Novel types of RiPP-modifying enzymes.

Novel discoveries in natural product biosynthesis reveal hidden bioactive compounds and expand our knowledge in enzymology. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly growing class of natural products featuring diverse non-canonical amino acids introduced by maturation enzymes as a class-defining characteristic. Underexplored RiPP sources, such as the human microbiome, the oceans, uncultured microorganisms, and plants are rich hunting grounds for novel enzymology. Unusual α- and ß-amino acids, peptide cleavages, lipidations, diverse macrocyclizations, and other features expand the range of chemical groups that are installed in RiPPs by often promiscuous enzymes. This review highlights the search for novelty in RiPP enzymology in the past two years, with respect to the discovery of new biochemical modifications but also towards novel applications.

Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

Direct Detection of the α-Carbon Radical Intermediate Formed by OspD: Mechanistic Insights into Radical S-Adenosyl-l-methionine Peptide Epimerization.

OspD is a radical S-adenosyl-l-methionine (SAM) peptide epimerase that converts an isoleucine (Ile) and valine (Val) of the OspA substrate to d-amino acids during biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP) natural product landornamide A. OspD is proposed to carry out this reaction via α-carbon (Cα) H-atom abstraction to form a peptidyl Cα radical that is stereospecifically quenched by hydrogen atom transfer (HAT) from a conserved cysteine (Cys). Here we use site-directed mutagenesis, freeze-quench trapping, isotopic labeling, and electron paramagnetic resonance (EPR) spectroscopy to provide new insights into the OspD catalytic mechanism including the direct observation of the substrate peptide Cα radical intermediate. The putative quenching Cys334 was changed to serine to generate an OspD C334S variant impaired in HAT quenching. The reaction of reduced OspD C334S with SAM and OspA freeze-quenched at 15 s exhibits a doublet EPR signal characteristic of a Cα radical coupled to a single ß-H. Using isotopologues of OspA deuterated at either Ile or Val, or both Ile and Val, reveals that the initial Cα radical intermediate forms exclusively on the Ile of OspA. Time-dependent freeze quench coupled with EPR spectroscopy provided evidence for loss of the Ile Cα radical concomitant with gain of a Val Cα radical, directly demonstrating the N-to-C directionality of epimerization by OspD. These results provide direct evidence for the aforementioned OspD-catalyzed peptide epimerization mechanism via a central Cα radical intermediate during RiPP maturation of OspA, a mechanism that may extend to other proteusin peptide epimerases.

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.

Site-specific bioorthogonal protein labelling by tetrazine ligation using endogenous ß-amino acid dienophiles.

The tetrazine ligation is an inverse electron-demand Diels-Alder reaction widely used for bioorthogonal modifications due to its versatility, site specificity and fast reaction kinetics. A major limitation has been the incorporation of dienophiles in biomolecules and organisms, which relies on externally added reagents. Available methods require the incorporation of tetrazine-reactive groups by enzyme-mediated ligations or unnatural amino acid incorporation. Here we report a tetrazine ligation strategy, termed TyrEx (tyramine excision) cycloaddition, permitting autonomous dienophile generation in bacteria. It utilizes a unique aminopyruvate unit introduced by post-translational protein splicing at a short tag. Tetrazine conjugation occurs rapidly with a rate constant of 0.625 (15) M-1 s-1 and was applied to produce a radiolabel chelator-modified Her2-binding Affibody and intracellular, fluorescently labelled cell division protein FtsZ. We anticipate the labelling strategy to be useful for intracellular studies of proteins, as a stable conjugation method for protein therapeutics, as well as other applications.

Characterization of an Unusual α-Oxoamine Synthase Off-Loading Domain from a Cyanobacterial Type I Fatty Acid Synthase.

Type I fatty acid synthases (FASs) are known from higher eukaryotes and fungi. We report the discovery of FasT, a rare type I FAS from the cyanobacterium Chlorogloea sp. CCALA695. FasT possesses an unusual off-loading domain, which was heterologously expressed in E. coli and found to act as an α-oxoamine synthase (AOS) in vitro. Similar to serine palmitoyltransferases from sphingolipid biosynthesis, the AOS off-loading domain catalyzes a decarboxylative Claisen condensation between l-serine and a fatty acyl thioester. While the AOS domain was strictly specific for l-serine, thioesters with saturated fatty acyl chains of six carbon atoms and longer were tolerated, with the highest activity observed for stearoyl-coenzyme A (C18 ). Our findings suggest a novel route to α-amino ketones via the direct condensation of iteratively produced long-chain fatty acids with l-serine by a FAS with a cis-acting AOS off-loading domain.

Modular Oxime Formation by a trans-AT Polyketide Synthase.

