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This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD 600 ). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Many complex terpenoids, predominantly isolated from plants and fungi, show drug-like physicochemical properties. Recent advances in genome mining revealed actinobacteria as an almost untouched treasure trove of terpene biosynthetic gene clusters (BGCs). In this study, we characterized a terpene BGC with an unusual architecture. The selected BGC includes, among others, genes encoding a terpene cyclase fused to a truncated reductase domain and a cytochrome P450 monooxygenase (P450) that is split over three gene fragments. Functional characterization of the BGC in a heterologous host led to the identification of several new members of the trans -eunicellane family of diterpenoids, the euthailols, that feature unique oxidation patterns. A combination of bioinformatic analyses, structural modeling studies, and heterologous expression revealed a dual function of the pathway-encoded hypothetical protein that acts as an isomerase and an oxygenase. Moreover, in the absence of other tailoring enzymes, a P450 hydroxylates the eunicellane scaffold at a position that is not modified in other eunicellanes. Surprisingly, both the modifications installed by the hypothetical protein and one of the P450s exhibit partial redundancy. Bioactivity assays revealed that some of the euthailols show growth inhibitory properties against Gram-negative ESKAPE pathogens. The characterization of the euthailol BGC in this study provides unprecedented insights into the partial functional redundancy of tailoring enzymes in complex diterpenoid biosynthesis and highlights hypothetical proteins as an important and largely overlooked family of tailoring enzymes involved in the maturation of complex terpenoids.

Pogostemon cablin (Blanco) Benth (Patchouli) is an aromatic herb extensively used in pharmaceutical and cosmetic industries. Sesquiterpenes are the characteristic constitutes in patchouli which are synthesized in the glandular trichomes on leaves and stems. Gibberellic acid (GA), a tetracyclic diterpenoid, plays a crucial role in the formation of glandular trichome. However, the diterpene biosynthesis remains largely unknown in patchouli. Here we identified a small diterpene synthases (diTPSs) family comprising three class II diTPSs (PatCPS1-3) and three class I diTPSs (PatKSL1 and PatGLS1-2). These diTPSs are functionally characterized using a yeast heterologous expression system. PatCPS1 was identified as an ent-copalyl diphosphate synthase (ent-CPS), in combination with PatKSL1, yield ent-kaurene, the precursor of GA, indicating their involvement in primary metabolism. PatCPS2 converted GGPP into (+)-8, 13-copalyl diphosphate (CPP). No activity was detected for PatCPS3, PatGLS1 and PatGLS2. Three ohnologs of PatCPS1 were further characterized to explore the possible functional differentiation of ent-CPS during the evolution of tetraploid hybrid patchouli genome. GC-MS analysis showed all ohnologs are functional ent-CPSs, demonstrating the functional conservation of PatCPS1 during evolution. Expression profiling by qRT-PCR showed PatCPS1 and PatKSL1 are ubiquitously expressed in all tissues, consistent with their involvement in primary metabolism. Conversely, PatCPS2 and PatCPS3 were predominantly expressed in the above ground parts, indicating a role in specialized metabolism. In summary, these findings clarify the early stages of GA biosynthesis in patchouli and provide gene elements for further metabolic engineering of sesquiterpenes via diterpenoids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Fusicoccane (FC)-type diterpenoids are a class of diterpenoids characterized by a unique 5-8-5 ring system and exhibit diverse biological activities. Recently, we identified a novel FC-type diterpene synthase MgMS, which produces a myrothec-15(17)-en-7-ol (1) hydrocarbon skeleton, however, its tailoring congeners have not been elucidated. Here, we discovered two additional gene clusters Bn and Np, each encoding a highly homologous terpene synthase to MgMS but distinct tailoring enzymes. Heterologous expression of the terpene synthases BnMS and NpMS yielded the same product as MgMS. Subsequent introduction of three P450 enzymes MgP450, BnP450 and NpP450 from individual gene clusters resulted in four new FC-type diterpenoids 2-5. Notably, MgP450 serves as the first enzyme responsible for hydroxylation of the C19 methyl group, whereas NpP450 functions as a multifunctional P450 enzyme involved in the oxidations at C5, C6, and C19 positions of the 5-8-5 tricyclic skeleton. C5 oxidation of the hydrocarbon skeleton 1 led to broadening of the NMR signals and incomplete spectra, which was resolved by high-temperature NMR spectral analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

