Discovery of a cytochrome P450 enzyme catalyzing the formation of spirooxindole alkaloid scaffold.

Spirooxindole alkaloids feature a unique scaffold of an oxindole ring sharing an atom with a heterocyclic moiety. These compounds display an extensive range of biological activities such as anticancer, antibiotics, and anti-hypertension. Despite their structural and functional significance, the establishment and rationale of the spirooxindole scaffold biosynthesis are yet to be elucidated. Herein, we report the discovery and characterization of a cytochrome P450 enzyme from kratom (Mitragyna speciosa) responsible for the formation of the spirooxindole alkaloids 3-epi-corynoxeine (3R, 7R) and isocorynoxeine (3S, 7S) from the corynanthe-type (3R)-secoyohimbane precursors. Expression of the newly discovered enzyme in Saccharomyces cerevisiae yeast allows for the efficient in vivo and in vitro production of spirooxindoles. This discovery highlights the versatility of plant cytochrome P450 enzymes in building unusual alkaloid scaffolds and opens a gateway to access the prestigious spirooxindole pharmacophore and its derivatives.

Leveraging synthetic biology and metabolic engineering to overcome obstacles in plant pathway elucidation.

Major hurdles in plant biosynthetic pathway elucidation and engineering include the need for rapid testing of enzyme candidates and the lack of complex substrates that are often not accumulated in the plant, amenable to synthesis, or commercially available. Linking metabolic engineering with gene discovery in both yeast and plant holds great promise to expedite the elucidation process and, at the same time, provide a platform for the sustainable production of plant metabolites. In this review, we highlight how synthetic biology and metabolic engineering alleviated longstanding obstacles in plant pathway elucidation. Recent advances in developing these chassis that showcase established and emerging strategies in accelerating biosynthetic gene discovery will also be discussed.

Discovery and Characterization of Oxidative Enzymes Involved in Monoterpenoid Indole Alkaloid Biosynthesis.

Monoterpene indole alkaloid (MIA) constitutes a structurally diverse plant natural product group with remarkable pharmacological activities. Many MIAs have been routinely used as potent drugs for several diseases, including leukemia (vinblastine), lung cancer (camptothecin), and malaria (quinine). Nevertheless, MIAs are biosynthesized at extremely low abundance in plants and, in many cases, require additional chemical functionalizations before their therapeutic uses. As oxygenations and oxidative rearrangements are critical throughout MIAs' structural scaffolding and modifications, the discovery and engineering of oxidative enzymes play essential roles in understanding and boosting the supplies of MIAs. Recent advances in omics technologies and synthetic biology have provided unprecedented amount of biochemical data and tools, paving a wide pathway for discovering, characterizing, and engineering enzymes involved in MIA biosynthesis. Here, we discuss the latest progress in understanding the roles of oxidative enzymes in MIA metabolism and describe a bioinformatic and biochemical pipeline to identify, characterize, and make use of these plant biocatalysts.

Germacrene A Synthases for Sesquiterpene Lactone Biosynthesis Are Expressed in Vascular Parenchyma Cells Neighboring Laticifers in Lettuce.

Sesquiterpene lactone (STL) and natural rubber (NR) are characteristic isoprenoids in lettuce (Lactuca sativa). Both STL and NR co-accumulate in laticifers, pipe-like structures located along the vasculature. NR-biosynthetic genes are exclusively expressed in laticifers, but cell-type specific expression of STL-biosynthetic genes has not been studied. Here, we examined the expression pattern of germacrene A synthase (LsGAS), which catalyzes the first step in STL biosynthesis in lettuce. Quantitative PCR and Illumina read mapping revealed that the transcripts of two GAS isoforms (LsGAS1/LsGAS2) are expressed two orders of magnitude (~100-200) higher in stems than laticifers. This result implies that the cellular site for LsGAS1/2 expression is not in laticifers. To gain more insights, promoters of LsGAS1/2 were cloned and fused to ß-glucuronidase (GUS), followed by transformations of lettuce with these promoter-GUS constructs. In in situ GUS assays, the GUS expression driven by the LsGAS1/2 promoters was tightly associated with vascular bundles. High-resolution microsections showed that GUS signals are not present in laticifers but are detected in the vascular parenchyma cells neighboring the laticifers. These results suggest that expression of LsGAS1/2 occurs in the parenchyma cells neighboring laticifers, while the resulting STL metabolites accumulate in laticifers. It can be inferred that active metabolite-trafficking occurs from the parenchyma cells to laticifers in lettuce.

Cytochrome P450 Enzymes as Key Drivers of Alkaloid Chemical Diversification in Plants.

