Phytoalexin sakuranetin attenuates endocytosis and enhances resistance to rice blast.

Phytoalexin sakuranetin functions in resistance against rice blast. However, the mechanisms underlying the effects of sakuranetin remains elusive. Here, we report that rice lines expressing resistance (R) genes were found to contain high levels of sakuranetin, which correlates with attenuated endocytic trafficking of plasma membrane (PM) proteins. Exogenous and endogenous sakuranetin attenuates the endocytosis of various PM proteins and the fungal effector PWL2. Moreover, accumulation of the avirulence protein AvrCO39, resulting from uptake into rice cells by Magnaporthe oryzae, was reduced following treatment with sakuranetin. Pharmacological manipulation of clathrin-mediated endocytic (CME) suggests that this pathway is targeted by sakuranetin. Indeed, attenuation of CME by sakuranetin is sufficient to convey resistance against rice blast. Our data reveals a mechanism of rice against M. oryzae by increasing sakuranetin levels and repressing the CME of pathogen effectors, which is distinct from the action of many R genes that mainly function by modulating transcription.

Discovery of sesquiterpenoids from an actinomycete Crossiella cryophila through genome mining and heterologous expression.

Genome mining of the Actinomycete Crossiella cryophila facilitated the discovery of a minimal terpenoid biosynthetic gene cluster of cry consisting of a class I terpene cyclase CryA and a CYP450 monooxygenase CryB. Heterologous expression of cry allowed the isolation and characterization of two new sesquiterpenoids, ent-viridiflorol (1) and cryophilain (2). Notably, cryophilain (2) possesses a 5/7/3-fused tricyclic skeleton bearing a distinctive bridgehead hydroxy group. The combined in vivo and in vitro experiments revealed that CryA, the first ent-viridiflorol terpene cyclase, catalyzes farnesyl diphosphate to form the 5/7/3 sesquiterpene core scaffold and P450 CryB serves as a tailoring enzyme responsible for installing a hydroxy group at the bridgehead carbon.

Climbing the Oxidase Phase Ladder by Using Dioxygen as the Sole Oxidant: The Case Study of Costunolide.

Natural sesquiterpenoid lactones are prominent scaffolds in drug discovery. Despite the progress made in their synthesis, their extensive oxidative decoration makes their chemo- and stereoselective syntheses highly challenging. Herein, we report our effort to mimic part of the oxidase phase used in the costunolide pathway to achieve the protecting-group-free total synthesis of santamarine, dehydrocostus lactone, estafiatin, and nine more related natural sesquiterpenoid lactones by using dioxygen as the sole oxidant.

Targeting Vasohibins to Promote Axon Regeneration.

Treatments accelerating axon regeneration in the nervous system are still clinically unavailable. However, parthenolide promotes adult sensory neurons' axon growth in culture by inhibiting microtubule detyrosination. Here, we show that overexpression of vasohibins increases microtubule detyrosination in growth cones and compromises growth in culture and in vivo. Moreover, overexpression of these proteins increases the required parthenolide concentrations to promote axon regeneration. At the same time, the partial knockdown of endogenous vasohibins or their enhancer SVBP in neurons facilitates axon growth, verifying them as pharmacological targets for promoting axon growth. In vivo, repeated intravenous application of parthenolide or its prodrug di-methyl-amino-parthenolide (DMAPT) markedly facilitates the regeneration of sensory, motor, and sympathetic axons in injured murine and rat nerves, leading to acceleration of functional recovery. Moreover, orally applied DMAPT was similarly effective in promoting nerve regeneration. Thus, pharmacological inhibition of vasohibins facilitates axon regeneration in different species and nerves, making parthenolide and DMAPT the first promising drugs for curing nerve injury.

GC-MS-based Metabolomics Unravels Metabolites across Larval Development and Diapause of a Specialist Insect.

