Implications of RNA pseudouridylation for cancer biology and therapeutics: a narrative review.

BACKGROUND: Pseudouridine (Ψ), a C5-glycoside isomer of uridine, stands as one of the most prevalent RNA modifications in all RNA types. Distinguishing from the C-N bond linking uridine to ribose, the link between Ψ and ribose is a C-C bond, endowing Ψ modified RNA distinct properties and functions in various biological processes. The conversion of uridine to Ψ is governed by pseudouridine synthases (PUSs). RNA pseudouridylation is implicated in cancer biology and therapeutics. OBJECTIVES: In this review, we will summarize the methods for detecting Ψ, the process of Ψ generation, the impact of Ψ modification on RNA metabolism and gene expression, the roles of dysregulated Ψ and pseudouridine synthases in cancers, and the underlying mechanism. METHODS: We conducted a comprehensive search of PubMed from its inception through February 2024. The search terms included "pseudouridine"; "pseudouridine synthase"; "PUS"; "dyskerin"; "cancer"; "tumor"; "carcinoma"; "malignancy"; "tumorigenesis"; "biomarker"; "prognosis" and "therapy". We included studies published in peer-reviewed journals that focused on Ψ detection, specific mechanisms involving Ψ and PUSs, and prognosis in cancer patients with high Ψ expression. We excluded studies lacking sufficient methodological details or appropriate controls. RESULTS: Ψ has been recognized as a significant biomarker in cancer diagnosis and prognosis. Abnormal Ψ modifications mediated by various PUSs result in dysregulated RNA metabolism and impaired RNA function, promoting the development of various cancers. Overexpression of PUSs is common in cancer cells and predicts poor prognosis. PUSs inhibition arrests cell proliferation and enhances apoptosis in cancer cells, suggesting PUS-targeting cancer therapy may be a potential strategy in cancer treatment. DISCUSSION: High Ψ levels in serum, urine, and saliva may suggest cancer, but do not specify the type, requiring additional lab markers and imaging for accurate diagnosis. Standardized detection methods are also crucial for reliable results. PUSs are linked to cancer, but more researches are needed to understand their mechanisms in different cancers. Anticancer treatments targeting PUSs are still under developed.

RluA is the major mRNA pseudouridine synthase in Escherichia coli.

Pseudouridine (Ψ) is an ubiquitous RNA modification, present in the tRNAs and rRNAs of species across all domains of life. Conserved pseudouridine synthases modify the mRNAs of diverse eukaryotes, but the modification has yet to be identified in bacterial mRNAs. Here, we report the discovery of pseudouridines in mRNA from E. coli. By testing the mRNA modification capacity of all 11 known pseudouridine synthases, we identify RluA as the predominant mRNA-modifying enzyme. RluA, a known tRNA and 23S rRNA pseudouridine synthase, modifies at least 31 of the 44 high-confidence sites we identified in E. coli mRNAs. Using RNA structure probing data to inform secondary structures, we show that the target sites of RluA occur in a common sequence and structural motif comprised of a ΨURAA sequence located in the loop of a short hairpin. This recognition element is shared with previously identified target sites of RluA in tRNAs and rRNA. Overall, our work identifies pseudouridine in key mRNAs and suggests the capacity of Ψ to regulate the transcripts that contain it.

H2O2 sulfenylates CHE, linking local infection to the establishment of systemic acquired resistance.

In plants, a local infection can lead to systemic acquired resistance (SAR) through increased production of salicylic acid (SA). For many years, the identity of the mobile signal and its direct transduction mechanism for systemic SA synthesis in initiating SAR have been debated. We found that in Arabidopsis thaliana, after a local infection, the conserved cysteine residue of the transcription factor CCA1 HIKING EXPEDITION (CHE) undergoes sulfenylation in systemic tissues, which enhances its binding to the promoter of the SA-synthesis gene ISOCHORISMATE SYNTHASE1 (ICS1) and increases SA production. Furthermore, hydrogen peroxide (H2O2) produced through NADPH oxidases is the mobile signal that sulfenylates CHE in a concentration-dependent manner. Accumulation of SA and the previously reported signal molecules, such as N-hydroxypipecolic acid (NHP), then form a signal amplification loop to establish SAR.

