An updated catalogue of diverse type II polyketide synthase biosynthetic gene clusters captured from large-scale nucleotide databases.

Nature serves as a rich source of molecules with immense chemical diversity. Aptly named, these 'natural products' boast a wide variety of environmental, medicinal and industrial applications. Type II polyketides, in particular, confer substantial medicinal benefits, including antibacterial, antifungal, anticancer and anti-inflammatory properties. These molecules are produced by enzyme assemblies known as type II polyketide synthases (PKSs), which use domains such as the ketosynthase chain-length factor and acyl carrier protein to produce polyketides with varying lengths, cyclization patterns and oxidation states. In this work, we use a novel bioinformatic workflow to identify biosynthetic gene clusters (BGCs) that code for the core type II PKS enzymes. This method does not rely on annotation and thus was able to unearth previously 'hidden' type II PKS BGCs. This work led us to identify over 6000 putative type II PKS BGCs spanning a diverse set of microbial phyla, nearly double those found in most recent studies. Notably, many of these newly identified BGCs were found in non-actinobacteria, which are relatively underexplored as sources of type II polyketides. Results from this work lay an important foundation for future bioprospecting and engineering efforts that will enable sustainable access to diverse and structurally complex molecules with medicinally relevant properties.

Frequency Changes in Terminal Alkynes Provide Strong, Sensitive, and Solvatochromic Raman Probes of Biochemical Environments.

The C≡C stretching frequencies of terminal alkynes appear in the "clear" window of vibrational spectra, so they are attractive and increasingly popular as site-specific probes in complicated biological systems like proteins, cells, and tissues. In this work, we collected infrared (IR) absorption and Raman scattering spectra of model compounds, artificial amino acids, and model proteins that contain terminal alkyne groups, and we used our results to draw conclusions about the signal strength and sensitivity to the local environment of both aliphatic and aromatic terminal alkyne C≡C stretching bands. While the IR bands of alkynyl model compounds displayed surprisingly broad solvatochromism, their absorptions were weak enough that alkynes can be ruled out as effective IR probes. The same solvatochromism was observed in model compounds' Raman spectra, and comparisons to published empirical solvent scales (including a linear regression against four meta-aggregated solvent parameters) suggested that the alkyne C≡C stretching frequency mainly reports on local electronic interactions (i.e., short-range electron donor-acceptor interactions) with solvent molecules and neighboring functional groups. The strong solvatochromism observed here for alkyne stretching bands introduces an important consideration for Raman imaging studies based on these signals. Raman signals for alkynes (especially those that are π-conjugated) can be exceptionally strong and should permit alkynyl Raman signals to function as probes at very low concentrations, as compared to other widely used vibrational probe groups like azides and nitriles. We incorporated homopropargyl glycine into a transmembrane helical peptide via peptide synthesis, and we installed p-ethynylphenylalanine into the interior of the Escherichia coli fatty acid acyl carrier protein using a genetic code expansion technique. The Raman spectra from each of these test systems indicate that alkynyl C≡C bands can act as effective and unique probes of their local biomolecular environments. We provide guidance for the best possible future uses of alkynes as solvatochromic Raman probes, and while empirical explanations of the alkyne solvatochromism are offered, open questions about its physical basis are enunciated.

ActinoBase: tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria.

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.

Heterologous Expression, Purification, and Characterization of Type II Polyketide Synthase Acyl Carrier Proteins.

The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs.

The Cytochrome P450 OxyA from the Kistamicin Biosynthesis Cyclization Cascade is Highly Sensitive to Oxidative Damage.

Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilize a cysteine thiolate-ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of the active intermediate formed within P450s during their active cycle, it is remarkable that these enzymes can avoid auto-oxidation and retain the axial cysteine ligand in the deprotonated-and thus highly acidic-thiolate form. While little is known about the process of heme incorporation during P450 folding, there is an overwhelming preference for one heme orientation within the P450 active site. Indeed, very few structures to date contain an alternate heme orientation, of which two are OxyA homologs from glycopeptide antibiotic (GPA) biosynthesis. Given the apparent preference for the unusual heme orientation shown by OxyA enzymes, we investigated the OxyA homolog from kistamicin biosynthesis (OxyAkis), which is an atypical GPA. We determined that OxyAkis is highly sensitive to oxidative damage by peroxide, with both UV and EPR measurements showing rapid bleaching of the heme signal. We determined the structure of OxyAkis and found a mixed population of heme orientations present in this enzyme. Our analysis further revealed the possible modification of the heme moiety, which was only present in samples where the alternate heme orientation was present in the protein. These results suggest that the typical heme orientation in cytochrome P450s can help prevent potential damage to the heme-and hence deactivation of the enzyme-during P450 catalysis. It also suggests that some P450 enzymes involved in GPA biosynthesis may be especially prone to oxidative damage due to the heme orientation found in their active sites.

Engineered Chimeras Unveil Swappable Modular Features of Fatty Acid and Polyketide Synthase Acyl Carrier Proteins.

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein, we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the Escherichia coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based cross-linking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step toward the strategic engineering of "un-natural" natural products.

Probing the structure and function of acyl carrier proteins to unlock the strategic redesign of type II polyketide biosynthetic pathways.

Type II polyketide synthases (PKSs) are protein assemblies, encoded by biosynthetic gene clusters in microorganisms, that manufacture structurally complex and pharmacologically relevant molecules. Acyl carrier proteins (ACPs) play a central role in biosynthesis by shuttling malonyl-based building blocks and polyketide intermediates to catalytic partners for chemical transformations. Because ACPs serve as central hubs in type II PKSs, they can also represent roadblocks to successfully engineering synthases capable of manufacturing 'unnatural natural products.' Therefore, understanding ACP conformational dynamics and protein interactions is essential to enable the strategic redesign of type II PKSs. However, the inherent flexibility and transience of ACP interactions pose challenges to gaining insight into ACP structure and function. In this review, we summarize how the application of chemical probes and molecular dynamic simulations has increased our understanding of the structure and function of type II PKS ACPs. We also share how integrating these advances in type II PKS ACP research with newfound access to key enzyme partners, such as the ketosynthase-chain length factor, sets the stage to unlock new biosynthetic potential.

Widening the bottleneck: Heterologous expression, purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli.

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

MIBiG 2.0: a repository for biosynthetic gene clusters of known function.

Fueled by the explosion of (meta)genomic data, genome mining of specialized metabolites has become a major technology for drug discovery and studying microbiome ecology. In these efforts, computational tools like antiSMASH have played a central role through the analysis of Biosynthetic Gene Clusters (BGCs). Thousands of candidate BGCs from microbial genomes have been identified and stored in public databases. Interpreting the function and novelty of these predicted BGCs requires comparison with a well-documented set of BGCs of known function. The MIBiG (Minimum Information about a Biosynthetic Gene Cluster) Data Standard and Repository was established in 2015 to enable curation and storage of known BGCs. Here, we present MIBiG 2.0, which encompasses major updates to the schema, the data, and the online repository itself. Over the past five years, 851 new BGCs have been added. Additionally, we performed extensive manual data curation of all entries to improve the annotation quality of our repository. We also redesigned the data schema to ensure the compliance of future annotations. Finally, we improved the user experience by adding new features such as query searches and a statistics page, and enabled direct link-outs to chemical structure databases. The repository is accessible online at https://mibig.secondarymetabolites.org/.

Colorimetric Assay Reports on Acyl Carrier Protein Interactions.

The ability to produce new molecules of potential pharmaceutical relevance via combinatorial biosynthesis hinges on improving our understanding of acyl-carrier protein (ACP)-protein interactions. However, the weak and transient nature of these interactions makes them difficult to study using traditional spectroscopic approaches. Herein we report that converting the terminal thiol of the E. coli ACP 4'-phosphopantetheine arm into a mixed disulfide with 2-nitro-5-thiobenzoate ion (TNB-) activates this site to form a selective covalent cross-link with the active site cysteine of a cognate ketoacyl synthase (KS). The concomitant release of TNB2-, which absorbs at 412 nm, provides a visual and quantitative measure of mechanistically relevant ACP-KS interactions. The colorimetric assay can propel the engineering of biosynthetic routes to novel chemical diversity by providing a high-throughput screen for functional hybrid ACP-KS partnerships as well as the discovery of novel antimicrobial agents by enabling the rapid identification of small molecule inhibitors of ACP-KS interactions.

