Current State-of-the-Art Toward Chemoenzymatic Synthesis of Polyketide Natural Products.

Polyketide natural products have significant promise as pharmaceutical targets for human health and as molecular tools to probe disease and complex biological systems. While the biosynthetic logic of polyketide synthases (PKS) is well-understood, biosynthesis of designer polyketides remains challenging due to several bottlenecks, including substrate specificity constraints, disrupted protein-protein interactions, and protein solubility and folding issues. Focusing on substrate specificity, PKSs are typically interrogated using synthetic thioesters. PKS assembly lines and their products offer a wealth of information when studied in a chemoenzymatic fashion. This review provides an overview of the past two decades of polyketide chemoenzymatic synthesis and their contributions to the field of chemical biology. These synthetic strategies have successfully yielded natural product derivatives while providing critical insights into enzymatic promiscuity and mechanistic activity.

Targeted Enzyme Modifications Enable Regioselective Biosynthesis of Fluorinated Polyketides.

In attempts to enhance natural products as therapeutic agents, fluorination has emerged as a new tool for synthetic biologists and chemists. In recent articles published in Nature Chem. and Nature Chem. Bio., Grininger, Chang, and co-workers leveraged their expertise in engineering polyketide biosynthesis to incorporate fluorine into polyketide scaffolds.

Development of Genetically Encoded Biosensors for Reporting the Methyltransferase-Dependent Biosynthesis of Semisynthetic Macrolide Antibiotics.

Clarithromycin is an improved semisynthetic analogue of the naturally occurring macrolide, erythromycin. The subtle modification of a methyl group on the C-6 hydroxyl group endows the molecule with improved acid stability and results in a clinically useful antibiotic. Here, we show that the effector specificity of the biosensor protein, MphR, can be evolved to selectively recognize clarithromycin and therefore report on the production of this molecule in vivo. In addition, a crystal structure of the evolved variant reveals the molecular basis for selectivity and provides a guide for the evolution of a new metabolic function using this biosensor.

Protein engineering for natural product biosynthesis and synthetic biology applications.

As protein engineering grows more salient, many strategies have emerged to alter protein structure and function, with the goal of redesigning and optimizing natural product biosynthesis. Computational tools, including machine learning and molecular dynamics simulations, have enabled the rational mutagenesis of key catalytic residues for enhanced or altered biocatalysis. Semi-rational, directed evolution and microenvironment engineering strategies have optimized catalysis for native substrates and increased enzyme promiscuity beyond the scope of traditional rational approaches. These advances are made possible using novel high-throughput screens, including designer protein-based biosensors with engineered ligand specificity. Herein, we detail the most recent of these advances, focusing on polyketides, non-ribosomal peptides and isoprenoids, including their native biosynthetic logic to provide clarity for future applications of these technologies for natural product synthetic biology.

Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase.

Polyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.

Transcription factor-based biosensors: a molecular-guided approach for natural product engineering.

Natural products and their derivatives offer a rich source of chemical and biological diversity; however, traditional engineering of their biosynthetic pathways to improve yields and access to unnatural derivatives requires a precise understanding of their enzymatic processes. High-throughput screening platforms based on allosteric transcription-factor based biosensors can be leveraged to overcome the screening bottleneck to enable searching through large libraries of pathway/strain variants. Herein, the development and application of engineered allosteric transcription factor-based biosensors is described that enable optimization of precursor availability, product titers, and downstream product tailoring for advancing the natural product bioeconomy. We discuss recent successes for tailoring biosensor design, including computationally-based approaches, and present our future outlook with the integration of cell-free technologies and de novo protein design for rapidly generating biosensor tools.

Synthetic biology, combinatorial biosynthesis, and chemo­enzymatic synthesis of isoprenoids.

Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds. Often, this process is facilitated by the substrate or product promiscuity of the component enzymes, which can be leveraged to produce novel isoprenoids. To complement rational enhancements and even re-programming of isoprenoid biosynthesis, high-throughput approaches that rely on searching through large enzymatic libraries are being developed. This review summarizes recent advances and strategies related to isoprenoid synthetic biology, combinatorial biosynthesis, and chemo-enzymatic synthesis, focusing on the past 5 years. Emerging applications of cell-free biosynthesis and high-throughput tools are included that culminate in a discussion of the future outlook and perspective of isoprenoid biosynthetic engineering.

Synthetic biology enabling access to designer polyketides.

The full potential of polyketide discovery has yet to be reached owing to a lack of suitable technologies and knowledge required to advance engineering of polyketide biosynthesis. Recent investigations on the discovery, enhancement, and non-natural use of these biosynthetic gene clusters via computational biology, metabolic engineering, structural biology, and enzymology-guided approaches have facilitated improved access to designer polyketides. Here, we discuss recent successes in gene cluster discovery, host strain engineering, precursor-directed biosynthesis, combinatorial biosynthesis, polyketide tailoring, and high-throughput synthetic biology, as well as challenges and outlooks for rapidly generating useful target polyketides.

Cheminformatics Analysis and Modeling with MacrolactoneDB.

Macrolactones, macrocyclic lactones with at least twelve atoms within the core ring, include diverse natural products such as macrolides with potent bioactivities (e.g. antibiotics) and useful drug-like characteristics. We have developed MacrolactoneDB, which integrates nearly 14,000 existing macrolactones and their bioactivity information from different public databases, and new molecular descriptors to better characterize macrolide structures. The chemical distribution of MacrolactoneDB was analyzed in terms of important molecular properties and we have utilized three targets of interest (Plasmodium falciparum, Hepatitis C virus and T-cells) to demonstrate the value of compiling this data. Regression machine learning models were generated to predict biological endpoints using seven molecular descriptor sets and eight machine learning algorithms. Our results show that merging descriptors yields the best predictive power with Random Forest models, often boosted by consensus or hybrid modeling approaches. Our study provides cheminformatics insights into this privileged, underexplored structural class of compounds with high therapeutic potential.

Development of a Genetically Encoded Biosensor for Detection of Polyketide Synthase Extender Units in Escherichia coli.

The scaffolds of polyketides are constructed via assembly of extender units based on malonyl-CoA and its derivatives that are substituted at the C2-position with diverse chemical functionality. Subsequently, a transcription-factor-based biosensor for malonyl-CoA has proven to be a powerful tool for detecting malonyl-CoA, facilitating the dynamic regulation of malonyl-CoA biosynthesis and guiding high-throughput engineering of malonyl-CoA-dependent processes. Yet, a biosensor for the detection of malonyl-CoA derivatives has yet to be reported, severely restricting the application of high-throughput synthetic biology approaches to engineering extender unit biosynthesis and limiting the ability to dynamically regulate the biosynthesis of polyketide products that are dependent on such α-carboxyacyl-CoAs. Herein, the FapR biosensor was re-engineered and optimized for a range of mCoA concentrations across a panel of E. coli strains. The effector specificity of FapR was probed by cell-free transcription-translation, revealing that a variety of non-native and non-natural acyl-thioesters are FapR effectors. This FapR promiscuity proved sufficient for the detection of the polyketide extender unit methylmalonyl-CoA in E. coli, providing the first reported genetically encoded biosensor for this important metabolite. As such, the previously unknown broad effector promiscuity of FapR provides a platform to develop new tools and approaches that can be leveraged to overcome limitations of pathways that construct diverse α-carboxyacyl-CoAs and those that are dependent on them, including biofuels, antibiotics, anticancer drugs, and other value-added products.

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production.

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene (ADH) pathway serve to leverage consecutive kinases to convert exogenous alcohols into pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway-isopentenyl phosphate kinase from Thermoplasma acidophilum-towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide unnatural pyrophosphates and provide a critical first step in characterizing the rate-limiting steps in the artificial ADH pathway.

An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism.

Isoprenoids are constructed in nature using hemiterpene building blocks that are biosynthesized from lengthy enzymatic pathways with little opportunity to deploy precursor-directed biosynthesis. Here, an artificial alcohol-dependent hemiterpene biosynthetic pathway was designed and coupled to several isoprenoid biosynthetic systems, affording lycopene and a prenylated tryptophan in robust yields. This approach affords a potential route to diverse non-natural hemiterpenes and by extension isoprenoids modified with non-natural chemical functionality. Accordingly, the prototype chemo-enzymatic pathway is a critical first step toward the construction of engineered microbial strains for bioconversion of simple scalable building blocks into complex isoprenoid scaffolds.

Engineering the Substrate Specificity of a Modular Polyketide Synthase for Installation of Consecutive Non-Natural Extender Units.

