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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.

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.

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.

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.

Main conclusion: Using virus-induced gene silencing, we demonstrated that the enzymes GES, ISY, and MLPL are responsible for nepetalactone biosynthesis in Nepeta cataria. Abstract: Nepetalactone is the main iridoid that is found in the Nepeta genus and is well-known for its psychoactive effect on house cats. Moreover, there is a burgeoning interest into the effect of nepetalactone on insects. Although the enzymes for nepetalactone biosynthesis have been biochemically assayed in vitro, validation of the role that these enzymes have in planta has not been demonstrated. Virus-induced gene silencing (VIGS) is a silencing method that relies on transient transformation and is an approach that has been particularly successful when applied to a variety of non-model plants. Here, we use a recently designed visual-marker dependent VIGS system to demonstrate that the nepetalactone biosynthetic enzymes GES, ISY, and MLPL impact nepetalactone biosynthesis in Nepeta cataria.

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.

Microorganisms and plants represent major sources of natural compounds with a plethora of bioactive properties. Among these, plant natural products (PNPs) remain indispensable to human health. With few exceptions, PNP-based pharmaceuticals come from plant specialized metabolisms and display a structure far too complex for a profitable production by total chemical synthesis. Accordingly, their industrial processes of supply are still mostly based on the extraction of final products or precursors directly from plant materials. This implies that particular contexts (e.g. pandemics, climate changes) and natural resource overexploitation are main drivers for the high production cost and recurrent supply shortages. Recently, biotechnological manufacturing alternatives gave rise to a multitude of benchmark studies implementing the production of important PNPs in various heterologous hosts. Here, we spotlight unprecedented advancements in the field of metabolic engineering dedicated to the heterologous production of a prominent series of PNPs that were achieved during the year 2020. We also discuss how the knowledge accumulated in recent years could pave the way for a broader manufacturing palette of natural products from a wide range of natural resources.

Synthetic Biology and Metabolic Engineering in Plants and Microbes: Part A, the new volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods, synthetic biology, and metabolic engineering in plants and microbes, and includes sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways.Continues the legacy of this premier serial with quality chapters authored by leaders in the field Contains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbesPresents sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways

Synthetic Biology and Metabolic Engineering in Plants and Microbes, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods, synthetic biology, and metabolic engineering in plants and microbes, and includes sections on such topics as the usage of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways.Continues the legacy of this premier serial with quality chapters authored by leaders in the field of enzymologyContains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbesIncludes sections on such topics as the uses of integrases in microbial engineering, biosynthesis and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways

Synthetic Biology and Metabolic Engineering in Plants and Microbes, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods, synthetic biology, and metabolic engineering in plants and microbes, and includes sections on such topics as the usage of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways.Continues the legacy of this premier serial with quality chapters authored by leaders in the field of enzymologyContains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbesIncludes sections on such topics as the uses of integrases in microbial engineering, biosynthesis and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways

Synthetic Biology and Metabolic Engineering in Plants and Microbes: Part A, the new volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods, synthetic biology, and metabolic engineering in plants and microbes, and includes sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways.Continues the legacy of this premier serial with quality chapters authored by leaders in the field Contains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbesPresents sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways

Synthetic Biology and Metabolic Engineering in Plants and Microbes: Part A, the new volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods, synthetic biology, and metabolic engineering in plants and microbes, and includes sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways.Continues the legacy of this premier serial with quality chapters authored by leaders in the field Contains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbesPresents sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways

Synthetic Biology and Metabolic Engineering in Plants and Microbes, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods, synthetic biology, and metabolic engineering in plants and microbes, and includes sections on such topics as the usage of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways.Continues the legacy of this premier serial with quality chapters authored by leaders in the field of enzymologyContains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbesIncludes sections on such topics as the uses of integrases in microbial engineering, biosynthesis and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

The secoiridoids are the main class of specialized metabolites present in olive (Olea europaea L.) fruit. In particular, the secoiridoid oleuropein strongly influences olive oil quality because of its bitterness, which is a desirable trait. In addition, oleuropein possesses a wide range of pharmacological properties, including antioxidant, anti-inflammatory, and anti-cancer activities. In accordance, obtaining high oleuropein varieties is a main goal of molecular breeding programs. Here we use a transcriptomic approach to identify candidate genes belonging to the secoiridoid pathway in olive. From these candidates, we have functionally characterized the olive homologue of iridoid synthase (OeISY), an unusual terpene cyclase that couples an NAD (P)H-dependent 1,4-reduction step with a subsequent cyclization, and we provide evidence that OeISY likely generates the monoterpene scaffold of oleuropein in olive fruits. OeISY, the first pathway gene characterized for this type of secoiridoid, is a potential target for breeding programs in a high value secoiridoid-accumulating species.