By utilizing the conformational selectivity of biosynthesis and the flexibility of chemical synthesis, researchers have formulated metabolic engineering-based semi-synthetic approaches that initiate with the final product's structure and identify key biosynthesis intermediates. Nonetheless, these tailored semi-synthetic routes focused on end-products, neglecting the possibility of biobased intermediates as a platform for derivatization. To address this challenge, this studyproposed a novel strategy resembling chemosynthesis-style divergent exploration to amplify the significance of biobased intermediates, in the case of geranylgeraniol (GGOH). Using the novel bifunctional terpene synthase PTTC066 and systematic metabolic engineering modifications, the engineered yeast straindemonstrated high GGOH production levels (3.32 g/L, 0.039 g/L/h). This platformenabled the semi-synthesis of various pharmaceuticals, including the anti-ulcer drug teprenone, the osteoporosis treatment drug menaquinone-4, and introduced a novel route for synthesizingα-tocotrienol. This study offers a fresh outlook on semi-synthetic approaches, opening avenues for improvements, substitutions, and innovations in industrial production processes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tiangang, Liu reports financial support was provided by Wuhan Hesheng Technology Co., Ltd. Wuhan University and Wuhan Hesheng Technology Co., Ltd have applied for several patents based on this work. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Three uncommon isospongian diterpenoids including a new one, 3-epi-kravanhin A (2), were isolated from the leaves of Amomum tsao-ko. Compounds 2 and 3 dose-dependently promoted GLP-1 secretion on STC-1 cells with promotion ratios of 109.7 % and 186.1 % (60 μM). Mechanism study demonstrated that the GLP-1 stimulative effects of 2 and 3 were closely related with Ca 2+ /CaMKII and PKA pathways, but irrelevant to GPBAR1 and GPR119 receptors. Moreover, compound 1 showed DPP-4 inhibitory activity with an IC 50 value of 311.0 μM. Molecular docking verified the binding affinity of 1 with DPP-4 by hydrogen bonds between the γ-lactone carbonyl (C-15) and Arg61 residue. Bioinformatics study indicated that compound 1 exerted antidiabetic effects by improving inflammation, oxidative stress and insulin resistance. This study first disclosed the presence of isospongian diterpenoids in A. tsao-ko, which showed antidiabetic potency by promoting GLP-1 secretion and inhibiting DPP-4 activity., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Key Message: Based on transport inhibition and genome-wide analysis, 123 ABC transporters of Euphorbia lathyris were identified, and it was found that the PDR family members ElABCG39 mediated ingenol efflux. Identification of ingenol biosynthetic enzymes and transporters in plant is fundamental to realize its biosynthesis in chassis cells. At present, several key enzymes of the ingenol biosynthesis pathway have been identified, while the mechanisms governing the accumulation or transport of ingenol to distinct plant tissue compartments remain elusive. In this study, transport inhibition analyses were performed, along with genome-wide identification of 123 genes encoding ABC proteins in Euphorbia lathyris L., eventually discovering that a PDR transporter ElABCG39 mediates ingenol transmembrane transport and is localized on the plasma membrane. Expression of this protein in yeast AD1-8 promoted the transmembrane efflux of ingenol with strong substrate specificity. Furthermore, in ElABCG39 RNAi transgenic hairy roots, ingenol transmembrane efflux was significantly reduced and hairy root growth was inhibited. The discovery of the first Euphorbia macrocyclic diterpene transporter ElABCG39 has not only further improved the ingenane diterpenoid biosynthesis regulatory network, but also provided a new key element for ingenol production in chassis cells., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Geranyllinalool, a natural diterpenoid found in plants, has a floral and woody aroma, making it valuable in flavors and fragrances. Currently, its synthesis primarily depends on chemical methods, which are environmentally harmful and economically unsustainable. Microbial synthesis through metabolic engineering has shown potential for producing geranyllinalool. However, achieving efficient synthesis remains challenging owing to the limited availability of terpenoid precursors in microorganisms. Thus, an artificial isopentenol utilization pathway (IUP) was constructed and introduced in Escherichia coli to enhance precursor availability and further improve terpenoid synthesis., Results: We first constructed an artificial IUP in E. coli to enhance the supply of precursor geranylgeranyl diphosphate (GGPP) and then screened geranyllinalool synthases from plants to achieve efficient synthesis of geranyllinalool (274.78 ± 2.48 mg/L). To further improve geranyllinalool synthesis, we optimized various cultivation factors, including carbon source, IPTG concentration, and prenol addition and obtained 447.51 ± 6.92 mg/L of geranyllinalool after 72 h of shaken flask fermentation. Moreover, a scaled-up production in a 5-L fermenter was investigated to give 2.06 g/L of geranyllinalool through fed-batch fermentation. To the best of our knowledge, this is the highest reported titer so far., Conclusions: Efficient synthesis of geranyllinalool in E. coli can be achieved through a two-step pathway and optimization of culture conditions. The findings of this study provide valuable insights into the production of other terpenoids in E. coli., (© 2024. The Author(s).)

Diterpenoid Phytoalexin Factor (DPF) is a key transcription factor involved in diterpenoid phytoalexin (DP) biosynthesis under non-stressed conditions in rice (Oryza sativa L.). Using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9, DPF knockout rice lines were generated. Treatments with abiotic stresses (copper chloride, ultraviolet light, and jasmonic acid) and biotic stress (blast fungus infection) to the knockout lines revealed that the DPF positively regulates stress-induced DP biosynthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Cyclocybe aegerita is a model mushroom belonging to the fungal phylum Basidiomycota. Among others, C. aegerita is known for its diverse terpenome, containing various volatile and nonvolatile terpenes and terpenoids. Here, we deepen the knowledge on their biosynthetic pathways by studying the terpene synthase Agr2 in detail. In contrast to previous studies, the heterologous production of Agr2 in the agaric host Coprinopsis cinerea revealed the production of two terpenes, one of which was the already known sesquiterpene viridiflorene. The other one was a so far unknown diterpene that had to be isolated and purified by means of preparative RP-HPLC for structure elucidation. 1D- and 2D-NMR experiments revealed the compound as the novel diterpene cyclocybene, pointing to the bifunctionality of Agr2 to produce both a sesquiterpene and a diterpene.

The cyclisation mechanism of the fungal fusicoccane (FC)-type diterpene synthase (DTS) TadA was investigated by extensive isotopic labelling experiments, and the pH-dependency of the product selectivity of this enzyme was explored. These studies provide new insights into the cyclisation mechanisms of FC-type DTSs.

Fusicoccadiene synthase from Phomopsis amygdala (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate, and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are connected by a flexible 69-residue linker. The PT domain mediates oligomerization to form predominantly octamers, with cyclase domains randomly splayed out around the PT core. Surprisingly, despite the random positioning of cyclase domains, substrate channeling is operative in catalysis since most of the GGPP generated by the PT remains on the enzyme for cyclization. Here, we demonstrate that covalent linkage of the PT and cyclase domains is not required for GGPP channeling, although covalent linkage may improve channeling efficiency. Moreover, GGPP competition experiments with other diterpene cyclases indicate that the PaFS PT and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryoelectron microscopy structure of the 600-kD "linkerless" construct, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the PT octamer and exhibit fascinating quaternary structural flexibility. These results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the PT octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place., Competing Interests: Competing interests statement:The authors declare no competing interest.

Andrographis paniculata is known for its diterpenoid medicinal compounds with antibacterial and anti-inflammatory properties. However, it faces production and cultivation challenges due to low temperatures (LTs). Cytochrome P450 monooxygenases (CYPs) are key enzymes in diterpenoid accumulation. Nevertheless, the functions and LT-related expression patterns of diterpenoid pathway CYPs in Andrographis paniculata remain poorly understood. In this study, 346 CYPs were discovered in Andrographis paniculata . Among them, 328 CYPs belonged to 42 known subfamilies. The remaining 17 CYPs might have represented novel subfamilies unique to this species. A total of 65 candidate CYPs associated with diterpenoid modification were identified. Of these, 50 were transmembrane proteins, and 57 were localized to chloroplasts. The CYP71 subfamily was the most abundant and had the highest motif diversity. Promoters of all candidate CYPs commonly contained elements responsive to gibberellins (GAs), methyl jasmonate (MeJA), and abiotic stresses. Notably, the XP_051152769 protein, corresponding to a CYP gene over 40,000 bp in length, featured an extraordinarily long intron (40,751 nts). Functional elements within this intron were related to LT, GAs, and dehydration pathways. Based on the promoter element arrangement and subfamily classification, 10 representative candidate CYPs were selected. Under LT stress, significant expression changes were observed in three representative CYPs : CYP71D , ent-kaurenoic acid oxidase ( KAO ), and ent - kaurene oxidase ( KO ). KAO and KO were significantly upregulated during early LT stress. KAO and KO interacted with each other and jointly interacted with GA20OX2-like. CYP71D acted as a negative response factor to LT stress. Among the 37 proteins interacting with CYP71D, 95% were CYPs. This study provides a critical preliminary foundation for investigating the functions of diterpenoid pathway CYPs in Andrographis paniculata , thereby facilitating the development of LT-tolerant cultivars.

Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 10 5  m -1 ·s -1 , thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 10 4  M -1 ·s -1 . LC-MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Lecithin:retinol acyltransferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs. HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Alternaria species (Deuteromycetes, Ascomycota) as ubiquitous fungi and prolific producers of a variety of toxic compounds are a part of microbiomes of plants, humans, and animals, mainly causing disease, allergic reactions, and toxicosis. However, some species have also been reported as endophytic microorganisms with highly bioactive metabolites. Our previous results indicate that potentially endophytic Alternaria species from Cupressaceae produce bioactive metabolites that possibly contribute to plant holobiont's health. Here, a possible mechanism behind this bioactivity is elucidated. As some endophytic fungi are reported to produce cytotoxic taxane diterpenoids, eight potentially endophytic Alternaria isolates from our collection were analyzed for the presence of the key genes of the paclitaxel (Taxol) biosynthetic pathway, i.e., taxadin synthase (ts), 10-deacetylbaccatin III-10-O-acetyltransferase (dbat), and C-13-phenylpropanoid side-chain CoA acyltransferase (bapt). The presence of all genes, i.e., ts, dbat, and bapt, was detected by PCR in six isolates and dbat and bapt in two isolates. Chemical analyses of the fermentation broths by TLC and HPLC chromatography and IR spectroscopy indicated the synthesis of the final product, i.e., paclitaxel. So, we introduce the synthesis of taxane diterpenoids as a possible mechanism by which Alternaria occupies the plant niches and protects the plant holobiont in the presence of competing microorganisms., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Geranylgeraniol (GGOH) is a crucial component in fragrances and essential oils, and a valuable precursor of vitamin E. It is primarily extracted from the oleoresin of Bixa orellana , but is challenged by long plant growth cycles, severe environmental pollution, and low extraction efficiency. Chemically synthesized GGOH typically comprises a mix of isomers, making the separation process both challenging and costly. Advancements in synthetic biology have enabled the construction of microbial cell factories for GGOH production. In this study, Yarrowia lipolytica was engineered to efficiently synthesize GGOH by expressing heterologous phosphatase genes, enhancing precursor supplies of farnesyl diphosphate, geranylgeranyl pyrophosphate, and acetyl-CoA, and downregulating the squalene synthesis pathway by promoter engineering. Additionally, optimizing fermentation conditions and reducing reactive oxygen species significantly increased the GGOH titer to 3346.47 mg/L in a shake flask. To the best of our knowledge, this is the highest reported GGOH titer in shaking flasks to date, setting a new benchmark for terpenoid production.

The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and-according to a structural model-active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Sclareolide, a natural product with bioactive and fragrant properties, is not only utilized in the food, healthcare, and cosmetics industries but also serves as a precursor for the production of ambroxide and some bioactive compounds. Currently, there are three primary methods for producing sclareolide: direct extraction from plants, chemical synthesis using sclareol as a precursor, and the biotransformation of sclareol. Here, we established a platform for producing sclareolide through a modular coculture system with Saccharomyces cerevisiae and Cryptococcus albidus ATCC 20918. S. cerevisiae was engineered for de novo sclareol biosynthesis from glucose, while C. albidus enabled the production of sclareolide via sclareol biotransformation. To enhance the supply of sclareol, a recombinant yeast strain was constructed through metabolic engineering to produce 536.2 mg/L of sclareol. Further improvement of the coculture system for sclareolide production was achieved by incorporating Triton X-100 facilitated intermediate permeability, inoculation proportion adjustment, and culture temperature optimization. These refinements culminated in a sclareolide yield of 626.3 mg/L. This study presents a novel streamlined and efficient approach for sclareolide preparation, showcasing the potential of the microbial consortium in sustainable bioproduction.

Background: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug., Result: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L., Conclusion: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation., (© 2024. The Author(s).)

Brazilian Cerrado is recognised as a biodiversity hotspot due to the presence of endemic species with great biological potential. Particularly, Lomatozona artemisiifolia , is a rare species found in the Cerrado region in midwestern Brazil. Efforts have been made for its conservation in the Cerrado, such as the use of in vitro micropropagation, demanding a comparative analysis between grown plants and those collected from nature. For this purpose, we performed the chemical study of L. artemisiifolia by LC-ESI-MS/MS and molecular networking analysis in the Global Natural Products Social Molecular Networking (GNPS) with in silico annotation using Network Annotation Propagation (NAP), which led to the observation of labdane diterpenes and flavonoid subclasses as the most representative specialised metabolites of this plant. In addition, molecular networking and chemometric analysis were correlated, allowing the metabolite profile emerging from field growth and micropropagation conditions to be observed.

More and more diterpenoids have attracted extensive attention due to the diverse chemical structures and excellent biological activities, and have been developed into clinical drugs or consumer products. The vast majority of diterpenoids are derived from plants. With the long-term development of plant medicinal materials, the natural resources of many plant diterpenoids are decreasing, and the biosynthetic mechanism of key active components has increasingly become a research hotspot. Using synthetic biology to engineer microorganisms into "cell factories" to produce the desired compounds is an essential means to solve these problems. In this review, we depict the plant-derived diterpenoids from chemical structure, biological activities, and biosynthetic pathways. We use representative plant diterpenes as examples to expound the research progress on their biosynthesis, and summarize the heterologous production of plant diterpenoids in microorganisms in recent years, hoping to lay the foundation for the development and application of plant diterpenoids in the future.

Isopentenyl diphosphate isomerase (IDI), a key enzyme in the biosynthetic pathway of diterpenoid alkaloids (DAs), plays an essential regulatory role in the synthesis and accumulation of DAs. In this study, the coding sequence (CDS) of AcIDI1 was isolated from the mother roots of Aconitum carmichaelii Debx. (GeneBank accession number OR915879). Bioinformatics analysis showed that the CDS of AcIDI1 was 894 bp, encoding a protein with 297 amino acids and the putative protein localized in the chloroplast. AcIDI1 exhibited significant homology with sequences encoding IDI in other species, and was most closely related to Aconitum vilmorinianum. Furthermore, the fusion protein has been successfully expressed in Escherichia coli (E. coli), providing a basis for future functional studies of AcIDI1. The expression pattern of AcIDI1 was analyzed by real-time quantitative PCR (qPCR), which demonstrates that AcIDI1 is a tissue-specific gene in the roots of A. carmichaelii and exhibits high expression in both daughter and mother roots. By comparing the expression levels of AcIDI1 in three tissues of the roots of A. carmichaelii at different growth stages, we propose that the mother roots (MRs) are the centers of resources allocation. The roots of A. carmichaelii continuously absorb the energy from external environment, while resources transfer behavior from MRs to both daughter roots (DRs) and axillary buds (ABs) occurs as the plant grows. This study establishes a foundation for applying the IDI gene to regulate the biosynthesis and accumulation of DAs in A. carmichaelii., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses. These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence. Here, we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana (Bs) and Fusarium graminearum (Fg) at the metabolite level. We identify hordedanes, a previously undescribed set of labdane-related diterpenoids with antimicrobial properties, as critical players in these interactions. Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 600-kb gene cluster. Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes, including one of the most functionally decorated products 19-β-hydroxy-hordetrienoic acid (19-OH-HTA). Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but, unexpectedly, show reduced Bs colonization. By contrast, colonization by Fusarium graminearum, another fungal pathogen of barley and wheat, is 4-fold higher in the mutants completely lacking hordedanes. Accordingly, 19-OH-HTA enhances both germination and growth of Bs, whereas it inhibits other pathogenic fungi, including Fg. Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs. Taken together, these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Cultivated rice (Oryza sativa) produces a variety of diterpenoid-type phytoalexins. Diterpene synthase genes that are responsible for the biosynthesis of momilactones, phytocassanes, and oryzalexins have been identified in O. sativa cv. Nipponbare. OsKSL10 (Os12t0491800 in RAP and LOC_Os12g30824 in MSU) was previously identified as an enzyme catalyzing the conversion of ent-copalyl diphosphate to ent-sandaracopimaradiene for the production of oryzalexins A to F. Our previous study on Oryza rufipogon, a wild progenitor of Asian cultivated rice, showed that both OrKSL10 and OrKSL10ind from O. rufipogon accessions W1943 and W0106, respectively, closely related to the japonica and indica subspecies, converted ent-copalyl diphosphate to ent-miltiradiene. Thus, the functional conversion of ent-miltiradiene synthase into ent-sandaracopimaradiene synthase is implied to have occurred through natural amino acid mutations, the details of which have not been elucidated. In this study, we show that introduction of A654G substitution into OrKSL10 significantly alters its function into more closely resembling that of OsKSL10. Moreover, double substitution V546I/A654G almost completely converts the function of OrKSL10 into that of OsKSL10. On the other hand, the reversed substitution I546V/G654A was insufficient to convert the function of OsKSL10 into OrKSL10, indicating the introduction of additional substitution S522I is required for the functionality of OsKSL10. Lastly, point mutations at the 654 A residue in OrKSL10 suggest that hydrophobic side chains at this position have a negative influence on the production of ent-sandaracopimaradiene., (© 2024 Federation of European Biochemical Societies.)

New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis . This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.

Covering: up to the end of 2023Cephalotane diterpenoids are a unique class of natural products exclusive to the genus Cephalotaxus , featuring a rigid 7,6,5,6-fused tetracyclic architecture. The study of cephalotanes dates back to the 1970s, when harringtonolide (1), a Cephalotaxus troponoid with a peculiar norditerpenoid carbon skeleton, was first discovered. In recent years, prototype C 20 diterpenoids proposed as cephalotane were disclosed, which triggered intense studies on this diterpenoid family. To date, a cumulative total of 105 cephalotane diterpenoids with great structural diversity and biological importance have been isolated. In addition, significant advances have been made in the field of total synthesis and biosynthesis of cephalotanes in recent years. This review provides a complete overview of the chemical structures, bioactivities, biosynthetic aspects, and completed total synthesis of all the isolated cephalotane diterpenoids, which will help guide future research on this class of compounds.

Eunicellane diterpenoids, containing a typical 6,10-bicycle, are bioactive compounds widely present in marine corals, but rarely found in bacteria and plants. The intrinsic macrocycle exhibits innate structural flexibility resulting in dynamic conformational changes. However, the mechanisms controlling flexibility remain unknown. The discovery of a terpene synthase, MicA, that is responsible for the biosynthesis of a nearly non-flexible eunicellane skeleton, enable us to propose a feasible theory about the flexibility in eunicellane structures. Parallel studies of all eunicellane synthases in nature discovered to date, including 2Z-geranylgeranyl diphosphate incubations and density functional theory-based Boltzmann population computations, reveale that a trans-fused bicycle with a 2Z-configuration alkene restricts conformational flexibility resulting in a nearly non-flexible eunicellane skeleton. The catalytic route and the enzymatic mechanism of MicA are also elucidated by labeling experiments, density functional theory calculations, structural analysis of the artificial intelligence-based MicA model, and mutational studies., (© 2024. The Author(s).)

Vitamin A is an important nutrient for multiple physiological functions. To elucidate the role of vitamin A in vivo, vitamin A-deficient diets have been often used in mice to establish a vitamin A-deficiency model. However, the information on the appropriate feeding periods and time course of changes in vitamin A content in organs after the start of vitamin A-deficient diet feeding is lacking. This study aimed to assess the retinoids levels in liver and white adipose tissue in mice fed a vitamin A-deficient diet for ≤8 weeks. High-performance liquid chromatography was used to measure the retinoids levels in liver and white adipose tissue every 2 weeks for ≤8 weeks. Vitamin A-deficient diet feeding significantly decreased retinol in the liver over 6 weeks, but retinyl palmitate, a main storage form of vitamin A, was not changed over 8 weeks. The plasma retinol level remained constant throughout the experiment. In white adipose tissue, retinyl palmitate gradually decreased over 8 weeks. These results indicate that vitamin A-deficient diet feeding longer than 6 weeks reduced retinol in liver and retinyl palmitate in white adipose tissue over 8 weeks, although it is not enough for the induction of a whole-body vitamin A deficiency.

Two terpene synthases from the pleuromutilin producing fungus Clitopilus passeckerianus were functionally characterised. The first enzyme CpTS1 produces the new diterpene clitopilene with a novel 6-6-5-5 tetracyclic skeleton, while the second enzyme CpTS2 makes the new sesquiterpene isopentalenene. The CpTS1 reaction mechanism was studied in depth using experimental and theoretical approaches.

Chamaecyparis obtusa and C. obtusa var. formosana of the Cupressaceae family are well known for their fragrance and excellent physical properties. To investigate the biosynthesis of unique diterpenoid compounds, diterpene synthase genes for specialized metabolite synthesis were cloned from C. obtusa and C. obtusa var. formosana. Using an Escherichia coli co-expression system, eight diterpene synthases (diTPSs) were characterized. CoCPS and CovfCPS are class II monofunctional (+)-copalyl diphosphate synthases [(+)-CPSs]. Class I monofunctional CoLS and CovfLS convert (+)-copalyl diphosphate [(+)-CPP] to levopimaradiene, CoBRS, CovfBRS1, and CovfBRS3 convert (+)-CPP to (-)-beyerene, and CovfSDS converts (+)-CPP to (-)-sandaracopimaradiene. These enzymes are all monofunctional diterpene syntheses in Cupressaceae family of gymnosperm, and differ from those in Pinaceae. The discovery of the enzyme responsible for the biosynthesis of tetracyclic diterpene (-)-beyerene was characterized for the first time. Diterpene synthases with different catalytic functions exist in closely related species within the Cupressaceae family, indicating that this group of monofunctional diterpene synthases is particularly prone to the evolution of new functions and development of species-specific specialized diterpenoid constituents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cembranoids and labdanes are two important types of diterpenes in tobacco (Nicotiana genus) that are predominantly found in the leaf and flower glandular trichome secretions. This is the first systematic review of the biosynthesis, chemical structures, bioactivities, and utilisation values of cembranoid and labdane diterpenes in tobacco. A total of 131 natural cembranoid diterpenes have been reported in tobacco since 1962; these were summarised and classified according to their chemical structure characteristics as isopropyl cembranoids (1-88), seco-cembranoids (89-103), chain cembranoids (104-123), and polycyclic cembranoids (124-131). Forty natural labdane diterpenes reported since 1961 were also summarised and divided into epoxy side chain labdanes (132-150) and epoxy-free side chain labdanes (151-171). Tobacco cembranoid and labdane diterpenes are both formed via the methylerythritol 4-phosphate pathway and are synthesised from geranylgeranyl diphosphate. Their biosynthetic pathways and the four key enzymes (cembratrienol synthase, cytochrome P450 hydroxylase, copalyl diphosphate synthase, and Z-abienol cyclase) that affect their biosynthesis have been described in detail. A systematic summary of the bioactivity and utilisation values of the cembranoid and labdane diterpenes is also provided. The agricultural bioactivities associated with cembranoid and labdane diterpenes include antimicrobial and insecticidal activities as well as induced resistance, while the medical bioactivities include cytotoxic and neuroprotective activities. Further research into the cembranoid and labdane diterpenes will help to promote their development and utilisation as plant-derived pesticides and medicines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cyathus olla, belonging to the genus Cyathus within the order Agaricales, is renowned for its bird's nest-like fruiting bodies and has been utilized in folk medicine. However, its genome remains poorly understood. To investigate genomic diversity within the genus Cyathus and elucidate biosynthetic pathways for medicinal compounds, we generated a high-quality genome assembly of C. olla with fourteen chromosomes. The comparative genome analysis revealed variations in both genomes and specific functional genes within the genus Cyathus. Phylogenomic and gene family variation analyses provided insights into evolutionary divergence, as well as genome expansion and contraction in individual Cyathus species and 36 typical Basidiomycota. Furthermore, analysis of LTR-RT and Ka/Ks revealed apparent whole-genome duplication (WGD) events its genome. Through genome mining and metabolite profiling, we identified the biosynthetic gene cluster (BGC) for cyathane diterpenes from C. olla. Furthermore, we predicted 32 BGCs, containing 41 core genes, involved in other bioactive metabolites. These findings represent a valuable genomic resource that will enhance our understanding of Cyathus species genetic diversity. The genome analysis of C. olla provides insights into the biosynthesis of medicinal compounds and establishes a fundamental basis for future investigations into the genetic basis of chemodiversity in this significant medicinal fungus., (© 2024. The Author(s).)

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αβγ domains. Active sites of ent-CPS exist at the interface of β and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or βγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its βγ domains. Domain-swapping experiments showed that replacing βγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of βγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without βγ domains., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Labdane-related diterpenoids (LRDs), a subgroup of terpenoids, exhibit structural diversity and significant commercial and pharmacological potential. LRDs share the characteristic decalin-labdanic core structure that derives from the cycloisomerization of geranylgeranyl diphosphate (GGPP). Labdanes derive their name from the oleoresin known as 'Labdanum', 'Ladano', or 'Aladano', used since ancient Greek times. Acetylated labdanes, rarely identified in plants, are associated with enhanced biological activities. Chemical analysis of Cistus creticus subsp. creticus revealed labda-7,13(E)-dien-15-yl acetate and labda-7,13(E)-dien-15-ol as major constituents. In addition, novel labdanes such as cis-abienol, neoabienol, ent-copalol, and one as yet unidentified labdane-type diterpenoid were detected for the first time. These compounds exhibit developmental regulation, with higher accumulation observed in young leaves. Using RNA-sequencing (RNA-seq) analysis of young leaf trichomes, it was possible to identify, clone, and eventually functionally characterize labdane-type diterpenoid synthase (diTPS) genes, encoding proteins responsible for the production of labda-7,13(E)-dien-15-yl diphosphate (endo-7,13-CPP), labda-7,13(E)-dien-15-yl acetate, and labda-13(E)-ene-8α-ol-15-yl acetate. Moreover, the reconstitution of labda-7,13(E)-dien-15-yl acetate and labda-13(E)-ene-8α-ol-15-yl acetate production in yeast is presented. Finally, the accumulation of LRDs in different plant tissues showed a correlation with the expression profiles of the corresponding genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

To better assess the practical value and avoid potential risks of the traditionally medicinal and edible basidiomycete Schizophyllum commune , which may arise from undescribed metabolites, a combination of elicitors was introduced for the first time to discover products from cryptic and low-expressed gene clusters under laboratory cultivation. Treating S. commune NJFU21 with the combination of five elicitors led to the upregulated production of a class of unusual linear diterpene-derived variants, including eleven new ones ( 1 - 11 ), along with three known ones ( 12 - 14 ). The structures and stereochemistry were determined by 1D and 2D NMR, HRESIMS, ECD, OR and VCD calculations. Notably, the elongation terminus of all the diterpenes was decorated by an unusual butenedioic acid moiety. Compound 1 was a rare monocyclic diterpene, while 2 - 6 possessed a tetrahydrofuran moiety. The truncated metabolites 4 , 5 and 13 belong to the trinorditerpenes. All the diterpenes displayed approximately 70% scavenging of hydroxyl radicals at 50 μM and null cytotoxic activity at 10 μM. In addition, compound 1 exhibited potent antifungal activity against the plant pathogenic fungi Colletotrichum camelliae , with MIC values of 8 μg/mL. Our findings indicated that this class of diterpenes could provide valuable protectants for cosmetic ingredients and the lead compounds for agricultural fungicide development.

Cembratriene-ol (CBT-ol), a plant-derived macrocyclic diterpene with notable insecticidal activity, has attracted considerable attention with respect to the development of sustainable and green biopesticides. Currently, CBT-ol production is limited by an inefficient and costly plant extraction strategy. Herein, CBT-ol production was enhanced by redesigning the CBT-ol biosynthetic pathway in Candida tropicalis, with subsequent truncation of CBT-ol synthase further increasing CBT-ol production. Moreover, bottlenecks in the CBT-ol biosynthetic pathway were eliminated by adjusting the gene dosage of the rate-limiting enzymes. Ultimately, the resulting strain C. tropicalis CPPt-03D produced 129.17 mg/L CBT-ol in shaking flasks (a 144-fold increase relative to that of the initial strain C01-CD) with CBT-ol production reaching 1,425.76 mg/L in a 5-L bioreactor, representing the highest CBT-ol titer reported to date. These findings provide a green process and promising platform for the industrial production of CBT-ol and lays the foundation for organic farming., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xianzhong Chen reports financial support was provided by the National Natural Science Foundation of China. Xianzhong Chen and Wei Shen reports financial support was provided by the Key Research and Development Program of China. Lihua Zhang reports financial support was provided by the Key Scientific Research Project of Higher Education Institutions in Henan province. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Kalmegh (Andrographis paniculata) spatiotemporally produces medicinally-important ent-labdane-related diterpenoids (ent-LRDs); andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), neoandrographolide (NAD). ApCPS1 and ApCPS2, the ent-copalyl pyrophosphate (ent-CPP)-producing class II diterpene synthases (diTPSs) were identified, but their contributions to ent-CPP precursor supply for ent-LRD biosynthesis were not well understood. Here, we characterized ApCPS4, an additional ent-CPP-forming diTPS. Further, we elucidated in planta function of the ent-CPP-producing diTPSs (ApCPS1,2,4) by integrating transcript-metabolite co-profiles, biochemical analysis and gene functional characterization. ApCPS1,2,4 localized to the plastids, where diterpenoid biosynthesis occurs in plants, but ApCPS1,2,4 transcript expression patterns and ent-LRD contents revealed a strong correlation of ApCPS2 expression and ent-LRD accumulation in kalmegh. ApCPS1,2,4 upstream sequences differentially activated β-glucuronidase (GUS) in Arabidopsis and transiently-transformed kalmegh. Similar to higher expression of ApCPS1 in kalmegh stem, ApCPS1 upstream sequence activated GUS in stem/hypocotyl of Arabidopsis and kalmegh. However, ApCPS2,4 upstream sequences weakly activated GUS expression in Arabidopsis, which was not well correlated with ApCPS2,4 transcript expression in kalmegh tissues. Whereas, ApCPS2,4 upstream sequences could activate GUS expression at a considerable level in kalmegh leaf and roots/calyx, respectively, suggesting the involvement of transcriptional regulator(s) of ApCPS2,4 that might participate in kalmegh-specific diterpenoid pathway. Interestingly, ApCPS2-silenced kalmegh showed a drastic reduction in AD, DDAD and NAD contents and compromised defense against insect herbivore Spodoptera litura. However, ent-LRD contents and herbivore defense in ApCPS1 or ApCPS4-silenced plants remained largely unaltered. Overall, these results suggested an important role of ApCPS2 in producing ent-CPP for medicinal ent-LRD biosynthesis and defense against insect herbivore., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest associated with this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20 K197G (Erg20 G ) and Erg20 F96W, N127W (Erg20 WW ), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20 WWG mutant with over four times higher GPP accumulating capability than Erg20 WW , as observed through geraniol production levels. The Erg20 WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20 WWG -fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20 WWG -LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20 WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Miltiradiene serves as a crucial precursor in the synthesis of various high-value abietane-type diterpenes, exhibiting diverse pharmacological activities. Previous efforts to enhance miltiradiene production have primarily focused on the mevalonate acetate (MVA) pathway. However, limited emphasis has been placed on optimizing the supply of acetyl-CoA and NADPH. In this study, we constructed a platform yeast strain for miltiradiene production by reinforcing the biosynthetic pathway of geranylgeranyl diphosphate (GGPP) and acetyl-CoA, and addressing the imbalance between the supply and demand of the redox cofactor NADPH within the cytoplasm, resulting in an increase in miltiradiene yield to 1.31 g/L. Furthermore, we conducted modifications to the miltiradiene synthase fusion protein t Sm KSL1- Cf TPS1. Finally, the comprehensive engineering strategies and protein modification strategies culminated in 1.43 g/L miltiradiene in the engineered yeast under shake flask culture conditions. Overall, our work established efficient yeast cell factories for miltiradiene production, providing a foothold for heterologous biosynthesis of abietane-type diterpenes.

