Triumphs and Challenges of Natural Product Discovery in the Postgenomic Era.

Natural products have played significant roles as medicine and food throughout human history. Here, we first provide a brief historical overview of natural products, their classification and biosynthetic origins, and the microbiological and genetic methods used for their discovery. We also describe and discuss the technologies that revolutionized the field, which transitioned from classic genetics to genome-centric discovery approximately two decades ago. We then highlight the most recent advancements and approaches in the current postgenomic era, in which genome mining is a standard operation and high-throughput analytical methods allow parallel discovery of genes and molecules at an unprecedented pace. Finally, we discuss the new challenges faced by the field of natural products and the future of systematic heterologous expression and strain-independent discovery, which promises to deliver more molecules in vials than ever before.

Biofuels for a sustainable future.

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.

Engineering Cellular Metabolism.

Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation.

Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Integrating biological redesign: where synthetic biology came from and where it needs to go.

Synthetic biology seeks to extend approaches from engineering and computation to redesign of biology, with goals such as generating new chemicals, improving human health, and addressing environmental issues. Early on, several guiding principles of synthetic biology were articulated, including design according to specification, separation of design from fabrication, use of standardized biological parts and organisms, and abstraction. We review the utility of these principles over the past decade in light of the field's accomplishments in building complex systems based on microbial transcription and metabolism and describe the progress in mammalian cell engineering.

Acetate dependence of tumors.

Acetyl-CoA represents a central node of carbon metabolism that plays a key role in bioenergetics, cell proliferation, and the regulation of gene expression. Highly glycolytic or hypoxic tumors must produce sufficient quantities of this metabolite to support cell growth and survival under nutrient-limiting conditions. Here, we show that the nucleocytosolic acetyl-CoA synthetase enzyme, ACSS2, supplies a key source of acetyl-CoA for tumors by capturing acetate as a carbon source. Despite exhibiting no gross deficits in growth or development, adult mice lacking ACSS2 exhibit a significant reduction in tumor burden in two different models of hepatocellular carcinoma. ACSS2 is expressed in a large proportion of human tumors, and its activity is responsible for the majority of cellular acetate uptake into both lipids and histones. These observations may qualify ACSS2 as a targetable metabolic vulnerability of a wide spectrum of tumors.

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes.

Plastid-derived signals are known to coordinate expression of nuclear genes encoding plastid-localized proteins in a process termed retrograde signaling. To date, the identity of retrograde-signaling molecules has remained elusive. Here, we show that methylerythritol cyclodiphosphate (MEcPP), a precursor of isoprenoids produced by the plastidial methylerythritol phosphate (MEP) pathway, elicits the expression of selected stress-responsive nuclear-encoded plastidial proteins. Genetic and pharmacological manipulations of the individual MEP pathway metabolite levels demonstrate the high specificity of MEcPP as an inducer of these targeted stress-responsive genes. We further demonstrate that abiotic stresses elevate MEcPP levels, eliciting the expression of the aforementioned genes. We propose that the MEP pathway, in addition to producing isoprenoids, functions as a stress sensor and a coordinator of expression of targeted stress-responsive nuclear genes via modulation of the levels of MEcPP, a specific and critical retrograde-signaling metabolite.

Complete biosynthesis of the potent vaccine adjuvant QS-21.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

Mechanotransduction and extracellular matrix homeostasis.

Soft connective tissues at steady state are dynamic; resident cells continually read environmental cues and respond to them to promote homeostasis, including maintenance of the mechanical properties of the extracellular matrix (ECM) that are fundamental to cellular and tissue health. The mechanosensing process involves assessment of the mechanics of the ECM by the cells through integrins and the actomyosin cytoskeleton, and is followed by a mechanoregulation process, which includes the deposition, rearrangement or removal of the ECM to maintain overall form and function. Progress towards understanding the molecular, cellular and tissue-level effects that promote mechanical homeostasis has helped to identify key questions for future research.

