A mass spectrometry-guided genome mining approach for natural product peptidogenomics.

Peptide natural products show broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce here natural product peptidogenomics (NPP), a new MS-guided genome-mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo tandem MS (MS(n)) structures to genomics-based structures following biosynthetic logic. In this study, we show that NPP enabled the rapid characterization of over ten chemically diverse ribosomal and nonribosomal peptide natural products of previously unidentified composition from Streptomycete bacteria as a proof of concept to begin automating the genome-mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which are from well-characterized model Streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the \u3e20 distinct compound classes is also reviewed, and commonalities are discussed

Combinatorial biosynthesis for the generation of new-to-nature peptide antimicrobials

Natural peptide products are a valuable source of important therapeutic agents, including antibiotics, antivirals and crop protection agents. Aided by an increased understanding of structure-activity relationships of these complex molecules and the biosynthetic machineries that produce them, it has become possible to re-engineer complete machineries and biosynthetic pathways to create novel products with improved pharmacological properties or modified structures to combat antimicrobial resistance. In this review, we will address the progress that has been made using non-ribosomally produced peptides and ribosomally synthesized and post-translationally modified peptides as scaffolds for designed biosynthetic pathways or combinatorial synthesis for the creation of novel peptide antimicrobials

Discovery and insight into the unique tailoring of the paeninodin lasso peptide from paenibacillus dendritiformis C454

Lasso peptides, such as microcin J25, BI-32169, lariatin and capistruin, are a structurally unique and pharmacologically relevant class of RiPPs (ribosomally synthesized and posttranslationally modified peptides) natural products. Compared with other intensively modified RiPPs, such as lantibiotics, lasso peptides only have a unique knotted topology in which the tail of the peptide is threaded through an N-terminal macrolactam ring and trapped by steric hindrance of bulky side chains stabilizing the entropically disfavored lasso structure. Except for this unusual knot structure, further posttranslational modifications on lasso peptides are very rare. Besides, lasso peptides have so far only been isolated from Proteo- and Actinobacterial sources. In this thesis, the lasso gene cluster from the Firmicute P. dendritiformis was investigated. Paeninodin, a new lasso peptide with an unusual phosphorylation at the side chain of the last serine was discovered by expression of this cluster in a heterologous host. The Paeninodin lasso peptide was isolated from a culture pellet. Mass spectrometric, carboxypeptidase Y assays and IM-MS studies proved paeninodin to be a new representative of lasso peptides. Morever, the biosynthetic pathway of modified lasso peptide was delineated through in vivo and in vitro studies. The kinase turned out to be a novel lasso peptide precursor kinase with wide substrate specificity. These results provide a way for the generation of novel lasso peptide analogs and, thereby, would facilitate lasso peptide engineering in the future

Advancing a systems cell-free metabolic engineering approach to natural product synthesis and discovery

Next generation DNA sequencing has led to an accumulation of a putative biosynthetic gene clusters for many natural product classes of interest. In vivo extraction and heterologous expression do not have sufficient throughput to validate predicted enzyme functions and inform future annotations. Further, engineering the production of new natural products is laborious and limited by the trade-offs between cell growth and product synthesis. Conversely, cell-free platforms, particularly those capable of cell-free protein synthesis (CFPS), facilitate rapid screening of enzyme function and prototyping of metabolic pathways. However, the protein content and metabolic activity of many cell-free systems are poorly defined, increasing variability between lysates and impeding systematic engineering. Here, the strength of untargeted peptidomics as an enabling tool for the engineering of cell-free systems is established based upon its ability to measure both global protein abundances and newly synthesized peptides. Synthesis of peptide natural products was found to be more robust in purified enzyme CFPS systems compared to crude lysates; however, non-specific peptide degradation, detected through peptidomics, remains a concern. Crude cell-free systems were determined be better suited to small molecule production, due to the extensive metabolic networks they were found to possess. Perturbations of these networks, carried out through changes to growth media, were observed through shotgun proteomics and informed engineering of phenol biosynthesis in a crude Escherichia coli lysate. Implementing shotgun proteomics as an analytical tool for cell-free systems will increase reproducibility and further the development of a platform for high-throughput functional genomics and metabolic engineering

Biomolecular NMR studies of the structure and biosynthesis of RiPP natural products