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.

Distribution and diversity of 'Tectomicrobia', a deep-branching uncultivated bacterial lineage harboring rich producers of bioactive metabolites.

Genomic and functional analyses of bacterial sponge symbionts belonging to the uncultivated candidate genus 'Entotheonella' has revealed them as the prolific producers of bioactive compounds previously identified from their invertebrate hosts. These studies also suggested 'Entotheonella' as the first members of a new candidate phylum, 'Tectomicrobia'. Here we analyzed the phylogenetic structure and environmental distribution of this as-yet sparsely populated phylum-like lineage. The data show that 'Entotheonella' and other 'Tectomicrobia' are not restricted to marine habitats but widely distributed among terrestrial locations. The inferred phylogenetic trees suggest several intra-phylum lineages with diverse lifestyles. Of these, the previously described 'Entotheonella' lineage can be more accurately divided into at least three different candidate genera with the terrestrial 'Candidatus Prasianella', the largely terrestrial 'Candidatus Allonella', the 'Candidatus Thalassonella' comprising sponge-associated members, and the more widely distributed 'Candidatus Entotheonella'. Genomic characterization of 'Thalassonella' members from a range of sponge hosts did not suggest a role as providers of natural products, despite high genomic similarity to 'Entotheonella' regarding primary metabolism and implied lifestyle. In contrast, the analysis revealed a correlation between the revised 'Entotheonella' 16S rRNA gene phylogeny and a specific association with sponges and their natural products. This feature might serve as a discovery method to accelerate the identification of new chemically rich 'Entotheonella' variants, and led to the identification of the first 'Entotheonella' symbiont in a non-tetractinellid sponge, Psammocinia sp., indicating a wide host distribution of 'Entotheonella'-based chemical symbiosis.

Heterocomplex structure of a polyketide synthase component involved in modular backbone halogenation.

Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid biosynthesis. The cytotoxic oocydin-type polyketides contain a vinyl chloride moiety introduced during polyketide chain elongation. Required for modular polyketide backbone halogenation are a non-heme iron and ɑ-ketoglutarate-dependent halogenase OocP and OocQ lacking characterized homologs. This work provides structural insights into these unusual PKS components and their interactions via a high-resolution X-ray crystallography structure of the heterocomplex. By mapping the protein-protein interactions and comparison with structures of similar halogenases, we illustrate the potential of this heterodimer complex as a replacement for the conserved homodimeric structure of homologous enzymes. The OocPQ protein pair has thus evolved as a means of stabilizing the halogenase and facilitating chemical transformations with great synthetic utility.

Structural and Biochemical Insights into Post-Translational Arginine-to-Ornithine Peptide Modifications by an Atypical Arginase.

Landornamide A is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product with antiviral activity. Its biosynthetic gene cluster encodes─among other maturases─the peptide arginase OspR, which converts arginine to ornithine units in an unusual post-translational modification. Peptide arginases are a recently discovered RiPP maturase family with few characterized representatives. They show little sequence similarity to conventional arginases, a well-characterized enzyme family catalyzing the hydrolysis of free arginine to ornithine and urea. Peptide arginases are highly promiscuous and accept a variety of substrate sequences. The molecular basis for binding the large peptide substrate and for the high promiscuity of peptide arginases remains unclear. Here, we report the first crystal structure of a peptide arginase at a resolution of 2.6 Å. The three-dimensional structure reveals common features and differences between conventional arginases and the peptide arginase: the binuclear metal cluster and the active-site environment strongly resemble each other, while the quaternary structures diverge. Kinetic analyses of OspR with various substrates provide new insights into the order of biosynthetic reactions during the post-translational maturation of landornamide A. These results provide the basis for pathway engineering to generate derivatives of landornamide A and for the general application of peptide arginases as biosynthetic tools for peptide engineering.

Emulating nonribosomal peptides with ribosomal biosynthetic strategies.

Peptide natural products are important lead structures for human drugs and many nonribosomal peptides possess antibiotic activity. This makes them interesting targets for engineering approaches to generate peptide analogues with, for example, increased bioactivities. Nonribosomal peptides are produced by huge mega-enzyme complexes in an assembly-line like manner, and hence, these biosynthetic pathways are challenging to engineer. In the past decade, more and more structural features thought to be unique to nonribosomal peptides were found in ribosomally synthesised and posttranslationally modified peptides as well. These streamlined ribosomal pathways with modifying enzymes that are often promiscuous and with gene-encoded precursor proteins that can be modified easily, offer several advantages to produce designer peptides. This review aims to provide an overview of recent progress in this emerging research area by comparing structural features common to both nonribosomal and ribosomally synthesised and posttranslationally modified peptides in the first part and highlighting synthetic biology strategies for emulating nonribosomal peptides by ribosomal pathway engineering in the second part.