Author Correction: Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

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

The expansion of agriculture has shaped the recent evolutionary history of a specialized squash pollinator.

The expansion of agriculture is responsible for the mass conversion of biologically diverse natural environments into managed agroecosystems dominated by a handful of genetically homogeneous crop species. Agricultural ecosystems typically have very different abiotic and ecological conditions from those they replaced and create potential niches for those species that are able to exploit the abundant resources offered by crop plants. While there are well-studied examples of crop pests that have adapted into novel agricultural niches, the impact of agricultural intensification on the evolution of crop mutualists such as pollinators is poorly understood. We combined genealogical inference from genomic data with archaeological records to demonstrate that the Holocene demographic history of a wild specialist pollinator of Cucurbita (pumpkins, squashes, and gourds) has been profoundly impacted by the history of agricultural expansion in North America. Populations of the squash bee Eucera pruinosa experienced rapid growth in areas where agriculture intensified within the past 1,000 y, suggesting that the cultivation of Cucurbita in North America has increased the amount of floral resources available to these bees. In addition, we found that roughly 20% of this bee species' genome shows signatures of recent selective sweeps. These signatures are overwhelmingly concentrated in populations from eastern North America where squash bees were historically able to colonize novel environments due to human cultivation of Cucurbita pepo and now exclusively inhabit agricultural niches. These results suggest that the widespread cultivation of crops can prompt adaptation in wild pollinators through the distinct ecological conditions imposed by agricultural environments.

Evolutionarily diverse origins of deformed wing viruses in western honey bees.

Novel transmission routes can allow infectious diseases to spread, often with devastating consequences. Ectoparasitic varroa mites vector a diversity of RNA viruses, having switched hosts from the eastern to western honey bees (Apis cerana to Apis mellifera). They provide an opportunity to explore how novel transmission routes shape disease epidemiology. As the principal driver of the spread of deformed wing viruses (mainly DWV-A and DWV-B), varroa infestation has also driven global honey bee health declines. The more virulent DWV-B strain has been replacing the original DWV-A strain in many regions over the past two decades. Yet, how these viruses originated and spread remains poorly understood. Here, we use a phylogeographic analysis based on whole-genome data to reconstruct the origins and demography of DWV spread. We found that, rather than reemerging in western honey bees after varroa switched hosts, as suggested by previous work, DWV-A most likely originated in East Asia and spread in the mid-20th century. It also showed a massive population size expansion following the varroa host switch. By contrast, DWV-B was most likely acquired more recently from a source outside East Asia and appears absent from the original varroa host. These results highlight the dynamic nature of viral adaptation, whereby a vector's host switch can give rise to competing and increasingly virulent disease pandemics. The evolutionary novelty and rapid global spread of these host-virus interactions, together with observed spillover into other species, illustrate how increasing globalization poses urgent threats to biodiversity and food security.

High-throughput metabolic engineering: advances in small-molecule screening and selection.

Metabolic engineering for the overproduction of high-value small molecules is dependent upon techniques in directed evolution to improve production titers. The majority of small molecules targeted for overproduction are inconspicuous and cannot be readily obtained by screening. We provide a review on the development of high-throughput colorimetric, fluorescent, and growth-coupled screening techniques, enabling inconspicuous small-molecule detection. We first outline constraints on throughput imposed during the standard directed evolution workflow (library construction, transformation, and screening) and establish a screening and selection ladder on the basis of small-molecule assay throughput and sensitivity. An in-depth analysis of demonstrated screening and selection approaches for small-molecule detection is provided. Particular focus is placed on in vivo biosensor-based detection methods that reduce or eliminate in vitro assay manipulations and increase throughput. We conclude by providing our prospectus for the future, focusing on transcription factor-based detection systems as a natural microbial mode of small-molecule detection.

Reciprocal regulation of cardiac ß-oxidation and pyruvate dehydrogenase by insulin.