American Trypanosomiasis, also known as Chagas disease, is caused by the protozoan Trypanosoma cruzi and exhibits limited options for treatment. Natural products offer various structurally complex metabolites with biological activities, including those with anti- T. cruzi potential. The discovery and development of prototypes based on natural products frequently display multiple phases that could be facilitated by machine learning techniques to provide a fast and efficient method for selecting new hit candidates. Using Random Forest and k-Nearest Neighbors, two models were constructed to predict the biological activity of natural products from plants against intracellular amastigotes of T. cruzi . The diterpenoid andrographolide was identified from a virtual screening as a promising hit compound. Hereafter, it was isolated from Cymbopogon schoenanthus and chemically characterized by spectral data analysis. Andrographolide was evaluated against trypomastigote and amastigote forms of T. cruzi , showing IC 50 values of 29.4 and 2.9 μM, respectively, while the standard drug benznidazole displayed IC 50 values of 17.7 and 5.0 μM, respectively. Additionally, the isolated compound exhibited a reduced cytotoxicity (CC 50 = 92.8 μM) against mammalian cells and afforded a selectivity index (SI) of 32, similar to that of benznidazole (SI = 39). From the in silico analyses, we can conclude that andrographolide fulfills many requirements implemented by DND i to be a hit compound. Therefore, this work successfully obtained machine learning models capable of predicting the activity of compounds against intracellular forms of T. cruzi .

Ethnopharmacological Relevance: Aconitum carmichaelii is widely used in traditional Chinese medicine clinics as a bulk medicinal material. It has been used in China for more than two thousand years. Nevertheless, the stems and leaves of this plant are usually discarded as non-medicinal parts, even though they have a large biomass and exhibit therapeutic properties. Thus, it is crucial to investigate metabolites of different parts of Aconitum carmichaelii and explore the relationship between metabolites and toxicity to unleash the utilization potential of the stems and leaves., Aim of the Study: Using plant metabolomics, we aim to correlate different metabolites in various parts of Aconitum carmichaelii with toxicity, thereby screening for toxicity markers. This endeavor seeks to offer valuable insights for the development of Aconitum carmichaelii stem and leaf-based applications., Materials and Methods: UHPLC-Q-Orbitrap MS/MS-based plant metabolomics was employed to analyze metabolites of the different parts of Aconitum carmichaelii. The cardiotoxicity and hepatotoxicity of the extracts from different parts of Aconitum carmichaelii were also investigated using zebrafish as animal model. Toxicity markers were subsequently identified by correlating toxicity with metabolites., Results: A total of 113 alkaloids were identified from the extracts of various parts of Aconitum carmichaelii, with 64 different metabolites in stems and leaves compared to daughter root (Fuzi), and 21 different metabolites in stems and leaves compared to mother root (Wutou). The content of aporphine alkaloids in the stems and leaves of Aconitum carmichaelii is higher than that in the medicinal parts, while the content of the diester-diterpenoid alkaloids is lower. Additionally, the medicinal parts of Aconitum carmichaelii exhibited cardiotoxicity and hepatotoxicity, while the stems and leaves have no obvious toxicity. Finally, through correlation analysis and animal experimental verification, mesaconitine, deoxyaconitine, and hypaconitine were used as toxicity markers., Conclusion: Given the low toxicity of the stems and leaves and the potential efficacy of aporphine alkaloids, the stems and leaves of Aconitum carmichaelii hold promise as a valuable medicinal resource warranting further development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Kauralexin A1 (KA1) is a key intermediate of the kauralexin A series metabolites of maize phytoalexins. However, their application is severely limited by their low abundance in maize. In this study, an efficient biosynthetic pathway was constructed to produce KA1 in Saccharomyces cerevisiae . Also, metabolic and enzyme engineering strategies were applied to construct the high-titer strains, such as chassis modification, screening synthases, the colocalization of enzymes, and multiple genomic integrations. First, the KA1 precursor ent -kaurene was synthesized using the efficient diterpene synthase GfCPS/KS from Fusarium fujikuroi , and optimized to reach 244.36 mg/L in shake flasks, which displayed a 200-fold increase compared to the initial strain. Then, the KA1 was produced under the catalysis of ZmCYP71Z18 from Zea mays and SmCPR1 from Salvia miltiorrhiza , and the titer was further improved by integrating the fusion protein into the genome. Finally, an ent -kaurene titer of 763.23 mg/L and a KA1 titer of 42.22 mg/L were achieved through a single-stage fed-batch fermentation in a 5 L bioreactor. This is the first report of the heterologous biosynthesis of maize diterpene phytoalexins in S. cerevisiae , which lays a foundation for further pathway reconstruction and biosynthesis of the kauralexin A series maize phytoalexins.

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., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plants synthesize abundant terpenes through glandular trichomes (GTs), thereby protecting themselves from environmental stresses and increasing the economic value in some medicinal plants. However, the potential mechanisms for simultaneously regulating terpenes synthesis and GTs development remain unclear. Here, we showed that terpenes in Conyza blinii could be synthesized through capitate GTs. By treating with appropriate intensity of UV-B, the density of capitate GTs and diterpene content can be increased. Through analyzing corresponding transcriptome, we identified a MYB transcription factor CbMYB108 as a positive regulator of both diterpene synthesis and capitate GT density. Transiently overexpressing/silencing CbMYB108 on C. blinii leaves could increase diterpene synthesis and capitate GT density. Further verification showed that CbMYB108 upregulated CbDXS and CbGGPPS expression in diterpene synthesis pathway. Moreover, CbMYB108 could also upregulated the expression of CbTTG1, key WD40 protein confirmed in this study to promote GT development, rather than through interaction between CbMYB108 and CbTTG1 proteins. Thus, results showed that the UV-B-induced CbMYB108 owned dual-function of simultaneously improving diterpene synthesis and GT development. Our research lays a theoretical foundation for cultivating C. blinii with high terpene content, and broadens the understanding of the integrated mechanism on terpene synthesis and GT development in plants., (© 2024 John Wiley & Sons Ltd.)