Chemotaxis and autoinducer-2 signalling mediate colonization and contribute to co-existence of Escherichia coli strains in the murine gut.

Bacteria communicate and coordinate their behaviour at the intra- and interspecies levels by producing and sensing diverse extracellular small molecules called autoinducers. Autoinducer 2 (AI-2) is produced and detected by a variety of bacteria and thus plays an important role in interspecies communication and chemotaxis. Although AI-2 is a major autoinducer molecule present in the mammalian gut and can influence the composition of the murine gut microbiota, its role in bacteria-bacteria and bacteria-host interactions during gut colonization remains unclear. Combining competitive infections in C57BL/6 mice with microscopy and bioinformatic approaches, we show that chemotaxis (cheY) and AI-2 signalling (via lsrB) promote gut colonization by Escherichia coli, which is in turn connected to the ability of the bacteria to utilize fructoselysine (frl operon). We further show that the genomic diversity of E. coli strains with respect to AI-2 signalling allows ecological niche segregation and stable co-existence of different E. coli strains in the mammalian gut.

Genome-based discovery and total synthesis of janustatins, potent cytotoxins from a plant-associated bacterium.

Host-associated bacteria are increasingly being recognized as underexplored sources of bioactive natural products with unprecedented chemical scaffolds. A recently identified example is the plant-root-associated marine bacterium Gynuella sunshinyii of the chemically underexplored order Oceanospirillales. Its genome contains at least 22 biosynthetic gene clusters, suggesting a rich and mostly uncharacterized specialized metabolism. Here, in silico chemical prediction of a non-canonical polyketide synthase cluster has led to the discovery of janustatins, structurally unprecedented polyketide alkaloids with potent cytotoxicity that are produced in minute quantities. A combination of MS and two-dimensional NMR experiments, density functional theory calculations of 13C chemical shifts and semiquantitative interpretation of transverse rotating-frame Overhauser effect spectroscopy data were conducted to determine the relative configuration, which enabled the total synthesis of both enantiomers and assignment of the absolute configuration. Janustatins feature a previously unknown pyridodihydropyranone heterocycle and an unusual biological activity consisting of delayed, synchronized cell death at subnanomolar concentrations.

N-Succinyltransferase Encoded by a Cryptic Siderophore Biosynthesis Gene Cluster in Streptomyces Modifies Structurally Distinct Antibiotics.

The antibiotic desertomycin A and its previously undescribed inactive N-succinylated analogue, desertomycin X, were isolated from Streptomyces sp. strain YIM 121038. Genome sequencing and analysis readily identified the desertomycin biosynthetic gene cluster (BGC), which lacked genes encoding acyltransferases that would account for desertomycin X formation. Scouting the genome for putative N-acyltransferase genes led to the identification of a candidate within a cryptic siderophore BGC (csb) encoding a putative homologue of the N6'-hydroxylysine acetyltransferase IucB. Expression of the codon-optimized gene designated csbC in Escherichia coli yielded the recombinant protein that was able to N-succinylate desertomycin A as well as several other structurally distinct antibiotics harboring amino groups. Some antibiotics were rendered antibiotically inactive due to the CsbC-catalyzed succinylation in vitro. Unlike many known N-acyltransferases involved in antibiotic resistance, CsbC could not efficiently acetylate the same antibiotics. When expressed in E. coli, CsbC provided low-level resistance to kanamycin and ampicillin, suggesting that it may play a role in antibiotic resistance in natural habitats, where the concentration of antibiotics is usually low. IMPORTANCE In their natural habitats, bacteria encounter a plethora of organic compounds, some of which may be represented by antibiotics produced by certain members of the microbial community. A number of antibiotic resistance mechanisms have been described, including those specified by distinct genes encoding proteins that degrade, modify, or expel antibiotics. In this study, we report identification and characterization of an enzyme apparently involved in the biosynthesis of a siderophore, but also having the ability of modify and thereby inactivate a wide variety of structurally diverse antibiotics. This discovery sheds light on additional capabilities of bacteria to withstand antibiotic treatment and suggests that enzymes involved in secondary metabolism may have an additional function in the natural environment.

Structure of a Promiscuous Thioesterase Domain Responsible for Branching Acylation in Polyketide Biosynthesis.