Tracking carrier protein motions with Raman spectroscopy.

Engineering microbial biosynthetic pathways represents a compelling route to gain access to expanded chemical diversity. Carrier proteins (CPs) play a central role in biosynthesis, but the fast motions of CPs make their conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we present a low-resource method to directly reveal carrier protein-substrate interactions. Chemoenzymatic loading of commercially available, alkyne-containing substrates onto CPs enables rapid visualization of the molecular cargo's local environment using Raman spectroscopy. This method could clarify the foundations of the chain sequestration mechanism, facilitate the rapid characterization of CPs, and enable visualization of the vectoral processing of natural products both in vitro and in vivo.

Studying trans-acting enzymes that target carrier protein-bound amino acids during nonribosomal peptide synthesis.

Nonribosomal peptide biosynthesis is a complex enzymatic assembly responsible for producing a great diversity of bioactive peptide natural products. Due to the recurring arrangement of catalytic domains within these machineries, great interest has been shown in reengineering these pathways to produce novel, designer peptide products. However, in order to realize such ambitions, it is first necessary to develop a comprehensive understanding of the selectivity, mechanisms, and structure of these complex enzymes, which in turn requires significant in vitro experiments. Within nonribosomal biosynthesis, some modifications are performed by enzymatic domains that are not linked to the main nonribosomal peptide synthetase but rather act in trans: these systems offer great potential for redesign, but in turn require detailed study. In this chapter, we present an overview of in vitro experiments that can be used to characterize examples of such trans-interacting enzymes from nonribosomal peptide biosynthesis: Cytochrome P450 monooxygenases and flavin-dependent halogenases.

Vibrant symbiosis: Achieving reciprocal science outreach through biological art.

Scientific outreach efforts traditionally involve formally trained scientists teaching the general public about the methods, significance, and excitement of science. We recently experimented with an alternative "symbiotic outreach" model that prioritizes building a reciprocal relationship between formally trained and "outsider" scientists to facilitate active two-way communication. Herein, we present the results of our outreach effort involving college students and adults with intellectual and developmental disabilities working together to make biological and multimedia art. By discussing the steps others can take to cultivate reciprocal outreach within their local communities, we hope to lower the barrier for widespread adoption of similar approaches and ultimately to decrease the gap between formally trained scientists and the general public.

A standardized workflow for submitting data to the Minimum Information about a Biosynthetic Gene cluster (MIBiG) repository: prospects for research-based educational experiences.

Microorganisms utilize complex enzymatic pathways to biosynthesize structurally complex and pharmacologically relevant molecules. These pathways are encoded by gene clusters and are found in a diverse set of organisms. The Minimum Information about a Biosynthetic Gene cluster repository facilitates standardized and centralized storage of experimental data on these gene clusters and their molecular products, by utilizing user-submitted data to translate scientific discoveries into a format that can be analyzed computationally. This accelerates the processes of connecting genes to chemical structures, understanding biosynthetic gene clusters in the context of environmental diversity, and performing computer-assisted design of synthetic gene clusters. Here, we present a Standard Operating Procedure, Excel templates, a tutorial video, and a collection of relevant review literature to support scientists in their efforts to submit data into MiBIG. Further, we provide tools to integrate gene cluster annotation projects into the classroom environment, including workflows and assessment materials.

The Effect of Divalent Cations on the Thermostability of Type II Polyketide Synthase Acyl Carrier Proteins.

