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This review describes examples of the broadening industrial relevance of engineered secondary metabolism enzymes, natural products and analogs being made with these enzymes, and technology improvements that have enabled their development since 1999.
Abstract Nonstandard amino acids (nsAAs) that are l ‐phenylalanine derivatives with aryl ring functionalization have long been harnessed in natural product synthesis, therapeutic peptide synthesis, and diverse applications of genetic code expansion. Yet, to date, these chiral molecules have often been the products of poorly enantioselective and environmentally harsh organic synthesis routes. Here, we reveal the broad specificity of multiple natural pyridoxal 5′‐phosphate (PLP)‐dependent enzymes, specifically an l ‐threonine transaldolase, a phenylserine dehydratase, and an aminotransferase, toward substrates that contain aryl side chains with diverse substitutions. We exploit this tolerance to construct a one‐pot biocatalytic cascade that achieves high‐yield synthesis of 18 diverse l ‐phenylalanine derivatives from aldehydes under mild aqueous reaction conditions. We demonstrate the addition of a carboxylic acid reductase module to this cascade to enable the biosynthesis of l ‐phenylalanine derivatives from carboxylic acids that may be less expensive or less reactive than the corresponding aldehydes. Finally, we investigate the scalability of the cascade by developing a lysate‐based route for preparative‐scale synthesis of 4‐formyl‐ l ‐phenylalanine, a nsAA with a bio‐orthogonal handle that is not readily market‐accessible. Overall, this work offers an efficient, versatile, and scalable route with the potential to lower manufacturing costs and democratize synthesis for many valuable nsAAs.
Abstract Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli . Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus , RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.
Materials, methods of making, and methods of providing a trimetallic oxygen carrier for converting methane containing fuel to synthesis gas. The trimetallic oxygen carrier comprises CuxFeyMnzOt, where CuxFeyMnzOt is a chemical composition with 0 < x ≤ 3 and 0 < y ≤ 3 and 0 < z ≤ 3 and, 0 < t ≤ 5. For example, CuxFeyMnzOt may be one of CuMnFeO 4 , CuFe 0.5 Mn 1.5 O 4 , CuFeMn 2 O 4 , CuFe 2 MnO 4 , or Cu impregnated on FerMnsOu, Fe impregnated on CurMnsOu, Mn impregnated on CurFesOu where r > 0, s > 0 and u > 0 and combinations thereof. Reaction of trimetallic CuxFeyMnzOt with methane generates a product stream comprising at least 50 vol. % CO and H 2 .
The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate ( S )-reticuline to 4.6 g L –1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.
Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein’s catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. In conclusion, the reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb’s ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.
Abstract Halocyclization of alkenes is a powerful bond-forming tool in synthetic organic chemistry and a key step in natural product biosynthesis, but catalyzing halocyclization with high enantioselectivity remains a challenging task. Identifying suitable enzymes that catalyze enantioselective halocyclization of simple olefins would therefore have significant synthetic value. Flavin-dependent halogenases (FDHs) catalyze halogenation of arene and enol(ate) substrates. Herein, we reveal that FDHs engineered to catalyze site-selective aromatic halogenation also catalyze non-native bromolactonization of olefins with high enantioselectivity and near-native catalytic proficiency. Highly selective halocyclization is achieved by characterizing and mitigating the release of HOBr from the FDH active site using a combination of reaction optimization and protein engineering. The structural origins of improvements imparted by mutations responsible for the emergence of halocyclase activity are discussed. This expansion of FDH catalytic activity presages the development of a wide range of biocatalytic halogenation reactions.
Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.
The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.
Monensin A is a prototypical natural polyether polyketide antibiotic. It acts by binding a metal cation and facilitating its transport across the cell membrane. Biosynthesis of monensin A involves construction of a polyene polyketide backbone, subsequent epoxidation of the alkenes, and, lastly, formation of cyclic ethers via epoxide-opening cyclization. MonCI, a flavin-dependent monooxygenase, is thought to transform all three alkenes in the intermediate polyketide premonensin A into epoxides. Our crystallographic study has revealed that MonCI’s exquisite stereocontrol is due to the preorganization of the active site residues which allows only one specific face of the alkene to approach the reactive C(4a)-hydroperoxyflavin moiety. Furthermore, MonCI has an unusually large substrate-binding cavity that can accommodate premonensin A in an extended or folded conformation which allows any of the three alkenes to be placed next to C(4a)-hydroperoxyflavin. Importantly, MonCI, with its ability to perform multiple epoxidations on the same substrate in a stereospecific manner, demonstrates the extraordinary versatility of the flavin-dependent monooxygenase family of enzymes.
Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.
Correction to: Nature Communicationshttps://doi.org/10.1038/s41467-024-54578-3, published online 27 November 2024. The original version of this Article omitted a line in the Acknowledgements section: Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. This has been now added into the last line of the acknowledgements section in both the PDF and HTML versions of the Article.
