Structural Plasticity within 3-Hydroxy-3-Methylglutaryl Synthases Catalyzing the First Step of ß-Branching in Polyketide Biosynthesis Underpins a Dynamic Mechanism of Substrate Accommodation.

Understanding how enzymes have been repurposed by evolution to carry out new functions is a key goal of mechanistic enzymology. In this study we aimed to identify the adaptations required to allow the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) enzymes of primary isoprenoid assembly to function in specialized polyketide biosynthetic pathways, where they initiate ß-branching. This role notably necessitates that the HMG synthases (HMGSs) act on substrates tethered to noncatalytic acyl carrier protein (ACP) domains instead of coenzyme A, and accommodation of substantially larger chains within the active sites. Here, we show using a combination of X-ray crystallography and small-angle X-ray scattering, that a model HMGS from the virginiamycin system exhibits markedly increased flexibility relative to its characterized HMGCS counterparts. This mobility encompasses multiple secondary structural elements that define the dimensions and chemical nature of the active site, as well the catalytic residues themselves. This result was unexpected given the well-ordered character of the HMGS within the context of an HMGS/ACP complex, but analysis by synchrotron radiation circular dichroism demonstrates that this interaction leads to increased HMGS folding. This flexible to more rigid transition is notably not accounted for by AlphaFold2, which yielded a structural model incompatible with binding of the native substrates. Taken together, these results illustrate the continued necessity of an integrative structural biology approach combining crystallographic and solution-phase data for elucidating the mechanisms underlying enzyme remodeling, information which can inform strategies to replicate such evolution effectively in the laboratory.

Pyrrolizwilline, a unique bacterial alkaloid assembled by a nonribosomal peptide synthetase and non-enzymatic dimerization.

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of heterocyclic specialized metabolites characterized by a core structure comprising a hexahydro-1H-pyrrolizine. PAs are synthesized through two main pathways. In plants, assembly occurs via a homospermidine synthase, and in bacteria, through combined action of a nonribosomal peptide synthetase and a Baeyer-Villiger monooxygenase. While the toxic properties of plant-derived PAs and their prevalence in animal and human foods have been extensively studied, the biological roles and biosynthesis of more complex bacterial PAs are not well understood. Here, we report the identification and characterization of a bacterial biosynthetic gene cluster from Xenorhabdus hominickii, xhpA-G, which is responsible for producing the PA pseudo-dimer pyrrolizwilline. Analysis of X. hominickii promoter exchange mutants together with heterologous expression of xhpA-G in E. coli, revealed a set of pathway intermediates, two of which were chemically synthesized, as well as multiple derivatives. This information was leveraged to propose a detailed biosynthetic pathway to pyrrolizwilline. Furthermore, we have characterized the hydrolase XhpG, the key enzyme in the conversion of the pathway intermediate pyrrolizixenamide to pyrrolizwilline, using X-ray crystallography and small-angle X-ray scattering (SAXS).

Modular Cloning Tools for Streptomyces spp. and Application to the De Novo Biosynthesis of Flavokermesic Acid.

The filamentous Streptomyces are among the most prolific producers of bioactive natural products and are thus attractive chassis for the heterologous expression of native and designed biosynthetic pathways. Although suitable Streptomyces hosts exist, including genetically engineered cluster-free mutants, the approach is currently limited by the relative paucity of synthetic biology tools facilitating the de novo assembly of multicomponent gene clusters. Here, we report a modular system (MoClo) for Streptomyces including a set of adapted vectors and genetic elements, which allow for the construction of complete genetic circuits. Critical functional validation of each of the elements was obtained using the previously reported ß-glucuronidase (GusA) reporter system. Furthermore, we provide proof-of-principle for the toolbox inS. albus, demonstrating the efficient assembly of a biosynthetic pathway to flavokermesic acid (FK), an advanced precursor of the commercially valuable carminic acid.

Insights into docking in megasynthases from the investigation of the toblerol trans-AT polyketide synthase: many α-helical means to an end.

The fidelity of biosynthesis by modular polyketide synthases (PKSs) depends on specific moderate affinity interactions between successive polypeptide subunits mediated by docking domains (DDs). These sequence elements are notably portable, allowing their transplantation into alternative biosynthetic and metabolic contexts. Herein, we use integrative structural biology to characterize a pair of DDs from the toblerol trans-AT PKS. Both are intrinsically disordered regions (IDRs) that fold into a 3 α-helix docking complex of unprecedented topology. The C-terminal docking domain (CDD) resembles the 4 α-helix type (4HB) CDDs, which shows that the same type of DD can be redeployed to form complexes of distinct geometry. By carefully re-examining known DD structures, we further extend this observation to type 2 docking domains, establishing previously unsuspected structural relations between DD types. Taken together, these data illustrate the plasticity of α-helical DDs, which allow the formation of a diverse topological spectrum of docked complexes. The newly identified DDs should also find utility in modular PKS genetic engineering.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

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.