Genome-wide identification of oxidosqualene cyclase genes regulating natural rubber in Taraxacum kok-saghyz.

MAIN CONCLUSION: Nine TkOSC genes have been identified by genome-wide screening. Among them, TkOSC4-6 might be more crucial for natural rubber biosynthesis in Taraxacum kok-saghyz roots. Taraxacum kok-saghyz Rodin (TKS) roots contain large amounts of natural rubber, inulin, and valuable metabolites. Oxidosqualene cyclase (OSC) is a key member for regulating natural rubber biosynthesis (NRB) via the triterpenoid biosynthesis pathway. To explore the functions of OSC on natural rubber producing in TKS, its gene family members were identified in TKS genome via genome-wide screening. Nine TkOSCs were identified, which were mainly distributed in the cytoplasm. Their family genes experienced a neutral selection during the evolution process. Overall sequence homology analysis OSC proteins revealed 80.23% similarity, indicating a highly degree of conservation. Pairwise comparisons showed a multiple sequence similarity ranging from 57% to 100%. Protein interaction prediction revealed that TkOSCs may interact with baruol synthase, sterol 1,4-demethylase, lupeol synthase and squalene epoxidase. Phylogenetic analysis showed that OSC family proteins belong to two branches. TkOSC promoter regions contain cis-acting elements related to plant growth, stress response, hormones response and light response. Protein accumulation analysis demonstrated that TkOSC4, TkOSC5 and TkOSC6 proteins had strong expression levels in the root, latex and plumular axis. Comparison of gene expression patterns showed TkOSC1, TkOSC4, TkOSC5, TkOSC6, TkOSC7, TkOSC8 and TkOSC9 might be important in regulating NRB. Combination of gene and protein results revealed TkOSC4-6 might be more crucial, and the data might contribute to a more profound understanding of the roles of OSCs for NRB in TKS roots.

In-depth analysis of isochorismate synthase-derived metabolism in plant immunity: Identification of meta-substituted benzoates and salicyloyl-malate.

Isochorismate-derived metabolism enables biosynthesis of the plant defense hormone salicylic acid (SA) and its derivatives. In Arabidopsis thaliana, the stress-induced accumulation of SA depends on ISOCHORISMATE SYNTHASE1 (ICS1) and also requires the presumed isochorismate transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) and the GH3 enzyme avrPphB SUSCEPTIBLE3 (PBS3). By comparative metabolite and structural analyses, we identified several hitherto unreported ICS1- and EDS5-dependent, biotic stress-inducible Arabidopsis metabolites. These involve meta-substituted SA derivatives (5-formyl-SA, 5-carboxy-SA, 5-carboxymethyl-SA), their benzoic acid (BA) analogs (3-formyl-BA, 3-carboxy-BA, 3-carboxymethyl-BA), and besides the previously detected salicyloyl-aspartate (SA-Asp), the ester conjugate salicyloyl-malate (SA-Mal). SA functions as a biosynthetic precursor for SA-Mal and SA-Asp, but not for the meta-substituted SA- and BA-derivatives, which accumulate to moderate levels at later stages of bacterial infection. Interestingly, Arabidopsis leaves possess oxidizing activity to effectively convert meta-formyl- into meta-carboxy-SA/BAs. In contrast to SA, exogenously applied meta-substituted SA/BA-derivatives and SA-Mal exert a moderate impact on plant immunity and defence-related gene expression. While the isochorismate-derived metabolites are negatively regulated by the SA receptor NON-EXPRESSOR OF PR GENES1, SA conjugates (SA-Mal, SA-Asp, SA-glucose conjugates) and meta-substituted SA/BA-derivatives are oppositely affected by PBS3. Notably, our data indicate a PBS3-independent path to isochorismate-derived SA at later stages of bacterial infection, which does not considerably impact immune-related characteristics. Moreover, our results argue against a previously proposed role of EDS5 in the biosynthesis of the immune signal N-hydroxypipecolic acid and associated transport processes. We propose a significantly extended biochemical scheme of plant isochorismate metabolism that involves an alternative generation mode for benzoate- and salicylate-derivatives.

Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis.

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid's precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis. KEY POINTS: • Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis. • Perillic acid was first synthesized de novo in engineered microorganisms. • The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

ß-Tyrosine and its biosynthetic enzyme TAM1 are predominantly distributed in the ancestral subpopulation of japonica rice in Oryza rufipogon.

Intraspecific variation in specialized metabolites plays a crucial role in the adaptive response to diverse environments. Two major subspecies, japonica and indica, are observed in Asian cultivated rice (Oryza sativa L.). Previously, we identified (3R)-ß-tyrosine, a novel nonproteinogenic ß-amino acid in plants, along with the enzyme tyrosine aminomutase (TAM1), which is required for ß-tyrosine biosynthesis, in the japonica cultivar Nipponbare. Notably, TAM1 and ß-tyrosine were preferentially distributed in japonica cultivars compared with indica cultivars. Considering its phytotoxicity and antimicrobial activity, intraspecific variation in ß-tyrosine may contribute to the defensive potential of japonica rice. Investigation of the evolutionary trajectory of TAM1 and ß-tyrosine should enhance our understanding of the evolution of rice defense. However, their distribution patterns in O. rufipogon, the direct ancestor of O. sativa, remain unclear. Therefore, in this study, we extensively examined TAM1 presence/absence and ß-tyrosine content in 110 genetically and geographically diverse O. rufipogon accessions and revealed that they are characteristically observed in the ancestral subpopulation of japonica rice, while being absent or slightly accumulated in other subpopulations. Thus, we conclude that TAM1 and ß-tyrosine in japonica rice are likely derived from its ancestral subpopulation. Furthermore, the high and low TAM1 possession rates and ß-tyrosine content in japonica and indica rice, respectively, could be attributed to distribution patterns of TAM1 and ß-tyrosine in their ancestral subpopulations. This study provides fundamental insights into the evolution of rice defense.

[Cloning and functional characterization of oxidosqualene cyclase gene for α-amyrin synthesis].

Ursolic acid has gradually attracted much attention due to its unique pharmacological activities and valuable market value in recent years. Currently, ursolic acid is mostly extracted from loquat leaves, but the plant extraction method has low yield and high cost, and chemical synthesis is not readily available, so the biosynthesis method provides a new source for ursolic acid. α-amyrin acts as the main precursor for the synthesis of ursolic acid, and its yield is positively correlated with ursolic acid yield. Oxidosqualene cyclase(OSC) belongs to a multigene family which can catalyze the common precursor 2,3-oxidosqualene to generate different types of triterpene backbones, and plays a decisive role in the synthesis of triterpenoids. However, there are fewer reported key genes catalyzing the synthesis of α-amyrin in medicinal plants, and the yield and proportion of α-amyrin in the catalyzed products have always been a focus of research. In this study, ItOSC2, MdOSC1, AaOSC2 and CrAS, four enzymes capable of catalyzing the production of α-amyrin from 2,3-oxidosqualene, were cloned from Iris tectorum, Malus domestica, Artemisia annua and Catharanthus roseus, subject to sequence alignment and phylogenetic tree analyses, and transformed into Saccharomyces cerevisiae as plasmids. After 7 days of fermentation, the yield and proportions of α-amyrin, ß-amyrin and ergosterol were measured. Finally, AaOSC2 with the best ability to catalyze the generation of α-amyrin was filtered out, providing a key gene element for the later construction of engineered yeast strains with high production of α-amyrin and ursolic acid.

The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi.

Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, ß-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3ß-ol, and dammara-20,24-dien-3ß-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3ß-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and ß-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi.

Biocatalytic stereocontrolled head-to-tail cyclizations of unbiased terpenes as a tool in chemoenzymatic synthesis.