Natural products have been a rich source of new chemical matter with clinical applications. These compounds are broadly defined as small molecules generated by organisms from multiple kingdoms including plants, fungi, and prokaryotes. The outsized impact natural products have had on modern medicine has inspired ongoing efforts to find and characterize new natural products. The vast increase in sequenced genomes has allowed natural product discovery to move from a blind screening approach to a bioinformatics guided approach. Screening of extracts for bioactivity is not only laborious, but also leads to the rediscovery of known compounds. With a genome-mining approach, bioinformatic analysis of natural product gene clusters empowers researchers to identify producer strains and novel products. This eliminates the rediscovery problem and reduces the throughput necessary for novel natural product isolation. Within the large family of natural products, the Ribosomally-produced and Posttranslationally modified Peptides (RiPPs) are a particularly intriguing class. These compounds begin as a genetically encoded precursor, which is translated into a short (~50) residue peptide. This precursor peptide is then modified by genetically co-located enzymes to generate a mature natural product. The diverse, enzymatically-derived modifications to RiPP precursors include both sidechain and backbone modifications and generate a large and chemically diverse class of natural products. RiPPs are a potentially fruitful source of new chemical matter for clinical application for several reasons. First, the fact that RiPP precursors are genetically encoded means that there is a direct link between genetic sequence and chemical structure. Because of this, bioinformatic analysis allows relatively easy prediction of final chemical structure, and opens the possibility for analog generation through relatively simple genetic modifications. Secondly, recent advances in genome sequencing have greatly increased the known space of organismal genomes. As far as RiPPs are concerned, this offers plentiful opportunity for genome mining to identify novel RiPP biosynthetic gene clusters with potentially novel bioactivities. In addition to leveraging bioinformatics to discover novel RiPPs, this class of natural product has an increasingly diverse range of bioactivities. The wealth of information regarding known mechanisms of action of RiPPs serves as further justification for the discovery and characterization of novel RiPPs. In Chapter 1, I discuss the current state of understanding of the mechanisms of action of several classes of RiPP. This introductory chapter is an excerpt from a comprehensive review of the mechanisms of action of all known RiPPs. In Chapter 2, we report the bioinformatics-guided discovery and biosynthetic reconstitution of the lasso peptide fusilassin. This biosynthetic gene cluster was shown to possess exceptional plasticity, and was used to generate a number of non-native lasso peptides, including lasso peptides from separate pathways. The enzymes in this pathway also exhibited the greatest in vitro stability of known lasso peptides and served as a platform for a number of additional efforts aimed at characterizing interactions of biosynthetic enzymes and generating lasso peptide analog libraries. In Chapter3, we report the NMR-based characterization of protein-protein interactions governing the biosynthesis of the lasso peptide fusilassin. We accomplished the full backbone assignment of the FusE RiPP recognition element and utilized chemical shift perturbations to characterize the precursor peptide-FusE interactions. Solvent paramagnetic relaxation enhancements were used to characterize the interactions between FusE and the leader peptidase FusB. In Chapter 4, we described the discovery of two novel thioether-containing RiPPs discovered through genome mining. The sactipeptide huazacin was characterized by HRMS/MS and NMR spectroscopy and was shown to have bioactivity against Clostridia sp. Utilizing sequence homology and HRMS/MS, the natural product freyrasin was hypothesized to contain unprecedented alpha-to-beta carbon thioether linkages. This novel structure was confirmed by the complete assignment of freyrasin and TOCSY and NOESY-based experimental characterization of this novel post-translational modification. Based on the NMR structure elucidation, this natural product served as the first fully characterized member of a novel class of RiPPs termed the ranthipeptides. In Chapter 5 we utilize reactivity-based screening to characterize the post-translational modification of the citrulassin lasso peptides which contain a non-proteinogenic citrulline residue. Comparative genomic analyses and complementation experiments identified the arginine deiminase responsible for the installation of this functional groups. Solution NMR analysis generated a 3D solution NMR ensemble structure for this peptide. In Chapter 6 we report the discovery of two novel thiopeptide RiPPs. Genome-mining identified a minimal thiopeptide biosynthetic gene cluster with two predicted precursor peptides. These compounds were produced synthetically and chemoenzymatically and fully characterized by HRMS/MS and NMR. The structure elucidation of these compounds led to the reclassification of the thiopeptides as pyritides. In Chapter 7 genome mining approaches were used to identify and isolate a novel graspetide termed thatisin A. Results from HPLC purifications demonstrated a thermallydependent atropisomerization. Temperature-controlled NMR structure elucidation accomplished the structure elucidation of thatisin A and demonstrated the presence of a cis-proline residue located within a constrained macrocycle. Computational and MS-based methanolysis experiments further confirmed our proline isomerization conformational exchange hypothesis