Decrypting the programming of ß-methylation in virginiamycin M biosynthesis.

During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic ß-ketones into ß-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for ß-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second ß-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin ß-branching programming. Furthermore, variations in this control - whether natural or by design - open up avenues for diversifying polyketide structures towards high-value derivatives.

Engineering Modular Polyketide Biosynthesis in Streptomyces Using CRISPR/Cas: A Practical Guide.

The CRISPR/Cas system, which has been widely applied to organisms ranging from microbes to animals, is currently being adapted for use in Streptomyces bacteria. In this case, it is notably applied to rationally modify the biosynthetic pathways giving rise to the polyketide natural products, which are heavily exploited in the medical and agricultural arenas. Our aim here is to provide the potential user with a practical guide to exploit this approach for manipulating polyketide biosynthesis, by treating key experimental aspects including vector choice, design of the basic engineering components, and trouble-shooting.

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins.

The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites - a reduction in chain length of 14 carbons relative to the 51-membered parental compounds - but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Manipulating polyketide stereochemistry by exchange of polyketide synthase modules.

A key goal of modular polyketide synthase (PKS) engineering is to alter polyketide stereochemistry. Here we report that exchanging whole PKS modules is a more productive approach than swapping individual ketoreductase (KR) domains for introducing rare 'A2' and 'B2' stereochemistry into model polyketides, and identify four modular 'biobricks' for such synthetic biology efforts.

Towards improved understanding of intersubunit interactions in modular polyketide biosynthesis: Docking in the enacyloxin IIa polyketide synthase.

Modular polyketide synthases (PKSs) are molecular-scale assembly lines comprising multiple gigantic polypeptide subunits. Faithful ordering of the subunits is mediated by extreme C- and N-terminal regions called docking domains (DDs). Decrypting how specificity is achieved by these elements is important both for understanding PKS function and modifying it to generate useful polyketide analogues for biological evaluation. Here we report the biophysical and structural characterisation of all six PKS/PKS interfaces in the unusual, chimaeric cis-AT/trans-AT PKS pathway responsible for biosynthesis of the antibiotic enacyloxin IIa in Burkholderia ambifaria. Taken together with previous work, our data reveal that specificity is achieved in the enacyloxin PKS by deploying at least three functionally orthogonal classes of DDs. We also demonstrate for the first time that cis-AT PKS subunits incorporate DDs with intrinsically disordered character, reinforcing the utility of such regions for achieving both medium affinity and high specificity at PKS intersubunit junctions. Overall, this work substantially increases the number of orthogonal DDs available for creating novel, highly-specific interfaces within cis- and trans-AT PKSs and their hybrids. It also reveals unexpected sequence/structure relationships in PKS DDs, identifying major current limitations to both accurately predicting and categorising these useful protein-protein interaction motifs.

Author Correction: Insights into a dual function amide oxidase/macrocyclase from lankacidin biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Insights into a dual function amide oxidase/macrocyclase from lankacidin biosynthesis.

In the original version of this Article, the final concentration of riboflavin in the supplemented LB medium for recombinant LkcE expression was incorrectly stated as 1 g L-1 (this was the concentration of the stock solution) and should have read 10-50 mg L-1. This error has been corrected in both the PDF and HTML versions of the Article.

Insights into a dual function amide oxidase/macrocyclase from lankacidin biosynthesis.

Acquisition of new catalytic activity is a relatively rare evolutionary event. A striking example appears in the pathway to the antibiotic lankacidin, as a monoamine oxidase (MAO) family member, LkcE, catalyzes both an unusual amide oxidation, and a subsequent intramolecular Mannich reaction to form the polyketide macrocycle. We report evidence here for the molecular basis for this dual activity. The reaction sequence involves several essential active site residues and a conformational change likely comprising an interdomain hinge movement. These features, which have not previously been described in the MAO family, both depend on a unique dimerization mode relative to all structurally characterized members. Taken together, these data add weight to the idea that designing new multifunctional enzymes may require changes in both architecture and catalytic machinery. Encouragingly, however, our data also show LkcE to bind alternative substrates, supporting its potential utility as a general cyclization catalyst in synthetic biology.

Unpackaging the Roles of Streptomyces Natural Products.

Streptomyces are the principle source of antibiotics in clinical use, but what the bacteria use these molecules for remains largely a mystery. In this issue of Cell Chemical Biology, Hoefler et al. (2017) demonstrate a direct link between biosynthesis of the polyketide linearmycins and extracellular membrane vesicles.

Correction: Uncovering the structures of modular polyketide synthases.

Correction for 'Uncovering the structures of modular polyketide synthases' by Kira J. Weissman, Nat. Prod. Rep., 2015, 32, 436-453.