Plant-insect interactions are a driving force into ecosystem evolution and community dynamics. Many insect herbivores enter diapause, a developmental arrest stage in anticipation of adverse conditions, to survive and thrive through seasonal changes. Herein, we investigated the roles of medium- to non-polar metabolites during larval development and diapause in a specialist insect herbivore, Chlosyne lacinia, reared on Aldama robusta leaves. Varying metabolites were determined using gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Sesquiterpenes and steroids were the main metabolites putatively identified in A. robusta leaves, whereas C. lacinia caterpillars were characterized by triterpenes, steroids, fatty acids, and long-chain alkanes. We found out that C. lacinia caterpillars biosynthesized most of the identified steroids and fatty acids from plant-derived ingested metabolites, as well as all triterpenes and long-chain alkanes. Steroids, fatty acids, and long-chain alkanes were detected across all C. lacinia instars and in diapausing caterpillars. Sesquiterpenes and triterpenes were also detected across larval development, yet they were not detected in diapausing caterpillars, which suggested that these metabolites were converted to other molecules prior to the diapause stage. Our findings shed light on the chemical content variation across C. lacinia development and diapause, providing insights into the roles of metabolites in plant-insect interactions.

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases.

Sesquiterpenes comprise a diverse group of natural products with a wide range of applications in cosmetics, food, medicine, agriculture, and biofuels. Heterologous biosynthesis is increasingly employed for sesquiterpene production, aiming to overcome the limitations associated with chemical synthesis and natural extraction. Sesquiterpene synthases (STSs) play a crucial role in the heterologous biosynthesis of sesquiterpene. Under the catalysis of STSs, over 300 skeletons are produced through various cyclization processes (C1-C10 closure, C1-C11 closure, C1-C6 closure, and C1-C7 closure), which are responsible for the diversity of sesquiterpenes. According to the cyclization types, we gave an overview of advances in understanding the mechanism of STSs cyclization from the aspects of protein crystal structures and site-directed mutagenesis. We also summarized the applications of engineering STSs in the heterologous biosynthesis of sesquiterpene. Finally, the bottlenecks and potential research directions related to the STSs cyclization mechanism and application of modified STSs were presented.

Sesquiterpenes of the ectomycorrhizal fungus Pisolithus microcarpus alter root growth and promote host colonization.

Trees form symbioses with ectomycorrhizal (ECM) fungi, maintained in part through mutual benefit to both organisms. Our understanding of the signaling events leading to the successful interaction between the two partners requires further study. This is especially true for understanding the role of volatile signals produced by ECM fungi. Terpenoids are a predominant class of volatiles produced by ECM fungi. While several ECM genomes are enriched in the enzymes responsible for the production of these volatiles (i.e., terpene synthases (TPSs)) when compared to other fungi, we have limited understanding of the biochemical products associated with each enzyme and the physiological impact of specific terpenes on plant growth. Using a combination of phylogenetic analyses, RNA sequencing, and functional characterization of five TPSs from two distantly related ECM fungi (Laccaria bicolor and Pisolithus microcarpus), we investigated the role of these secondary metabolites during the establishment of symbiosis. We found that despite phylogenetic divergence, these TPSs produced very similar terpene profiles. We focused on the role of P. microcarpus terpenes and found that the fungus expressed a diverse array of mono-, di-, and sesquiterpenes prior to contact with the host. However, these metabolites were repressed following physical contact with the host Eucalyptus grandis. Exposure of E. grandis to heterologously produced terpenes (enriched primarily in γ -cadinene) led to a reduction in the root growth rate and an increase in P. microcarpus-colonized root tips. These results support a very early putative role of fungal-produced terpenes in the establishment of symbiosis between mycorrhizal fungi and their hosts.

Anti-Inflammatory Sesquiterpenes from Fruiting Bodies of Schizophyllum commune.

Schizophyllum commune, a fleshy fungus, is an important medicinal and food-homologous mushroom in China. In this work, eight undescribed sesquiterpenes schizomycins A-H (1-8) and one new meroterpenoid schizomycin I (9) together with three known analogues (10-12) were isolated from fruiting bodies of S. commune. Their planar structures were established by extensive spectroscopic and mass spectrometric data. The absolute configurations of compounds 1, 2, and 4 were determined by single crystal X-ray diffraction, and compounds 3 and 5-9 were confirmed by electronic circular dichroism calculations. Anti-inflammatory activities of all isolated compounds were evaluated for their inhibitory effects on IL-6 and IL-1ß production in RAW 264.7 cells. Among them, compound 7 exhibited significant IL-6 inhibitory activity with an IC50 value of 3.6 µM. The results of molecular docking showed that compound 7 interacts with amino acid residues (Gly117, Lys118, Asp120, Thr166, and Try168) of the IL-6 receptor protein through hydrogen bonding.

Photosynthetic production of α-farnesene by engineered Synechococcus elongatus UTEX 2973 from carbon dioxide.

Cyanobacteria are the prospective biosolar cell factories to produce a range of bioproducts through CO2 sequestration. Farnesene is a sesquiterpene with an array of applications in biofuels, pest management, cosmetics, flavours and fragrances. This is the first time a codon-optimized farnesene synthase (AFS) gene is engineered into the genomic neutral site of Synechococcus elongatus UTEX 2973 for farnesene synthesis through its endogenous methylerythritol phosphate (MEP) pathway, rendering UTEX AFS strain. Similarly, bottleneck gene(s) of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and/or fusion of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase (idispA) were engineered engendering UTEX AFS::dxs, UTEX AFS::idispA and UTEX AFS::dxs::idispA strains. UTEX AFS::dxs::idispA achieves farnesene productivity of 2.57 mg/L/day, the highest among engineered cyanobacterial strains studied so far. It demonstrates farnesene production, which is 31.3-times higher than the UTEX AFS strain. Moreover, the engineered strains show similar productivity over a three-month period, stipulating the genetic stability of the strains.

Structure-Based Engineering of a Sesquiterpene Cyclase to Generate an Alcohol Product: Conversion of epi-Isozizaene Synthase into α-Bisabolol Synthase.

The sesquiterpene cyclase epi-isozizaene synthase (EIZS) from Streptomyces coelicolor catalyzes the metal-dependent conversion of farnesyl diphosphate (FPP) into the complex tricyclic product epi-isozizaene. This remarkable transformation is governed by an active site contour that serves as a template for catalysis, directing the conformations of multiple carbocation intermediates leading to the final product. Mutagenesis of residues defining the active site contour remolds its three-dimensional shape and reprograms the cyclization cascade to generate alternative cyclization products. In some cases, mutagenesis enables alternative chemistry to quench carbocation intermediates, e.g., through hydroxylation. Here, we combine structural and biochemical data from previously characterized EIZS mutants to design and prepare F95S-F198S EIZS, which converts EIZS into an α-bisabolol synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C). We report the complete biochemical characterization of this double mutant as well as the 1.47 Å resolution X-ray crystal structure of its complex with three Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, which partially mimics a carbocation intermediate. Most notably, the two mutations together create an active site contour that stabilizes the bisabolyl carbocation intermediate and positions a water molecule for the hydroxylation reaction. Structural comparison with a naturally occurring α-bisabolol synthase reveals common active site features that direct α-bisabolol generation. In showing that EIZS can be redesigned to generate a sesquiterpene alcohol product instead of a sesquiterpene hydrocarbon product, we have expanded the potential of EIZS as a platform for the development of designer cyclases that could be utilized in synthetic biology applications.

Multi-omics revealed molecular mechanism of biphenyl phytoalexin formation in response to yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells.