Background - Nudibranchs comprise a group of > 6000 marine soft-bodied mollusk species known to use secondary metabolites (natural products) for chemical defense. The full diversity of these metabolites and whether symbiotic microbes are responsible for their synthesis remains unexplored. Another issue in searching for undiscovered natural products is that computational analysis of genomes of uncultured microbes can result in detection of novel biosynthetic gene clusters; however, their in vivo functionality is not guaranteed which limits further exploration of their pharmaceutical or industrial potential. To overcome these challenges, we used a fluorescent pantetheine probe, which produces a fluorescent CoA-analog employed in biosynthesis of secondary metabolites, to label and capture bacterial symbionts actively producing these compounds in the mantle of the nudibranch Doriopsilla fulva. Results - We recovered the genome of Candidatus Doriopsillibacter californiensis from the Ca. Tethybacterales order, an uncultured lineage of sponge symbionts not found in nudibranchs previously. It forms part of the core skin microbiome of D. fulva and is nearly absent in its internal organs. We showed that crude extracts of D. fulva contained secondary metabolites that were consistent with the presence of a beta-lactone encoded in Ca. D. californiensis genome. Beta-lactones represent an underexplored group of secondary metabolites with pharmaceutical potential that have not been reported in nudibranchs previously. Conclusions - Altogether, this study shows how probe-based, targeted sorting approaches can capture bacterial symbionts producing secondary metabolites in vivo.
Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to > 99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.
Humans around the globe have lived through an unprecedented time. The emergence and rapid spread of coronavirus disease 2019 (COVID-19) have resulted in the loss of millions of lives, and this number is still climbing every day and hour. As a consequence, there have been drastic changes over the past two years in how we live, communicate, and work, some of which may have changed forever: remote conferencing has become a new norm for businesses, schools, and academia; work-from-home and hybrid working is now more than just acceptable in many industries; most importantly, COVID-19 has raised global awareness of infectious diseases and pandemic responses to an exceptional level. Certainly, COVID-19 is not the only disease that is causing human suffering at this moment. Other highly contagious and lethal viruses, such as the human immunodeficiency virus (HIV), Ebola, and the recent addition to the list, Monkeypox, pose extraordinary challenges to modern civilizations. On the other hand, non-communicable diseases, such as heart disease, stroke, and cancer, are still claiming the majority of deaths on a global level (World Health Organization, 2020). Before the availability of synthetic drugs, civilizations worldwide relied on natural products extracted from the environment and various organisms for healthcare. Natural products and their derivatives continue to be critical components in the drug library available today. Indeed, almost a quarter of the new drugs approved by the US Food and Drug Administration (FDA) since 1981 are natural products, botanical mixtures, or their derivatives (Newman and Cragg, 2020). Recent developments in analytical characterization, genome mining, and engineering have further increased interest in drug discovery from the pool of natural products (Atanasov et al., 2021). This Research Topic celebrates a collection of studies at the intersection of natural product research and nanotechnological advances. As a successor and extension of the Research Topic in “Nanotechnology in Traditional Medicines and Natural Products” published last year (Zhang et al., 2021), the present collection focused more on recent developments in employing the synthesis and characterization of nanostructures for the delivery of functional natural products. Specifically, the use of carrier nanostructures to conjugate or encapsulate therapeutically active natural products with carrier nanostructures could enable their robust, targeted delivery tailored to either tackle a specific disease or unlock a drug administration pathway. Over the last decade, nanostructures such as liposomes (Pattni et al., 2015) and lipid nanoparticles (Hou et al., 2021) have been developed as versatile platforms for the inclusion and delivery of a wide range of therapeutical compounds; a significant advance in this aspect is the stabilization of mRNA strands for COVID-19 vaccination recently produced by Pfizer/BioNTech and Moderna (Tenchov et al., 2021). Such engineered nanomaterials are now widely used in the development of new drugs, while regulatory forces, such as the US FDA, remain cautious with the evaluation of each product on a case-by-case basis (Wang and Grainger, 2022). In this collection, Zeng et al. provided a timely review of natural products conjugated to and contained within nanomaterials for the ongoing COVID-19 pandemic. Natural products that are potentially useful in the mitigation of this disease, such as chloroquine and curcumin, supported in nanocarrier systems, have been discussed in detail. More broadly, Cheng et al. comprehensively reviewed the use of liposomes as a nanocarrier for natural products. This article provides a summary of natural product compounds and extracts used to form liposomal structures. Readers can further enjoy the practical aspects of these engineered drugs for the treatment of specific diseases as well as insights into future directions in this field.
Engineering microbes for the production of valuable natural products is often hindered by the regulation of native competing metabolic networks in host. This is particularly evident in the case of terpenoid synthesis in yeast, where the canonical terpenoid precursors are tightly coupled to the biosynthesis of sterols essential for yeast viability. One way to circumvent this limitation is by engineering product pathways less connected to the host native metabolism. Here, we introduce a two-step isopentenol utilization pathway (IUP) in Saccharomyces cerevisiae to augment the native mevalonate pathway by providing a shortcut to the synthesis of the common terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). As such, the IUP was capable of elevating the IPP/DMAPP pool by 147-fold compared with the native pathway. We further demonstrate that cofeeding isoprenol and prenol enhances geranyl diphosphate (GPP) content for monoterpene biosynthesis. More importantly, we established a synthetic three-step route for efficient synthesis of di-and tetraterpene precursor geranylgeranyl diphosphate (GGPP), circumventing the competition with farnesyl diphosphate (FPP) for sterol biosynthesis and elevating the GGPP level by 374-fold. We combine these IUP-supported precursor-forming platforms with downstream terpene synthases to harness their potential and improve the production of industrially relevant terpenoids by several fold. In conclusion, our exploration provides a universal and effective platform for supporting terpenoid synthesis in yeast.
Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.