Doctor of Philosophy

dissertationCyanobactins are peptide natural products that fall under the broad class of the ribosomally synthesized and posttranslationally modified peptides (RiPPs). Since they are synthesized by the ribosome, the biosynthesis of these peptides is genetically encoded. A precursor peptide gene carries the primary amino acid sequence of the natural product. The precursor peptide is surrounded by other genes, which encode posttranslational enzymes that decorate the primary sequence with elaborate structural motifs. Due to the genetically encoded origins of the cyanobactins, simple manipulations at the peptide sequence level are tolerable and lead to the creation of a diversity of natural products. The roots to this tolerance lie in the innate extreme broad-substrate nature of the posttranslational enzymes. Here, we explore the biochemical basis of this promiscuity and in vitro methodologies to create structurally elaborate peptidic motifs. In addition, the cyanobactin biosynthetic machinery is a rich source of enzymes capable of performing a wide array of intriguing chemistry and here we probe into some of these mechanisms. Put together, the broad-substrate nature coupled with the unique enzymology of the cyanobactin biosynthetic machinery provides a toolkit for the creation of designer peptide motifs. This work holds promise in the field of peptide-based drug discovery

Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed

Elucidation and control of substrate recognition during RiPP biosynthesis

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a rapidly growing class of natural products. RiPP precursor peptides can undergo extensive enzymatic tailoring to yield structurally and functionally diverse products. Cyclodehydratases are a type of RiPP modifying enzyme that catalyze phosphorylation of the peptide backbone and subsequent nucleophilic attack by the sidechains of Cys, Ser, or Thr to form azoline heterocycless (or azoles upon oxidation). The catalytic unit of the cyclodehydratase is a YcaO-family protein which is often accompanied by a partner protein from the E1-like superfamily (depending on the of type cyclodehydratase). Although primarily known for azoline formation, recent work suggests that YcaO proteins can use different nucleophiles and partner proteins to generate thioamide, macroamidine, and possibly other peptide posttranslational modifications. In Chapter 1, I comprehensively review the biosynthetic gene clusters (BGCs), natural products, functions, mechanisms, and applications of YcaO proteins and outline future directions for this protein superfamily. In Chapter 2, I report my investigations into the substrate recognition of canonical cyclodehydratases. Recent work suggested that unrelated RiPP modifying enzymes contain structurally similar precursor peptide binding domains. Using profile hidden Markov model comparisons, I discovered related and previously unrecognized peptide binding domains in proteins spanning the majority of known prokaryotic RiPP classes. This conserved domain was designated the RiPP precursor peptide recognition element (RRE). Through binding studies, I verified the RRE's role for three distinct RiPP classes: linear azole-containing peptides, thiopeptides, and lasso peptides. Because numerous RiPP biosynthetic enzymes act on peptide substrates, these findings have powerful predictive value as to which protein(s) drive substrate binding, thereby laying a foundation for further characterization of RiPP biosynthetic pathways and the rational engineering of new peptide binding activities. In Chapter 3, I use knowledge gained from precursor peptide binding studies to engineer a peptide that can be recognized and modified by two biosynthetic enzymes from different pathways. Combining enzymes from multiple pathways is an attractive approach for producing molecules with desired structural features, but this strategy thus far has been hampered by limited substrate tolerance of enzymes from unrelated pathways. Because RiPP biosynthetic enzymes modify their substrates by binding motifs located usually in the N-terminal leader region of precursor peptides, RiPP biosynthetic systems are highly amenable to the engineering of new compounds. I exploit this by designing chimeric leader peptides that can be bound and processed by multiple enzymes from unrelated RiPP pathways. Using this broadly applicable strategy, a cyclodehydratase was combined with enzymes from the sactipeptide and lanthipeptide RiPP classes to create new-to-nature hybrid RiPPs. These hybrids provide insight into biosynthetic timing and enzyme compatibility and establish a general platform for the engineering additional hybrid RiPPs

Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature

Covering: 1988 to 2012 This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed