Biosynthesis and synthetic biology of psychoactive natural products.

Psychoactive natural products play an integral role in the modern world. The tremendous structural complexity displayed by such molecules confers diverse biological activities of significant medicinal value and sociocultural impact. Accordingly, in the last two centuries, immense effort has been devoted towards establishing how plants, animals, and fungi synthesize complex natural products from simple metabolic precursors. The recent explosion of genomics data and molecular biology tools has enabled the identification of genes encoding proteins that catalyze individual biosynthetic steps. Once fully elucidated, the "biosynthetic pathways" are often comparable to organic syntheses in elegance and yield. Additionally, the discovery of biosynthetic enzymes provides powerful catalysts which may be repurposed for synthetic biology applications, or implemented with chemoenzymatic synthetic approaches. In this review, we discuss the progress that has been made toward biosynthetic pathway elucidation amongst four classes of psychoactive natural products: hallucinogens, stimulants, cannabinoids, and opioids. Compounds of diverse biosynthetic origin - terpene, amino acid, polyketide - are identified, and notable mechanisms of key scaffold transforming steps are highlighted. We also provide a description of subsequent applications of the biosynthetic machinery, with an emphasis placed on the synthetic biology and metabolic engineering strategies enabling heterologous production.

Total Synthesis of (-)-Strictosidine and Interception of Aryne Natural Product Derivatives "Strictosidyne" and "Strictosamidyne".

Monoterpene indole alkaloids are a large class of natural products derived from a single biosynthetic precursor, strictosidine. We describe a synthetic approach to strictosidine that relies on a key facially selective Diels-Alder reaction between a glucosyl-modified alkene and an enal to set the C15-C20-C21 stereotriad. DFT calculations were used to examine the origin of stereoselectivity in this key step, wherein two of 16 possible isomers are predominantly formed. These calculations suggest the presence of a glucosyl unit, also inherent in the strictosidine structure, guides diastereoselectivity, with the reactive conformation of the vinyl glycoside dienophile being controlled by an exo-anomeric effect. (-)-Strictosidine was subsequently accessed using late-stage synthetic manipulations and an enzymatic Pictet-Spengler reaction. Several new natural product analogs were also accessed, including precursors to two unusual aryne natural product derivatives termed "strictosidyne" and "strictosamidyne". These studies provide a strategy for accessing glycosylic natural products and a new platform to access monoterpene indole alkaloids and their derivatives.

Engineered mitochondrial production of monoterpenes in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) from plants encompass a broad class of structurally complex and medicinally valuable natural products. MIAs are biologically derived from the universal precursor strictosidine. Although the strictosidine biosynthetic pathway has been identified and reconstituted, extensive work is required to optimize production of strictosidine and its precursors in yeast. In this study, we engineered a fully integrated and plasmid-free yeast strain with enhanced production of the monoterpene precursor geraniol. The geraniol biosynthetic pathway was targeted to the mitochondria to protect the GPP pool from consumption by the cytosolic ergosterol pathway. The mitochondrial geraniol producer showed a 6-fold increase in geraniol production compared to cytosolic producing strains. We further engineered the monoterpene-producing strain to synthesize the next intermediates in the strictosidine pathway: 8-hydroxygeraniol and nepetalactol. Integration of geraniol hydroxylase (G8H) from Catharanthus roseus led to essentially quantitative conversion of geraniol to 8-hydroxygeraniol at a titer of 227 mg/L in a fed-batch fermentation. Further introduction of geraniol oxidoreductase (GOR) and iridoid synthase (ISY) from C. roseus and tuning of the relative expression levels resulted in the first de novo nepetalactol production. The strategies developed in this work can facilitate future strain engineering for yeast production of later intermediates in the strictosidine biosynthetic pathway.

Production of semi-biosynthetic nepetalactone in yeast.