Plants produce more than 20,000 nitrogen-containing heterocyclic metabolites called alkaloids. These chemicals serve numerous eco-physiological functions in the plants as well as medicines and psychedelic drugs for human for thousands of years, with the anti-cancer agent vinblastine and the painkiller morphine as the best-known examples. Cytochrome P450 monooxygenases (P450s) play a key role in generating the structural variety that underlies this functional diversity of alkaloids. Most alkaloid molecules are heavily oxygenated thanks to P450 enzymes' activities. Moreover, the formation and re-arrangement of alkaloid scaffolds such as ring formation, expansion, and breakage that contribute to their structural diversity and bioactivity are mainly catalyzed by P450s. The fast-expanding genomics and transcriptomics databases of plants have accelerated the investigation of alkaloid metabolism and many players behind the complexity and uniqueness of alkaloid biosynthetic pathways. Here we discuss recent discoveries of P450s involved in the chemical diversification of alkaloids and how these inform our approaches in understanding plant evolution and producing plant-derived drugs.

Discovering and harnessing oxidative enzymes for chemoenzymatic synthesis and diversification of anticancer camptothecin analogues.

Semi-synthetic derivatives of camptothecin, a quinoline alkaloid found in the Camptotheca acuminata tree, are potent anticancer agents. Here we discovered two C. acuminata cytochrome P450 monooxygenases that catalyze regio-specific 10- and 11-oxidations of camptothecin, and demonstrated combinatorial chemoenzymatic C-H functionalizations of the camptothecin scaffold using the new enzymes to produce a suite of anticancer drugs, including topotecan (Hycamtin®) and irinotecan (Camptosar®). This work sheds new light into camptothecin metabolism, and represents greener approaches for accessing clinically relevant camptothecin derivatives.

The Progesterone 5ß-Reductase/Iridoid Synthase Family: A Catalytic Reservoir for Specialized Metabolism across Land Plants.

Iridoids are plant-derived terpenoids with a rich array of bioactivities. The key step in iridoid skeleton formation is the reduction of 8-oxogeranial by certain members of the progesterone 5ß-reductase/iridoid synthase (PRISE) family of short-chain alcohol dehydrogenases. Other members of the PRISE family have previously been implicated in the biosynthesis of the triterpenoid class of cardenolides, which requires the reduction of progesterone. Here, we explore the occurrence and activity of PRISE across major lineages of plants. We observed trace activities toward either 8-oxogeranial or progesterone in all PRISEs, including those from nonseed plants and green algae. Phylogenetic analysis, coupled with enzymatic assays, show that these activities appear to have become specialized in specific angiosperm lineages. This broad analysis of the PRISE family provides insight into how these enzymes evolved in plants and also suggests that iridoid synthase activity is an ancestral trait in all land plants, which might have contributed to the rise of iridoid metabolites.

Quantitation of Select Terpenes/Terpenoids and Nicotine Using Gas Chromatography-Mass Spectrometry with High-Temperature Headspace Sampling.

Plants are the main sources of many high-value bioactive terpenoids used in the medical, fragrance, and food industries. Increasing demand for these bioactive plants and their derivative products (e.g., cannabis and extracts thereof) requires robust approaches to verify feedstock, identify product adulteration, and ensure product safety. Reported here are single-laboratory validation details for a robust testing method to quantitate select terpenes and terpenoids in dry plant materials and terpenoid-containing vaping liquids (e.g., a derivative product) using high-temperature headspace gas chromatography-mass spectrometry, with glycerol used as a headspace solvent. Validated method recoveries were 75-103%, with excellent repeatability (relative standard deviation (RSD) < 5%) and intermediate precision (RSD < 12%). The use of high-temperature headspace (180 °C) permitted terpene and terpenoid profiles to be monitored at temperatures consistent with vaping conditions.

Catalytic Plasticity of Germacrene A Oxidase Underlies Sesquiterpene Lactone Diversification.

