in a single step. By intramolecularly stitching together the side chains of lysine and tryptophan, StrB provides a new route for biosynthesizing macrocyclic peptides.

  View details for DOI 10.1038/NCHEM.2237

  View details for Web of Science ID 000353347900017

  View details for PubMedID 25901822

  View details for PubMedCentralID PMC4443489