The heart alters the rate and relative oxidation of fatty acids and glucose based on availability and energetic demand. Insulin plays a crucial role in this process diminishing fatty acid and increasing glucose oxidation when glucose availability increases. Loss of insulin sensitivity and metabolic flexibility can result in cardiovascular disease. It is therefore important to identify mechanisms by which insulin regulates substrate utilization in the heart. Mitochondrial pyruvate dehydrogenase (PDH) is the key regulatory site for the oxidation of glucose for ATP production. Nevertheless, the impact of insulin on PDH activity has not been fully delineated, particularly in the heart. We sought in vivo evidence that insulin stimulates cardiac PDH and that this process is driven by the inhibition of fatty acid oxidation. Mice injected with insulin exhibited dephosphorylation and activation of cardiac PDH. This was accompanied by an increase in the content of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), and, thus, mitochondrial import of fatty acids. Administration of the CPT1 inhibitor oxfenicine was sufficient to activate PDH. Malonyl-CoA is produced by acetyl-CoA carboxylase (ACC). Pharmacologic inhibition or knockout of cardiac ACC diminished insulin-dependent production of malonyl-CoA and activation of PDH. Finally, circulating insulin and cardiac glucose utilization exhibit daily rhythms reflective of nutritional status. We demonstrate that time-of-day-dependent changes in PDH activity are mediated, in part, by ACC-dependent production of malonyl-CoA. Thus, by inhibiting fatty acid oxidation, insulin reciprocally activates PDH. These studies identify potential molecular targets to promote cardiac glucose oxidation and treat heart disease.

Promiscuous feeding on multiple adult honey bee hosts amplifies the vectorial capacity of Varroa destructor.

Varroa destructor is a cosmopolitan pest and leading cause of colony loss of the European honey bee. Historically described as a competent vector of honey bee viruses, this arthropod vector is the cause of a global pandemic of Deformed wing virus, now endemic in honeybee populations in all Varroa-infested regions. Our work shows that viral spread is driven by Varroa actively switching from one adult bee to another as they feed. Assays using fluorescent microspheres were used to indicate the movement of fluids in both directions between host and vector when Varroa feed. Therefore, Varroa could be in either an infectious or naïve state dependent upon the disease status of their host. We tested this and confirmed that the relative risk of a Varroa feeding depended on their previous host's infectiousness. Varroa exhibit remarkable heterogeneity in their host-switching behavior, with some Varroa infrequently switching while others switch at least daily. As a result, relatively few of the most active Varroa parasitize the majority of bees. This multiple-feeding behavior has analogs in vectorial capacity models of other systems, where promiscuous feeding by individual vectors is a leading driver of vectorial capacity. We propose that the honeybee-Varroa relationship offers a unique opportunity to apply principles of vectorial capacity to a social organism, as virus transmission is both vectored and occurs through multiple host-to-host routes common to a crowded society.

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast.

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. METHODS: Apoe-/- and Mmp12-/-/Apoe-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined. RESULTS: Unexpectedly, death from aortic rupture was significantly higher in Mmp12-/-/Apoe-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12-/- mice pointed to complement overactivation. Treatment with IgG-FH1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice. CONCLUSIONS: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Foldy: An open-source web application for interactive protein structure analysis.

Foldy is a cloud-based application that allows non-computational biologists to easily utilize advanced AI-based structural biology tools, including AlphaFold and DiffDock. With many deployment options, it can be employed by individuals, labs, universities, and companies in the cloud without requiring hardware resources, but it can also be configured to utilize locally available computers. Foldy enables scientists to predict the structure of proteins and complexes up to 6000 amino acids with AlphaFold, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. In our manuscript, we detail Foldy's interface design, deployment strategies, and optimization for various user scenarios. We demonstrate its application through case studies including rational enzyme design and analyzing proteins with domains of unknown function. Furthermore, we compare Foldy's interface and management capabilities with other open and closed source tools in the field, illustrating its practicality in managing complex data and computation tasks. Our manuscript underlines the benefits of Foldy as a day-to-day tool for life science researchers, and shows how Foldy can make modern tools more accessible and efficient.