Engineered Saccharomyces cerevisiae as a Biosynthetic Platform of Nucleotide Sugars.

Glycosylation of biomolecules can greatly alter their physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Glycosylation reactions rely on the stepwise addition of sugars using nucleotide diphosphate (NDP)-sugars. Making these substrates readily available will greatly accelerate the characterization of new glycosylation reactions, elucidation of their underlying regulation mechanisms, and production of glycosylated molecules. In this work, we engineered Saccharomyces cerevisiae to heterologously express nucleotide sugar synthases to access a wide variety of uridine diphosphate (UDP)-sugars from simple starting materials (i.e., glucose and galactose). Specifically, activated glucose, uridine diphosphate d-glucose (UDP-d-Glc), can be converted to UDP-d-glucuronic acid (UDP-d-GlcA), UDP-d-xylose (UDP-d-Xyl), UDP-d-apiose (UDP-d-Api), UDP-d-fucose (UDP-d-Fuc), UDP-l-rhamnose (UDP-l-Rha), UDP-l-arabinopyranose (UDP-l-Arap), and UDP-l-arabinofuranose (UDP-l-Araf) using the corresponding nucleotide sugar synthases of plant and microbial origins. We also expressed genes encoding the salvage pathway to directly activate free sugars to achieve the biosynthesis of UDP-l-Arap and UDP-l-Araf. We observed strong inhibition of UDP-d-Glc 6-dehydrogenase (UGD) by the downstream product UDP-d-Xyl, which we circumvented using an induction system (Tet-On) to delay the production of UDP-d-Xyl to maintain the upstream UDP-sugar pool. Finally, we performed a time-course study using strains containing the biosynthetic pathways to produce five non-native UDP-sugars to elucidate their time-dependent interconversion and the role of UDP-d-Xyl in regulating UDP-sugar metabolism. These engineered yeast strains are a robust platform to (i) functionally characterize sugar synthases in vivo, (ii) biosynthesize a diverse selection of UDP-sugars, (iii) examine the regulation of intracellular UDP-sugar interconversions, and (iv) produce glycosylated secondary metabolites and proteins.

Computationally guided exploration of borosin biosynthetic strategies.

Borosins are ribosomally synthesized and post-translationally modified peptides containing backbone α- N -methylations. Identification of borosin precursor peptides is difficult because (1) there are no conserved sequence elements among borosin precursor peptides and (2) the biosynthetic gene clusters contain numerous domain architectures and peptide fusions. To tackle this problem, we updated the genome mining tool RODEO to automatically evaluate putative borosin BGCs and identify precursor peptides. Enabled by the new borosin module, we analyzed all borosin BGCs found in available sequence data and assigned precursor peptides to previously orphan borosin methyltransferases. Additionally, we bioinformatically predict and experimentally characterize a new fused borosin domain architecture, in which the modified core is N-terminal to the methyltransferase domain. Finally, we demonstrate that a borosin precursor peptide is the native substrate of shewasin A, a previously characterized pepsin-like aspartic peptidase whose native biological function was unknown.

Development of Corynebacterium glutamicum as a monoterpene production platform.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria.

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-D-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

Accessing Diverse Pyridine-Based Macrocyclic Peptides by a Two-Site Recognition Pathway.

Macrocyclic peptides are sought-after molecular scaffolds for drug discovery, and new methods to access diverse libraries are of increasing interest. Here, we report the enzymatic synthesis of pyridine-based macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides are a recently described class of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are related to the long-known thiopeptide natural products. RiPP precursors typically contain an N-terminal leader region that is physically engaged by the biosynthetic proteins that catalyze modification of the C-terminal core region of the precursor peptide. We demonstrate that pyritide-forming enzymes recognize both the leader region and a C-terminal tripeptide motif, with each contributing to site-selective substrate modification. Substitutions in the core region were well-tolerated and facilitated the generation of a wide range of pyritide analogues, with variations in macrocycle sequence and size. A combination of the pyritide biosynthetic pathway with azole-forming enzymes was utilized to generate a thiazole-containing pyritide (historically known as a thiopeptide) with no similarity in sequence and macrocycle size to the naturally encoded pyritides. The broad substrate scope of the pyritide biosynthetic enzymes serves as a future platform for macrocyclic peptide lead discovery and optimization.

Structure Prediction and Synthesis of Pyridine-Based Macrocyclic Peptide Natural Products.

The structural and functional characterization of natural products is vastly outpaced by the bioinformatic identification of biosynthetic gene clusters (BGCs) that encode such molecules. Uniting our knowledge of bioinformatics and enzymology to predict and synthetically access natural products is an effective platform for investigating cryptic/silent BGCs. We report the identification, biosynthesis, and total synthesis of a minimalistic class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with the responsible BGCs encoding a subset of enzymes known from thiopeptide biosynthesis. On the basis of the BGC content, these RiPPs were predicted to undergo enzymatic dehydration of serine followed by [4+2]-cycloaddition to produce a trisubstituted, pyridine-based macrocycle. These RiPPs, termed "pyritides", thus contain the same six-membered, nitrogenous heterocycle that defines the thiopeptide RiPP class but lack the ubiquitous thiazole/thiazoline heterocycles, suggesting that thiopeptides should be reclassified as a more elaborate subclass of the pyritides. One pyritide product was obtained using an 11-step synthesis, and the structure verified by an orthogonal chemoenzymatic route using the precursor peptide and cognate pyridine synthase. This work exemplifies complementary bioinformatics, enzymology, and synthesis to characterize a minimalistic yet structurally intriguing scaffold that, unlike most thiopeptides, lacks growth-suppressive activity toward Gram-positive bacteria.

Reactivity-Based Screening for Citrulline-Containing Natural Products Reveals a Family of Bacterial Peptidyl Arginine Deiminases.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is processed by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminal sequence threaded through the macrocycle. This unique lariat topology, which typically provides considerable stability toward heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of Arg deimination to yield citrulline (Cit). Although a Cit-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for Arg deimination has remained unknown. Herein, we describe the use of reactivity-based screening to discriminate bacterial strains that produce Arg- versus Cit-bearing citrulassins, yielding 13 new lasso peptide variants. Partial phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the Cit-containing variants. Absence of this gene correlated strongly with lasso peptide variants only containing Arg (i.e., des-citrulassin). Heterologous expression of the PAD gene in a des-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in Arg deimination. The PADs were then bioinformatically surveyed to provide a deeper understanding of their taxonomic distribution and genomic contexts and to facilitate future studies that will evaluate any additional biochemical roles for the superfamily.

Bioinformatic Mapping of Radical S-Adenosylmethionine-Dependent Ribosomally Synthesized and Post-Translationally Modified Peptides Identifies New Cα, Cß, and Cγ-Linked Thioether-Containing Peptides.

Recently developed bioinformatic tools have bolstered the discovery of ribosomally synthesized and post-translationally modified peptides (RiPPs). Using an improved version of Rapid ORF Description and Evaluation Online (RODEO 2.0), a biosynthetic gene cluster mining algorithm, we bioinformatically mapped the sactipeptide RiPP class via the radical S-adenosylmethionine (SAM) enzymes that form the characteristic sactionine (sulfur-to-α carbon) cross-links between cysteine and acceptor residues. Hundreds of new sactipeptide biosynthetic gene clusters were uncovered, and a novel sactipeptide "huazacin" with growth-suppressive activity against Listeria monocytogenes was characterized. Bioinformatic analysis further suggested that a group of sactipeptide-like peptides heretofore referred to as six cysteines in forty-five residues (SCIFFs) might not be sactipeptides as previously thought. Indeed, the bioinformatically identified SCIFF peptide "freyrasin" was demonstrated to contain six thioethers linking the ß carbons of six aspartate residues. Another SCIFF, thermocellin, was shown to contain a thioether cross-linked to the γ carbon of threonine. SCIFFs feature a different paradigm of non-α carbon thioether linkages, and they are exclusively formed by radical SAM enzymes, as opposed to the polar chemistry employed during lanthipeptide biosynthesis. Therefore, we propose the renaming of the SCIFF family as radical non-α thioether peptides (ranthipeptides) to better distinguish them from the sactipeptide and lanthipeptide RiPP classes.

Enzymatic Reconstitution and Biosynthetic Investigation of the Lasso Peptide Fusilassin.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified natural product which possess a unique lariat knot conformation. The low entropy "threaded" conformation endows lasso peptides with considerable resistance to heat and proteolytic degradation, which are attractive properties for the development of peptide-based therapeutics. Despite their discovery nearly 30 years ago, the molecular mechanism underlying lasso peptide biosynthesis remains poorly characterized due to the low stability of the purified biosynthetic enzymes. Here, we report the biosynthetic reconstitution of a lasso peptide derived from Thermobifida fusca, termed fusilassin. Beyond robust catalytic activity, the fusilassin enzymes demonstrate extraordinary substrate tolerance during heterologous expression in E. coli and upon purification in cell-free biosynthetic reconstitution reactions. We provide evidence that the fusilassin biosynthetic enzymes are not capable of forming branched-cyclic products but can produce entirely unrelated lasso peptides. Finally, we leveraged our bioinformatic survey of all lasso peptides identified in GenBank to perform coevolutionary analysis of two requisite biosynthetic proteins. This effort correctly identified residues governing an important protein-protein interaction, illustrating how genomic insight can accelerate the characterization of natural product biosynthetic pathways. The fusilassin enzymes described within represent a model system for both designing future lasso peptides of biomedical importance and also for elucidating the molecular mechanisms that govern lasso peptide biosynthesis.

Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics.

Thiopeptides are members of the ribosomally synthesized and post-translationally modified peptide family of natural products. Most characterized thiopeptides display nanomolar potency toward Gram-positive bacteria by blocking protein translation with several being produced at the industrial scale for veterinary and livestock applications. Employing our custom bioinformatics program, RODEO, we expand the thiopeptide family of natural products by a factor of four. This effort revealed many new thiopeptide biosynthetic gene clusters with products predicted to be distinct from characterized thiopeptides and identified gene clusters for previously characterized molecules of unknown biosynthetic origin. To further validate our data set of predicted thiopeptide biosynthetic gene clusters, we isolated and characterized a structurally unique thiopeptide featuring a central piperidine and rare thioamide moiety. Termed saalfelduracin, this thiopeptide displayed potent antibiotic activity toward several drug-resistant Gram-positive pathogens. A combination of whole-genome sequencing, comparative genomics, and heterologous expression experiments confirmed that the thioamide moiety of saalfelduracin is installed post-translationally by the joint action of two proteins, TfuA and YcaO. These results reconcile the previously unknown origin of the thioamide in two long-known thiopeptides, thiopeptin and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic space, we provide a roadmap for the discovery of additional members of this natural product family.

RiPP antibiotics: biosynthesis and engineering potential.

The threat of antibiotic resistant bacterial infections continues to underscore the need for new treatment options. Historically, small molecule metabolites from microbes have provided a rich source of antibiotic compounds, and as a result, significant effort has been invested in engineering the responsible biosynthetic pathways to generate novel analogs with attractive pharmacological properties. Unfortunately, biosynthetic stringency has limited the capacity of non-ribosomal peptide synthetases and polyketide synthases from producing substantially different analogs in large numbers. Another class of natural products, the ribosomally synthesized and post-translationally modified peptides (RiPPs), have rapidly expanded in recent years with many natively displaying potent antibiotic activity. RiPP biosynthetic pathways are modular and intrinsically tolerant to alternative substrates. Several prominent RiPPs with antibiotic activity will be covered in this review with a focus on their biosynthetic plasticity. While only a few RiPP enzymes have been thoroughly investigated mechanistically, this knowledge has already been harnessed to generate new-to-nature compounds. Through the use of synthetic biology approaches, on-going efforts in RiPP engineering hold great promise in unlocking the potential of this natural product class.

In Vitro Biosynthetic Studies of Bottromycin Expand the Enzymatic Capabilities of the YcaO Superfamily.

The bottromycins belong to the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Bottromycins exhibit unique structural features, including a hallmark macrolactamidine ring and thiazole heterocycle for which divergent members of the YcaO superfamily have been biosynthetically implicated. Here we report the in vitro reconstitution of two YcaO proteins, BmbD and BmbE, responsible for the ATP-dependent cyclodehydration reactions that yield thiazoline- and macrolactamidine-functionalized products, respectively. We also establish the substrate tolerance for BmbD and BmbE and systematically dissect the role of the follower peptide, which we show serves a purpose similar to canonical leader peptides in directing the biosynthetic enzymes to the substrate. Lastly, we leverage the expanded capabilities of YcaO proteins to conduct an extensive bioinformatic survey to classify known YcaO chemistry. This analysis predicts new functions remain to be uncovered within the superfamily.

Mechanism of a Class C Radical S-Adenosyl-l-methionine Thiazole Methyl Transferase.

The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B-D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5'-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5'-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the ß-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.

Structural insights into enzymatic [4+2] aza-cycloaddition in thiopeptide antibiotic biosynthesis.

The [4+2] cycloaddition reaction is an enabling transformation in modern synthetic organic chemistry, but there are only limited examples of dedicated natural enzymes that can catalyze this transformation. Thiopeptides (or more formally thiazolyl peptides) are a class of thiazole-containing, highly modified, macrocyclic secondary metabolites made from ribosomally synthesized precursor peptides. The characteristic feature of these natural products is a six-membered nitrogenous heterocycle that is assembled via a formal [4+2] cycloaddition between two dehydroalanine (Dha) residues. This heteroannulation is entirely contingent on enzyme activity, although the mechanism of the requisite pyridine/dehydropiperidine synthase remains to be elucidated. The unusual aza-cylic product is distinct from the more common carbocyclic products of synthetic and biosynthetic [4+2] cycloaddition reactions. To elucidate the mechanism of cycloaddition, we have determined atomic resolution structures of the pyridine synthases involved in the biosynthesis of the thiopeptides thiomuracin (TbtD) and GE2270A (PbtD), in complex with substrates and product analogs. Structure-guided biochemical, mutational, computational, and binding studies elucidate active-site features that explain how orthologs can generate rigid macrocyclic scaffolds of different sizes. Notably, the pyridine synthases show structural similarity to the elimination domain of lanthipeptide dehydratases, wherein insertions of secondary structural elements result in the formation of a distinct active site that catalyzes different chemistry. Comparative analysis identifies other catalysts that contain a shared core protein fold but whose active sites are located in entirely different regions, illustrating a principle predicted from efforts in de novo protein design.

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) display a diverse range of structures and continue to expand as a natural product class. Accordingly, RiPPs exhibit a wide array of bioactivities, acting as broad and narrow spectrum growth suppressors, antidiabetics, and antinociception and anticancer agents. Because of these properties, and the complex repertoire of post-translational modifications (PTMs) that give rise to these molecules, RiPP biosynthesis has been intensely studied. RiPP biosynthesis often involves enzymes that perform unique chemistry with intriguing reaction mechanisms, which attract chemists and biochemists alike to study and re-engineer these pathways. One particular type of RiPP biosynthetic enzyme is the so-called radical S-adenosylmethionine (rSAM) enzyme, which utilizes radical-based chemistry to install several distinct PTMs. Here, we describe the rSAM enzymes characterized over the past decade that catalyze six reaction types from several RiPP biosynthetic pathways. We present the current state of mechanistic understanding and conclude with possible directions for future characterization of this enzyme family.

Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products.

Combining biosynthetic enzymes from multiple pathways is an attractive approach for producing molecules with desired structural features; however, progress has been hampered by the incompatibility of enzymes from unrelated pathways and intolerance toward alternative substrates. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a diverse natural product class that employs a biosynthetic logic that is highly amenable to engineering new compounds. RiPP biosynthetic proteins modify their substrates by binding to a motif typically located in the N-terminal leader region of the precursor peptide. Here, we exploit this feature by designing leader peptides that enable recognition and processing by multiple enzymes from unrelated RiPP pathways. Using this broadly applicable strategy, a thiazoline-forming cyclodehydratase was combined with enzymes from the sactipeptide and lanthipeptide families to create new-to-nature hybrid RiPPs. We also provide insight into design features that enable control over the hybrid biosynthesis to optimize enzyme compatibility and establish a general platform for engineering additional hybrid RiPPs.

Reconstitution and Substrate Specificity of the Radical S-Adenosyl-methionine Thiazole C-Methyltransferase in Thiomuracin Biosynthesis.

Thiomuracin is a thiopeptide antibiotic with potent activity toward Gram-positive drug-resistant bacteria. Thiomuracin is biosynthesized from a precursor peptide, TbtA, by a complex array of posttranslational modifications. One of several intriguing transformations is the C-methylation of thiazole, occurring at an unactivated sp2 carbon. Herein, we report the in vitro reconstitution of TbtI, the responsible radical S-adenosyl-methionine (rSAM) C-methyltransferase, which catalyzes the formation of 5-methylthiazole at a single site. Our studies demonstrate that a linear hexazole-bearing intermediate of TbtA is a substrate for TbtI whereas macrocyclized thiomuracin GZ is not. In determining the minimal substrate for TbtI, we found that the enzyme is functional when most of the leader peptide has been removed. The in vitro reconstitution of TbtI, a class C rSAM methyltransferase, further adds to the chemical versatility of rSAM enzymes, and informs on the complexity of thiomuracin biosynthesis.