The successful engineering of biosynthetic pathways hinges on understanding the factors that influence acyl carrier protein (ACP) stability and function. The stability and structure of ACPs can be influenced by the presence of divalent cations, but how this relates to primary sequence remains poorly understood. As part of a course-based undergraduate research experience, we investigated the thermostability of type II polyketide synthase (PKS) ACPs. We observed an approximate 40 °C range in the thermostability amongst the 14 ACPs studied, as well as an increase in stability (5 - 26 °C) of the ACPs in the presence of divalent cations. Distribution of charges in the helix II-loop-helix III region was found to impact the enthalpy of denaturation. Taken together, our results reveal clues as to how the sequence of type II PKS ACPs relates to their structural stability, information that can be used to study how ACP sequence relates to function.

Collaborating with Undergraduates To Contribute to Biochemistry Community Resources.

Course-based undergraduate research experiences (CUREs) have gained traction as effective ways to expand the impact of undergraduate research while fulfilling pedagogical goals. In this Perspective, we present innovative ways to incorporate fundamental benefits and principles of CUREs into a classroom environment through information/technology-based research projects that lead to student-generated contributions to digital community resources (CoRes). These projects represent an attractive class of CUREs because they are less resource-intensive than laboratory-based CUREs, and the projects align with the expectations of today's students to create rapid and publicly accessible contributions to society. We provide a detailed discussion of two example types of CoRe projects that can be implemented in courses to impact research and education at the chemistry-biology interface: bioinformatics annotations and development of educational tools. Finally, we present current resources available for faculty interested in incorporating CUREs or CoRe projects into their pedagogical practices. In sharing these stories and resources, we hope to lower the barrier for widespread adoption of CURE and CoRe approaches and generate discussions about how to utilize the classroom experience to make a positive impact on our students and the future of the field of biochemistry.

Utilizing Mechanistic Cross-Linking Technology to Study Protein-Protein Interactions: An Experiment Designed for an Undergraduate Biochemistry Lab.

Over the past decade, mechanistic crosslinking probes have been used to study protein-protein interactions in natural product biosynthetic pathways. This approach is highly interdisciplinary, combining elements of protein biochemistry, organic chemistry, and computational docking. The development of an experiment to engage undergraduate students in multidisciplinary research is described that leverages mechanistic crosslinking probes to study protein conformations and protein-protein interactions. This experiment provides students with a platform to learn chemoenzymatic synthesis, polyacrylamide gel electrophoresis, biochemical assays, and computational docking all while exploring a contemporary biochemical topic.

Uncovering protein-protein interactions through a team-based undergraduate biochemistry course.

How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.

Acyl Carrier Protein Cyanylation Delivers a Ketoacyl Synthase-Carrier Protein Cross-Link.

Acyl carrier proteins (ACPs) are central hubs in polyketide and fatty acid biosynthetic pathways, but the fast motions of the ACP's phosphopantetheine (Ppant) arm make its conformational dynamics difficult to capture using traditional spectroscopic approaches. Here we report that converting the terminal thiol of Escherichia coli ACP's Ppant arm into a thiocyanate activates this site to form a selective cross-link with the active site cysteine of its partner ketoacyl synthase (FabF). The reaction releases a cyanide anion, which can be detected by infrared spectroscopy. This represents a practical and generalizable method for obtaining and visualizing ACP-protein complexes relevant to biocatalysis and will be valuable in future structural and engineering studies.

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis.

The nonribosomal peptide synthetases (NRPSs) are one of the most promising resources for the production of new bioactive molecules. The mechanism of NRPS catalysis is based around sequential catalytic domains: these are organized into modules, where each module selects, modifies, and incorporates an amino acid into the growing peptide. The intermediates formed during NRPS catalysis are delivered between enzyme centers by peptidyl carrier protein (PCP) domains, which makes PCP interactions and movements crucial to NRPS mechanism. PCP movement has been linked to the domain alternation cycle of adenylation (A) domains, and recent complete NRPS module structures provide support for this hypothesis. However, it appears as though the A domain alternation alone is insufficient to account for the complete NRPS catalytic cycle and that the loaded state of the PCP must also play a role in choreographing catalysis in these complex and fascinating molecular machines.