Adaptive evolution of enzymes benefits from catalytic promiscuity. Sesquiterpene lactones (STLs) have diverged extensively in the Asteraceae, and studies of the enzymes for two representative STLs, costunolide and artemisinin, could provide an insight into the adaptive evolution of enzymes. Costunolide appeared early in Asteraceae evolution and is widespread, whereas artemisinin is a unique STL appearing in a single Asteraceae species, Artemisia annua Therefore, costunolide is a ubiquitous STL, while artemisinin is a specialized one. In costunolide biosynthesis, germacrene A oxidase (GAO) synthesizes germacrene A acid from germacrene A. Similarly, in artemisinin biosynthesis, amorphadiene oxidase (AMO) synthesizes artemisinic acid from amorphadiene. GAO promiscuity is suggested to drive the diversification of STLs. To examine the degree of GAO promiscuity, we expressed six sesquiterpene synthases from cotton (Gossypium arboretum), goldenrod (Solidago canadensis), valerian (Valeriana officinalis), agarwood (Aquilaria crassna), tobacco (Nicotiana tabacum), and orange (Citrus sinensis) in yeast to produce seven distinct sesquiterpene substrates (germacrene D, 5-epi-aristolochene, valencene, δ-cadinene, α- and δ-guaienes, and valerenadiene). GAO or AMO was coexpressed in these yeasts to evaluate the promiscuities of GAO and AMO. Remarkably, all sesquiterpenes tested were oxidized to sesquiterpene acids by GAO, but negligible activities were found from AMO. Hence, GAO apparently has catalytic potential to evolve into different enzymes for synthesizing distinct STLs, while the recently specialized AMO demonstrates rigid substrate specificity. Mutant GAOs implanted with active site residues of AMO showed substantially reduced stability, but their per enzyme activities to produce artemisinic acid increased by 9-fold. Collectively, these results suggest promiscuous GAOs can be developed as novel catalysts for synthesizing unique sesquiterpene derivatives.

Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle.

Vinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.

Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae.

The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs. Our previous discovery of GAO activity conserved across all major subfamilies of Asteraceae, including the phylogenetically basal lineage of Barnadesioideae, prompted further investigation of the presence of the gateway GAS in Barnadesioideae. Herein we isolated two terpene synthases (BsGAS1/BsGAS2) from the basal Barnadesia spinosa (Barnadesioideae) that displayed robust GAS activity when reconstituted in yeast and characterized in vitro. Despite the apparent lack of STLs in the Barnadesioideae, this work unambiguously confirms the presence of GAS in the basal genera of the Asteraceae. Phylogenetic analysis reveals that the two BsGASs fall into two distinct clades of the Asteraceae's GASs, and BsGAS1 clade is only retained in the evolutionary closer Cichorioideae subfamily, implicating BsGAS2 is likely the ancestral base of most GASs found in the lineages outside the Barnadesioideae. Taken together, these results show the enzymatic capacities of GAS and GAO emerged prior to the subsequent radiation of STL-producing Asteraceae subfamilies.

A lettuce (Lactuca sativa) homolog of human Nogo-B receptor interacts with cis-prenyltransferase and is necessary for natural rubber biosynthesis.

Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3-15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro.

Characterization of proanthocyanidin metabolism in pea (Pisum sativum) seeds.

BACKGROUND: Proanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied. RESULTS: PAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. 'Courier' and 'Solido' PAs were primarily prodelphinidin-types, whereas the PAs from 'LAN3017' were mainly the procyanidin-type. The mean degree of polymerization of 'LAN3017' PAs was also higher than those from 'Courier' and 'Solido'. Next-generation sequencing of 'Courier' seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis. CONCLUSION: Significant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in development to be used as substrates for polymerization into PAs. Biochemically competent recombinant PsANR and PsLAR activities were consistent with the pea seed coat PA profile composed of both cis- and trans-flavan-3-ols. Since the expression of PsLAR in Arabidopsis did not alter the PA subunit profile (which is only comprised of cis-flavan-3-ols), it necessitates further investigation of in planta metabolic flux through PsLAR.

De novo synthesis of high-value plant sesquiterpenoids in yeast.

Terpenoids comprise a structurally diverse group of natural products. Despite various and important uses of terpenoids (e.g., flavors, drugs, and nutraceuticals), most of them are, however, still extracted from plant sources, which suffer from high cost and low yield. Alternatively, terpenoids can be produced in microbes using their biosynthetic genes. With the explosion of sequence data, many genes for terpenoid metabolism can be characterized by biochemical approaches and used for the microbial production of terpenoids. However, substrates for in vitro studies of terpene synthases are costly, and the enzymatic synthesis of terpenoids in vitro using recombinant enzymes is insufficient to meet the chemical characterization need. Here, we describe the use of engineered yeast (EPY300) to evaluate in vivo production of sesquiterpenoids. Two sesquiterpene synthase genes (for valencene and 5-epi-aristolochene synthases) were expressed in EPY300 in native and N-terminal thioredoxin fusion forms. By using the thioredoxin fusion, valencene biosynthesis was slightly decreased; however, the production of 5-epi-aristolochene was increased by 10-fold, producing 420 µg mL(-1) of 5-epi-aristolochene. Accordingly, the thioredoxin-fused 5-epi-aristolochene was coexpressed with 5-epi-aristolochene dihydroxylase (cytochrome P450 monooxygenase) and its reductase in EPY300. This combinatorial expression yielded hydroxylated sesquiterpene, capsidiol, at ~250 µg mL(-1). Detailed experimental procedures and other considerations for this work are given.