KEY MESSAGE: Yeast extract-induced oxidative stress in Sorbus aucuparia suspension cells leads to the biosynthesis of various hormones, which activates specific signaling pathways that augments biphenyl phytoalexin production. Pathogen incursions pose a significant threat to crop yield and can have a pronounced effect on agricultural productivity and food security. Biphenyl phytoalexins are a specialized group of secondary metabolites that are mainly biosynthesized by Pyrinae plants as a defense mechanism against various pathogens. Despite previous research demonstrating that biphenyl phytoalexin production increased dramatically in Sorbus aucuparia suspension cells (SASCs) treated with yeast extract (YE), the underlying mechanisms remain poorly understood. To address this gap, we conducted an in-depth, multi-omics analysis of transcriptome, proteome, and metabolite (including biphenyl phytoalexins and phytohormones) dynamics in SASCs exposed to YE. Our results indicated that exposure to YE-induced oxidative stress in SASCs, leading to the biosynthesis of a range of hormones, including jasmonic acid (JA), jasmonic acid isoleucine (JA-ILE), gibberellin A4 (GA4), indole-3-carboxylic acid (ICA), and indole-3-acetic acid (IAA). These hormones activated specific signaling pathways that promoted phenylpropanoid biosynthesis and augmented biphenyl phytoalexin production. Moreover, reactive oxygen species (ROS) generated during this process also acted as signaling molecules, amplifying the phenylpropanoid biosynthesis cascade through activation of the mitogen-activated protein kinase (MAPK) pathway. Key genes involved in these signaling pathways included SaBIS1, SaBIS2, SaBIS3, SaPAL, SaB4H, SaOMT, SaUGT1, SaLOX2, SaPR1, SaCHIB1, SaCHIB2 and SaCHIB3. Collectively, this study provided intensive insights into biphenyl phytoalexin accumulation in YE-treated SASCs, which would inform the development of more efficient disease-resistance strategies in economically significant cultivars.

Bioprospecting of Artemisia genus: from artemisinin to other potentially bioactive compounds.

Species from genus Artemisia are widely distributed throughout temperate regions of the northern hemisphere and many cultures have a long-standing traditional use of these plants as herbal remedies, liquors, cosmetics, spices, etc. Nowadays, the discovery of new plant-derived products to be used as food supplements or drugs has been pushed by the exploitation of bioprospection approaches. Often driven by the knowledge derived from the ethnobotanical use of plants, bioprospection explores the existing biodiversity through integration of modern omics techniques with targeted bioactivity assays. In this work we set up a bioprospection plan to investigate the phytochemical diversity and the potential bioactivity of five Artemisia species with recognized ethnobotanical tradition (A. absinthium, A. alba, A. annua, A. verlotiorum and A. vulgaris), growing wild in the natural areas of the Verona province. We characterized the specialized metabolomes of the species (including sesquiterpenoids from the artemisinin biosynthesis pathway) through an LC-MS based untargeted approach and, in order to identify potential bioactive metabolites, we correlated their composition with the in vitro antioxidant activity. We propose as potential bioactive compounds several isomers of caffeoyl and feruloyl quinic acid esters (e.g. dicaffeoylquinic acids, feruloylquinic acids and caffeoylferuloylquinic acids), which strongly characterize the most antioxidant species A. verlotiorum and A. annua. Morevoer, in this study we report for the first time the occurrence of sesquiterpenoids from the artemisinin biosynthesis pathway in the species A. alba.

Analysis of root volatiles and functional characterization of a root-specific germacrene A synthase in Artemisia pallens.

MAIN CONCLUSION: The study demonstrated that Artemisia pallens roots can be a source of terpene-rich essential oil and root-specific ApTPS1 forms germacrene A contributing to major root volatiles. Davana (Artemisia pallens Bess) is a valuable aromatic herb within the Asteraceae family, highly prized for its essential oil (EO) produced in the aerial parts. However, the root volatile composition, and the genes responsible for root volatiles have remained unexplored until now. Here, we show that A. pallens roots possess distinct oil bodies and yields ~ 0.05% of EO, which is primarily composed of sesquiterpenes ß-elemene, neryl isovalerate, ß-selinene, and α-selinene, and trace amounts of monoterpenes ß-myrcene, D-limonene. This shows that, besides aerial parts, roots of davana can also be a source of unique EO. Moreover, we functionally characterized a terpene synthase (ApTPS1) that exhibited high in silico expression in the root transcriptome. The recombinant ApTPS1 showed the formation of ß-elemene and germacrene A with E,E-farnesyl diphosphate (FPP) as a substrate. Detailed analysis of assay products revealed that ß-elemene was the thermal rearrangement product of germacrene A. The functional expression of ApTPS1 in Saccharomyces cerevisiae confirmed the in vivo germacrene A synthase activity of ApTPS1. At the transcript level, ApTPS1 displayed predominant expression in roots, with significantly lower level of expression in other tissues. This expression pattern of ApTPS1 positively correlated with the tissue-specific accumulation level of germacrene A. Overall, these findings provide fundamental insights into the EO profile of davana roots, and the contribution of ApTPS1 in the formation of a major root volatile.

Modeling the environmental fate of bracken toxin ptaquiloside: Production, release and transport in the rhizosphere.

Plants produce a diverse array of toxic compounds which may be released by precipitation, explaining their wide occurrence in surrounding soil and water. This study presents the first mechanistic model for describing the generation and environmental fate of a natural toxin, i.e. ptaquiloside (PTA), a carcinogenic phytotoxin produced by bracken fern (Pteridium aquilinum L. Kuhn). The newly adapted DAISY model was calibrated based on two-year monitoring performed in the period 2018-2019 in a Danish bracken population located in a forest glade. Several functions related to the fate of PTA were calibrated, covering processes from toxin generation in the canopy, wash off by precipitation and degradation in the soil. Model results show a good description of observed bracken biomass and PTA contents, supporting the assumption that toxin production can be explained by the production of new biomass. Model results show that only 4.4 % of the PTA produced in bracken is washed off by precipitation, from both canopy and litter. Model simulations showed that PTA degrades rapidly once in the soil, especially during summer due to the high soil temperatures. Leaching takes place in form of pulses directly connected to precipitation events, with maximum simulated concentrations up to 4.39 µg L-1 at 50 cm depth. Macropore transport is mainly responsible for the events with the highest PTA concentrations, contributing to 72 % of the total mass of PTA leached. Based on the results, we identify areas with high density of bracken, high precipitation during the summer and soils characterized by fast transport, as the most vulnerable to surface and groundwater pollution by phytotoxins.

Identification and evolution of a diterpenoid phytoalexin oryzalactone biosynthetic gene in the genus Oryza.

The natural variation of plant-specialized metabolites represents the evolutionary adaptation of plants to their environments. However, the molecular mechanisms that account for the diversification of the metabolic pathways have not been fully clarified. Rice plants resist attacks from pathogens by accumulating diterpenoid phytoalexins. It has been confirmed that the composition of rice phytoalexins exhibits numerous natural variations. Major rice phytoalexins (momilactones and phytocassanes) are accumulated in most cultivars, although oryzalactone is a cultivar-specific compound. Here, we attempted to reveal the evolutionary trajectory of the diversification of phytoalexins by analyzing the oryzalactone biosynthetic gene in Oryza species. The candidate gene, KSLX-OL, which accounts for oryzalactone biosynthesis, was found around the single-nucleotide polymorphisms specific to the oryzalactone-accumulating cultivars in the long arm of chromosome 11. The metabolite analyses in Nicotiana benthamiana and rice plants overexpressing KSLX-OL indicated that KSLX-OL is responsible for the oryzalactone biosynthesis. KSLX-OL is an allele of KSL8 that is involved in the biosynthesis of another diterpenoid phytoalexin, oryzalexin S and is specifically distributed in the AA genome species. KSLX-NOL and KSLX-bar, which encode similar enzymes but are not involved in oryzalactone biosynthesis, were also found in AA genome species. The phylogenetic analyses of KSLXs, KSL8s, and related pseudogenes (KSL9s) indicated that KSLX-OL was generated from a common ancestor with KSL8 and KSL9 via gene duplication, functional differentiation, and gene fusion. The wide distributions of KSLX-OL and KSL8 in AA genome species demonstrate their long-term coexistence beyond species differentiation, suggesting a balancing selection between the genes.