Andrographis paniculata (Burm. f.) Nees is an important medicinal plant known for its bioactive compound andrographolide. NAC transcription factors (NAM, ATAF1/2, and CUC2) play a crucial role in secondary metabolite production, stress responses, and plant development through hormonal signaling. In this study, a putative partial transcript of three NAC family genes ( ApNAC83 , ApNAC21 22 and ApNAC02 ) was used to isolate full length genes using RACE. Bioinformatics analyses such as protein structure prediction, cis-acting regulatory elements, and gene ontology analysis were performed. Based on in silico predictions, the diterpenoid profiling of the plant's leaves (five-week-old) and the real-time PCR-based expression analysis of isolated NAC genes under abscisic acid (ABA) treatment were performed. Additionally, the expression analysis of isolated NAC genes under MeJA treatment and transient expression in Nicotiana tabacum was performed. Full-length sequences of three members of the NAC transcription factor family, ApNAC83 (1102 bp), ApNAC21 22 (996 bp), and ApNAC02 (1011 bp), were isolated and subjected to the promoter and gene ontology analysis, which indicated their role in transcriptional regulation, DNA binding, ABA-activated signaling, and stress management. It was observed that ABA treatment leads to a higher accumulation of andrographolide and 14-deoxyandrographolide content, along with the upregulation of ApNAC02 (9.6-fold) and the downregulation of ApNAC83 and ApNAC21 22 in the leaves. With methyl jasmonate treatment, ApNAC21 22 expression decreased, while ApNAC02 increased (1.9-fold), with no significant change being observed in ApNAC83 . The transient expression of the isolated NAC genes in a heterologous system ( Nicotiana benthamiana ) demonstrated their functional transcriptional activity, leading to the upregulation of the NtHMGR gene, which is related to the terpene pathway in tobacco. The expression analysis and heterologous expression of ApNAC21 22 and ApNAC02 indicated their role in andrographolide biosynthesis.

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, β-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, β-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases., (© 2024. The Author(s).)

Ethnopharmacological Relevance: Scoparia dulcis has been identified as a significant ethnopharmacological substance in the Li, Zhuang, and Dai ethnic groups of China. Traditional medicine use S. dulcis to treat numerous illnesses, most notably diabetes. The considerable antidiabetic properties of this herbal remedy have been established by several clinical investigations and animal experiments. The islet is the intended target of S. dulcis, although the cause of its activity and mechanism for diabetes treatment is unclear. The diterpenoids from S. dulcis have been shown in the literature to have significant hypoglycemic efficacy and to protect islet cells in vitro. Diterpenoids may be the components of this herbal remedy that preserve islets, but further research is needed., Aim of the Study: This study was projected to investigate the new diterpenoid scoparicol E from S. dulcis and examined its islet-protective effect and the potential mechanism both in vitro and in vivo., Methods: The structure of the novel diterpenoid scoparicol E was clarified by employing a wide range of spectroscopic methods. Using CCK-8 tests, cytotoxicity and antiapoptotic activity of scoparicol E were detected. Serum biochemical analysis and pathologic examination were performed to study the protective effect of scoparicol E against islet damage. The specific mechanism of action of scoparicol E was investigated through the mitochondrial membrane potential, Annexin V-FITC flow cytometry, and western blotting., Results: Scoparicol E reduced MLD-STZ-induced hyperglycemia in mice and increased insulin and islet apoptosis. Scoparicol E effectively suppressed the Bax/Bcl-2/Caspase-3 pathway, according to the in vivo western blot investigation. Scoparicol E showed significant antiapoptotic action in vitro. We also showed that scoparicol E might prevent islet cells from dying by inhibiting the Bax/Bcl-2/Caspase-3 pathway. The Annexin V-FITC flow cytometry results revealed that MIN6 cell apoptosis was considerably decreased following scoparicol E intervention, showing anti-islet cell apoptosis action. Furthermore, the Caspase-3-mediated apoptosis pathway depends on cytochrome c and the potential of the mitochondrial membrane. Scoparicol E prevented the release of cytochrome c, restored the mitochondrial membrane potential, and prevented MIN6 cell apoptosis., Conclusion: We demonstrated the new diterpenoid scoparicol E could protect islet cells apoptosis by modulating the Bax/Bcl-2/Caspase-3 pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The phlebotomine sandfly, Lutzomyia longipalpis , a major vector of the Leishmania parasite, uses terpene pheromones to attract conspecifics for mating. Examination of the L. longipalpis genome revealed a putative terpene synthase (TPS), which-upon heterologous expression in, and purification from, Escherichia coli -yielded a functional enzyme. The TPS, termed Ll TPS, converted geranyl diphosphate (GPP) into a mixture of monoterpenes with low efficiency, of which β-ocimene was the major product. ( E,E )-farnesyl diphosphate (FPP) principally produced small amounts of ( E )-β-farnesene, while ( Z,E )- and ( Z,Z )-FPP yielded a mixture of bisabolene isomers. None of these mono- and sesquiterpenes are known volatiles of L. longipalpis . Notably, however, when provided with ( E,E,E )-geranylgeranyl diphosphate (GGPP), Ll TPS gave sobralene as its major product. This diterpene pheromone is released by certain chemotypes of L. longipalpis , in particular those found in the Ceará state of Brazil. Minor diterpene components were also seen as products of the enzyme that matched those seen in a sandfly pheromone extract., Competing Interests: Competing interests statement:The authors declare no competing interest.

Main Conclusion: Protein modeling, carbocation docking, and molecular dynamics along with structure-based mutability landscapes provided insight into taxadiene synthase catalysis (first step of the anticancer Taxol biosynthesis), protein structure-function correlations, and promiscuity. Plant terpenes belong to one of the largest and most diverse classes of natural products. This diversity is driven by the terpene synthase enzyme family which comprises numerous different synthases, several of which are promiscuous. Taxadiene synthase (TXS) is a class I diterpene synthase that catalyzes the first step in the biosynthesis pathway of the diterpene Taxol, an anticancer natural product produced by the Taxus plant. Exploring the molecular basis of TXS catalysis and its promiscuous potential garnered interest as a necessary means for understanding enzyme evolution and engineering possibilities to improve Taxol biosynthesis. A catalytically active closed conformation TXS model was designed using the artificial intelligence system, AlphaFold, accompanied by docking and molecular dynamics simulations. In addition, a mutability landscape of TXS including 14 residues was created to probe for structure-function relations. The mutability landscape revealed no mutants with improved catalytic activity compared to wild-type TXS. However, mutations of residues V584, Q609, V610, and Y688 showed high degree of promiscuity producing cembranoid-type and/or verticillene-type major products instead of taxanes. Mechanistic insights into V610F, V584M, Q609A, and Y688C mutants compared to the wild type revealed the trigger(s) for product profile change. Several mutants spanning residues V584, Q609, Y688, Y762, Q770, and F834 increased production of taxa-4(20),11(12)-diene which is a more favorable substrate for Taxol production compared to taxa-4(5),11(12)-diene. Finally, molecular dynamics simulations of the TXS reaction cascade revealed residues involved in ionization, carbocation stabilization, and cyclization ushering deeper understanding of the enzyme catalysis mechanism., (© 2024. The Author(s).)