There is significant interest in diversifying the structures of polyketides to create new analogues of these bioactive molecules. This has traditionally been done by focusing on engineering the acyltransferase (AT) domains of polyketide synthases (PKSs) responsible for the incorporation of malonyl-CoA extender units. Non-natural extender units have been utilized by engineered PKSs previously; however, most of the work to date has been accomplished with ATs that are either naturally promiscuous and/or located in terminal modules lacking downstream bottlenecks. These limitations have prevented the engineering of ATs with low native promiscuity and the study of any potential gatekeeping effects by domains downstream of an engineered AT. In an effort to address this gap in PKS engineering knowledge, the substrate preferences of the final two modules of the pikromycin PKS were compared for several non-natural extender units and through active site mutagenesis. This led to engineering of the methylmalonyl-CoA specificity of both modules and inversion of their selectivity to prefer consecutive non-natural derivatives. Analysis of the product distributions of these bimodular reactions revealed unexpected metabolites resulting from gatekeeping by the downstream ketoreductase and ketosynthase domains. Despite these new bottlenecks, AT engineering provided the first full-length polyketide products incorporating two non-natural extender units. Together, this combination of tandem AT engineering and the identification of previously poorly characterized bottlenecks provides a platform for future advancements in the field.

Extender Unit Promiscuity and Orthogonal Protein Interactions of an Aminomalonyl-ACP Utilizing Trans-Acyltransferase from Zwittermicin Biosynthesis.

Trans-acting acyltransferases (trans-ATs) are standalone enzymes that select and deliver extender units to polyketide synthase assembly lines. Accordingly, there is interest in leveraging trans-ATs as tools to regioselectively diversify polyketide structures. Yet, little is known regarding the extender unit and acyl carrier protein (ACP) specificity of trans-ATs, particularly those that utilize unusual ACP-linked extender units. For example, the biosynthesis of the antibiotic zwittermicin involves the trans-AT ZmaF, which is responsible for installing a rare ACP-linked aminomalonyl extender unit. Here, we developed a method to access a panel of non-natural and non-native ACP-linked extender units and used it to probe the promiscuity of ZmaF, revealing one of the most promiscuous ATs characterized to date. Furthermore, we demonstrated that ZmaF is highly orthogonal with respect to its ACP specificity, and the ability of ZmaF to trans-complement noncognate PKS modules was also explored. Together, these results set the stage for further engineering ZmaF as a tool for polyketide diversification.

Engineering enzymatic assembly lines for the production of new antimicrobials.

A large portion of natural products are biosynthesized by the polyketide synthase and non-ribosomal peptide synthetase enzymatic assembly lines. Recent advancements in the study of these megasynthases has led to many new examples that demonstrate the production of non-natural natural products. The field is likely nearing the ability to design and build new biosynthetic pathways de novo. We discuss the various recent approaches taken towards this goal, focusing on the installation of new substrates, the swapping of enzymatic domains and modules, and the impact of metabolic engineering and synthetic biology. We also address the challenges remaining alongside the many successes in this area.

Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI.

Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations. Electrospray post-ionization can be used to circumvent many sample cleanup and desalting steps. SESI and IR-MALDESI are two examples of ionization methods that employ electrospray post-ionization at atmospheric pressure and temperature. By coupling the two techniques and doping the electrospray solvent with silver ions, olefinic terpenes of different classes and varying degrees of volatility were directly analyzed from a biological buffer system with no sample workup steps.

Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology.

Macrolides are a large group of natural products that display broad and potent biological activities and are biosynthesized by type I polyketide synthases (PKSs) and associated enzymatic machinery. There is an urgent need to access macrolides and unnatural macrolide derivatives for drug discovery, drug manufacture, and probe development. Typically, efforts to engineer the biosynthesis of macrolides and macrolide analogues in various microbial hosts are hampered by the complexity of macrolide biosynthetic pathways and our limited ability to rationally reprogram type I PKSs and post-PKS machinery. High-throughput approaches based on synthetic biology and directed evolution could overcome this problem by testing the function of large libraries of variants. Yet, methods that can identify mutant enzymes, pathways, and strains that produce the desired macrolide target are not generally available. Here we show that the promiscuous macrolide sensing transcription factor MphR is a powerful platform for engineering variants with tailored properties. We identified variants that displayed improved sensitivity toward erythromycin, tailored the inducer specificity, and significantly improved sensitivity to macrolides that were very poor inducers of the wild-type MphR biosensor. Designer macrolide biosensors should find broad utility and enable applications related to high-throughput synthetic biology and directed evolution of macrolide biosynthesis.

Polyketide Bioderivatization Using the Promiscuous Acyltransferase KirCII.

During polyketide biosynthesis, acyltransferases (ATs) are the essential gatekeepers which provide the assembly lines with precursors and thus contribute greatly to structural diversity. Previously, we demonstrated that the discrete AT KirCII from the kirromycin antibiotic pathway accesses nonmalonate extender units. Here, we exploit the promiscuity of KirCII to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by "click" chemistry.

Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering.

Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.