Atmospheric Degradation of Ecologically Important Biogenic Volatiles: Investigating the Ozonolysis of (E)-ß-Ocimene, Isomers of α and ß-Farnesene, α-Terpinene and 6-Methyl-5-Hepten-2-One, and Their Gas-Phase Products.

Biogenic volatile organic compounds (bVOCs), synthesised by plants, are important mediators of ecological interactions that can also undergo a series of reactions in the atmosphere. Ground-level ozone is a secondary pollutant generated through sunlight-driven reactions between nitrogen oxides (NOx) and VOCs. Its levels have increased since the industrial revolution and reactions involving ozone drive many chemical processes in the troposphere. While ozone precursors often originate in urban areas, winds may carry these hundreds of kilometres, causing ozone formation to also occur in less populated rural regions. Under elevated ozone conditions, ozonolysis of bVOCs can result in quantitative and qualitative changes in the gas phase, reducing the concentrations of certain bVOCs and resulting in the formation of other compounds. Such changes can result in disruption of bVOC-mediated behavioural or ecological interactions. Through a series of gas-phase experiments using Gas Chromatography Mass Spectrometry (GC-MS) and Proton Transfer Reaction Mass Spectrometry (PTR-MS), we investigated the products and their yields from the ozonolysis of a range of ubiquitous bVOCs, which were selected because of their importance in mediating ecological interactions such as pollinator and natural enemy attraction and plant-to-plant communication, namely: (E)-ß-ocimene, isomers of α and ß-farnesene, α-terpinene and 6-methyl-5-hepten-2-one. New products from the ozonolysis of these compounds were identified, and the formation of these compounds is consistent with terpene-ozone oxidation mechanisms. We present the degradation mechanism of our model bVOCs and identify their reaction products. We discuss the potential ecological implications of the degradation of each bVOC and of the formation of reaction products.

Terpene Synthase Gene Family in Chinese Chestnut (Castanea mollissima BL.) Harbors Two Sesquiterpene Synthase Genes Implicated in Defense against Gall Wasp Dryocosmus kuriphilus.

Chinese chestnut (Castanea mollissima BL.) is a well-known fruit tree that has been cultivated in East Asia for millennia. Leaves and buds of the plant can become seriously infested by the gall wasp Dryocosmus kuriphilus (GWDK), which results in gall formation and associated significant losses in fruit production. Herbivore-induced terpenes have been reported to play an important role in plant-herbivory interactions, and in this study, we show that upon herbivory by GWDK, four terpene-related compounds were significantly induced, while the concentrations of these four compounds in intact buds were relatively low. Among these compounds, (E)-nerolidol and (E, E)-α-farnesene have frequently been reported to be involved in plant herbivory defenses, which suggests direct and/or indirect functions in chestnut GWDK defenses. Candidate terpene synthase (TPS) genes that may account for (E)-nerolidol and (E, E)-α-farnesene terpene biosynthesis were characterized by transcriptomics and phylogenetic approaches, which revealed altered transcript levels for two TPSs: CmAFS, a TPS-g subfamily member, and CmNES/AFS, a TPS-b clade member. Both genes were dramatically upregulated in gene expression upon GWDK infestation. Furthermore, Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana showed that CmAFS catalyzed the formation of (E, E)-α-farnesene, while CmNES/AFS showed dual (E)-nerolidol and (E, E)-α-farnesene synthase activity. Biochemical assays of the recombinant CmAFS and CmNES/AFS proteins confirmed their catalytic activity in vitro, and the enzymatic products were consistent with two of the major volatile compounds released upon GWDK-infested chestnut buds. Subcellular localization demonstrated that CmAFS and CmNES/AFS were both localized in the cytoplasm, the primary compartment for sesquiterpene synthesis. In summary, we show that two novel sesquiterpene synthase genes CmAFS and CmNES/AFS are inducible by herbivory and can account for the elevated accumulation of (E, E)-α-farnesene and (E)-nerolidol upon GWDK infestation and may be implicated in chestnut defense against GWDK herbivores.