Microbial-based production of natural products provides a promising alternative to synthetic production and isolation from the native producer. The recently discovered NEPS1 cyclase/oxidase completes the biosynthetic pathway to nepetalactone, a biologically relevant iridoid known as both an insect repellent and cat attractant. In this work, we employ yeast-based whole-cell biocatalysis to produce semi-biosynthetic nepetalactone from a low-cost precursor via a four-step enzymatic process. The dependence of product yield on bioprocess parameters ranging from induction of gene expression to substrate loading was investigated. Subsequent factorial design and response surface methodology optimization approach enabled a 5.8-fold increase in nepetalactone titer to 153 mg/L. Our study provides insights into strategies for operating plasmid-based bioconversion of a fed substrate and sets the stage for scalable, microbial synthesis of nepetalactone.

Biosynthesis of Heptacyclic Duclauxins Requires Extensive Redox Modifications of the Phenalenone Aromatic Polyketide.

Duclauxins are dimeric and heptacyclic fungal polyketides with notable bioactivities. We characterized the cascade of redox transformations in the biosynthetic pathway of duclauxin from Talaromyces stipitatus. The redox reaction sequence is initiated by a cupin family dioxygenase DuxM that performs an oxidative cleavage of the peri-fused tricyclic phenalenone and affords a transient hemiketal-oxaphenalenone intermediate. Additional redox enzymes then morph the oxaphenoalenone into either an anhydride or a dihydrocoumarin-containing monomeric building block that is found in dimeric duxlauxins. Oxidative coupling between the monomers to form the initial C-C bond was shown to be catalyzed by a P450 monooxygenase, although the enzyme responsible for the second C-C bond formation was not found in the pathway. Collectively, the number and variety of redox enzymes used in the duclauxin pathway showcase Nature's strategy to generate structural complexity during natural product biosynthesis.

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,ß-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

ß-Terrecyclene synthase constructs the quadrane backbone in terrecyclic acid biosynthesis.

Quadrane sesquiterpenes featuring a distinctive tricyclic skeleton exhibit potent antimicrobial and anticancer activities. Although extensive studies have attempted to reveal the multistep carbocation rearrangement involved in the formation of the tricyclic quadrane scaffold, the exact biosynthetic pathway and chemical logic to generate the quadrane structure remains mysterious. Here we identified a novel sesquiterpene synthase that is capable of generating ß-terrecyclene possessing the quadrane scaffold and characterized the biosynthetic pathway of a representative fungal quadrane terrecyclic acid. Further mutagenesis coupled with isotopically sensitive branching studies of this ß-terrecyclene synthase provided insight into the mechanism involved in the formation of the quadrane scaffold.

Engineered Production of Strictosidine and Analogues in Yeast.

Monoterpene indole alkaloids (MIAs) are an expansive class of plant natural products, many of which have been named on the World Health Organization's List of Essential Medicines. Low production from native plant hosts necessitates a more reliable source of these drugs to meet global demand. Here, we report the development of a yeast-based platform for high-titer production of the universal MIA precursor, strictosidine. Our fed-batch platform produces ∼50 mg/L strictosidine, starting from the commodity chemicals geraniol and tryptamine. The microbially produced strictosidine was purified to homogeneity and characterized by NMR. Additionally, our approach enables the production of halogenated strictosidine analogues through the feeding of modified tryptamines. The MIA platform strain enables rapid access to strictosidine for reconstitution and production of downstream MIA natural products.

Cell-Free Total Biosynthesis of Plant Terpene Natural Products using an Orthogonal Cofactor Regeneration System.

Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD+-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD+ to NADH and a low ratio of NADP+ to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450; and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD+-dependent dehydrogenase to the one-pot mixture led to ~1 g/L of nepetalactone, the active cat- attractant in catnip.

Technology development for natural product biosynthesis in Saccharomyces cerevisiae.

The explosion of genomic sequence data and the significant advancements in synthetic biology have led to the development of new technologies for natural products discovery and production. Using powerful genetic tools, the yeast Saccharomyces cerevisiae has been engineered as a production host for natural product pathways from bacterial, fungal, and plant species. With an expanding library of characterized genetic parts, biosynthetic pathways can be refactored for optimized expression in yeast. New engineering strategies have enabled the increased production of valuable secondary metabolites by tuning metabolic pathways. Improvements in high-throughput screening methods have facilitated the rapid identification of variants with improved biosynthetic capabilities. In this review, we focus on the molecular tools and engineering strategies that have recently empowered heterologous natural product biosynthesis.