Thioesterases (TEs) are fundamentally important enzymes present in all bacteria and eukaryotes, where they have conserved functions in fatty acid biosynthesis and secondary metabolism. This work provides the first structural insights into a functionally distinct group of TEs that perform diverse acylations in polyketide and peptide biosynthesis (TEB s). Structural analysis of the oocydin (OocS) TEB domain facilitated identification and engineering of the active site to modulate acyl-group acceptance. In this way, we achieved higher reactivity using a structure-based approach, building a foundation for biocatalytic development of TEB -mediated O-acylation, a modification known to improve the bioactivity of oocydin-type polyketides. Lastly, the promiscuity of the OocS TEB motivated us to investigate, and ultimately provide evidence for, the production of longer chain branched oocydins in the native host Serratia plymuthica 4Rx13. This work frames the OocS TEB and homologs as invaluable synthetic biology tools for polyketide drug development.

Biosynthetic potential of the global ocean microbiome.

Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds2,3. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters ('Candidatus Eudoremicrobiaceae') that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments.

Marine Sponge Endosymbionts: Structural and Functional Specificity of the Microbiome within Euryspongia arenaria Cells.

Sponge microbiomes are typically profiled by analyzing the community DNA of whole tissues, which does not distinguish the taxa residing within sponge cells from extracellular microbes. To uncover the endosymbiotic microbiome, we separated the sponge cells to enrich the intracellular microbes. The intracellular bacterial community of sponge Euryspongia arenaria was initially assessed by amplicon sequencing, which indicated that it hosts three unique phyla not found in the extracellular and bulk tissue microbiomes. These three phyla account for 66% of the taxonomically known genera in the intracellular microbiome. The shotgun metagenomic analysis extended the taxonomic coverage to viruses and eukaryotes, revealing the most abundant signature taxa specific to the intracellular microbiome. Functional KEGG pathway annotation demonstrated that the endosymbiotic microbiome hosted the greatest number of unique gene orthologs. The pathway profiles distinguished the intra- and extracellular microbiomes from the tissue and seawater microbiomes. Carbohydrate-active enzyme analysis further discriminated each microbiome based on their representative and dominant enzyme families. One pathway involved in digestion system and family esterase had a consistently higher level in intracellular microbiome and could statistically differentiate the intracellular microbiome from the others, suggesting that triacylglycerol lipases could be the key functional component peculiar to the endosymbionts. The identified higher abundance of lipase-related eggNOG categories further supported the lipid-hydrolyzing metabolism of endosymbiotic microbiota. Pseudomonas members, reported as lipase-producing bacteria, were only in the endosymbiotic microbiome, meanwhile Pseudomonas also showed a greater abundance intracellularly. Our study aided a comprehensive sponge microbiome that demonstrated the taxonomic and functional specificity of endosymbiotic microbiota. IMPORTANCE Sponges host abundant microbial symbionts that can produce an impressive number of novel bioactive metabolites. However, knowledge on intracellular (endosymbiotic) microbiota is scarce. We characterize the composition and function of the endosymbiotic microbiome by separation of sponge cells and enrichment of intracellular microbes. We uncover a noteworthy number of taxa exclusively in the endosymbiotic microbiome. We unlock the unique pathways and enzymes of endosymbiotic taxa. This study achieves a more comprehensive sponge microbial community profile, which demonstrates the structural and functional specificity of the endosymbiotic microbiome. Our findings not only open the possibility to reveal the low abundant and the likely missed microbiota when directly sequencing the sponge bulk tissues, but also warrant future in-depth exploration within single sponge cells.

In Vivo Production of Diverse ß-Amino Acid-Containing Proteins.

The wide range of moieties installed in ribosomally synthesized and post-translationally modified peptides (RiPPs) suggests largely untapped potential for protein engineering. However, many RiPP maturases recognize target peptide precursors through an N-terminal leader sequence that is challenging to adapt to proteins. We have recently reported a family of enzymes that splice XYG sites in RiPPs to install α-keto-ß-amino acids. Backbone modifications influence diverse protein properties, yet the toolkit to install ß-amino acids is limited. Here we report their leader-independent incorporation into proteins in E. coli. Integrating an 11-residue splice tag into six different proteins permitted the site-selective introduction of ß-residues in vivo. The motif fusion at C-, N-terminal, and internal positions yielded various ß-residues. Our approach complements the few existing methods to introduce ß-amino acids or ketone-bearing moieties, suggesting diverse applications in chemical biology.

Structure and mechanism for iterative amide N-methylation in the biosynthesis of channel-forming peptide cytotoxins.

SignificanceThe channel-forming proteusins are bacterial helical peptides that allow permeation of positively charged ions to influence membrane potential and cellular physiology. We biochemically characterize the effect of two critical posttranslational modifications on the secondary structure of the peptide substrate. We determine how a methyl group can be added to the side chains of D-Asn residues in a peptide substrate and show how flanking residues influence selectivity. These studies should foster the development of small-molecule peptide ion channels as therapeutics.