PatWRKY71 transcription factor regulates patchoulol biosynthesis and plant defense response.

Patchoulol, a valuable compound belonging to the sesquiterpenoid family, is the primary component of patchouli oil produced by Pogostemon cablin (P. cablin). It has a variety of pharmacological and biological activities and is widely used in the medical and cosmetic industries. However, despite its significance, there is a lack of research on the transcriptional modulation of patchoulol biosynthesis.Salicylic acid (SA), is a vital plant hormone that serves as a critical signal molecule and plays an essential role in plant growth and defense. However, to date, no studies have explored the modulation of patchoulol biosynthesis by SA. In our study, we discovered that the application of SA can enhance the production of patchoulol. Utilizing transcriptome analysis of SA-treated P. cablin, we identified a crucial downstream transcription factor, PatWRKY71. The transcription level of PatWRKY71 was significantly increased with the use of SA. Furthermore, our research has revealed that PatWRKY71 was capable of binding to the promoter of PatPTS, ultimately leading to an increase in its expression. When PatWRKY71 was silenced by a virus, the expression of both PatWRKY71 and PatPTS was reduced, resulting in the down-regulation of patchoulol production. Through our studies, we discovered that heterologous expression of PatWRKY71 leads to an increase in the sensitivity of Arabidopsis to salt and Cd, as well as an outbreak of reactive oxygen species (ROS). Additionally, we uncovered the regulatory role of PatWRKY71 in both patchoulol biosynthesis and plant defense response. This discovery provided a theoretical basis for the improvement of the content of patchoulol and the resistance of P. cablin through genetic engineering.

Complete pathway elucidation and heterologous reconstitution of (+)-nootkatone biosynthesis from Alpinia oxyphylla.

(+)-Nootkatone is a natural sesquiterpene ketone widely used in food, cosmetics, pharmaceuticals, and agriculture. It is also regarded as one of the most valuable terpenes used commercially. However, plants contain trace amounts of (+)-nootkatone, and extraction from plants is insufficient to meet market demand. Alpinia oxyphylla is a well-known medicinal plant in China, and (+)-nootkatone is one of the main components within the fruits. By transcriptome mining and functional screening using a precursor-providing yeast chassis, the complete (+)-nootkatone biosynthetic pathway in Alpinia oxyphylla was identified. A (+)-valencene synthase (AoVS) was identified as a novel monocot-derived valencene synthase; three (+)-valencene oxidases AoCYP6 (CYP71BB2), AoCYP9 (CYP71CX8), and AoCYP18 (CYP701A170) were identified by constructing a valencene-providing yeast strain. With further characterisation of a cytochrome P450 reductase (AoCPR1) and three dehydrogenases (AoSDR1/2/3), we successfully reconstructed the (+)-nootkatone biosynthetic pathway in Saccharomyces cerevisiae, representing a basis for its biotechnological production. Identifying the biosynthetic pathway of (+)-nootkatone in A. oxyphylla unravelled the molecular mechanism underlying its formation in planta and also supported the bioengineering production of (+)-nootkatone. The highly efficient yeast chassis screening method could be used to elucidate the complete biosynthetic pathway of other valuable plant natural products in future.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.