Terpene synthesis stands at the forefront of modern synthetic chemistry and represents the state-of-the-art in the chemist's toolbox. Notwithstanding, these endeavors are inherently tied to the current availability of natural cyclic building blocks. Addressing this limitation, the stereocontrolled cyclization of abundant unbiased linear terpenes emerges as a valuable tool, which is still difficult to achieve with chemical catalysts. In this study, we showcase the remarkable capabilities of squalene-hopene cyclases (SHCs) in the chemoenzymatic synthesis of head-to-tail-fused terpenes. By combining engineered SHCs and a practical reaction setup, we generate ten chiral scaffolds with >99% ee and de, at up to decagram scale. Our mechanistic insights suggest how cyclodextrin encapsulation of terpenes may influence the performance of the membrane-bound enzyme. Moreover, we transform the chiral templates to valuable (mero)-terpenes using interdisciplinary synthetic methods, including a catalytic ring-contraction of enol-ethers facilitated by cooperative iodine/lipase catalysis.

Structural basis of pri-let-7 recognition by human pseudouridine synthase TruB1.

Let-7 was one of the first microRNAs (miRNAs) to be discovered and its expression promotes differentiation during development and function as tumor suppressors in various cancers. The maturation process of let-7 miRNA is tightly regulated by multiple RNA-binding proteins. For example, LIN28 binds to the terminal loops of the precursors of let-7 family and block their processing into mature miRNAs. Trim25 promotes the uridylation-mediated degradation of pre-let-7 modified by LIN28/TUT4. Recently, human pseudouridine synthase TruB1 has been reported to facilitate let-7 maturation by directly binding to pri-let-7 and recruiting Drosha-DGCR8 microprocessor. Through biochemical assay and structural investigation, we show that human TruB1 binds specifically the terminal loop of pri-let-7a1 at nucleotides 31-41, which folds as a small stem-loop architecture. Although TruB1 recognizes the terminal loop of pri-let-7a1 in a way similar to how E. coli TruB interacts with tRNA, a conserved KRKK motif in human and other higher eukaryotes adds an extra binding interface and strengthens the recognition of TruB1 for pri-let-7a1 through electrostatic interactions. These findings reveal the structural basis of TruB1-pri-let-7 interaction which may assists the elucidation of precise role of TruB1 in biogenesis of let-7.

Structural basis of chorismate isomerization by Arabidopsis ISOCHORISMATE SYNTHASE1.

Salicylic acid (SA) plays a crucial role in plant defense against biotrophic and semibiotrophic pathogens. In Arabidopsis (Arabidopsis thaliana), isochorismate synthase 1 (AtICS1) is a key enzyme for the pathogen-induced biosynthesis of SA via catalytic conversion of chorismate into isochorismate, an essential precursor for SA synthesis. Despite the extensive knowledge of ICS1-related menaquinone, siderophore, and tryptophan (MST) enzymes in bacteria, the structural mechanisms for substrate binding and catalysis in plant isochorismate synthase (ICS) enzymes are unknown. This study reveals that plant ICS enzymes catalyze the isomerization of chorismate through a magnesium-dependent mechanism, with AtICS1 exhibiting the most substantial catalytic activity. Additionally, we present high-resolution crystal structures of apo AtICS1 and its complex with chorismate, offering detailed insights into the mechanisms of substrate recognition and catalysis. Importantly, our investigation indicates the existence of a potential substrate entrance channel and a gating mechanism regulating substrate into the catalytic site. Structural comparisons of AtICS1 with MST enzymes suggest a shared structural framework with conserved gating and catalytic mechanisms. This work provides valuable insights into the structural and regulatory mechanisms governing substrate delivery and catalysis in AtICS1, as well as other plant ICS enzymes.

Comprehensive analysis to identify PUS7 as a prognostic biomarker from pan-cancer analysis to osteosarcoma validation.

AIM: Pseudouridylation has demonstrated the potential to control the development of numerous malignancies. PUS7(Pseudouridine Synthase 7) is one of the pseudouridine synthases, but the literature on this enzyme is limited to several cancer types. Currently, no investigation has been performed on the systematic pan-cancer analysis concerning PUS7 role in cancer diagnosis and prognosis. METHODS: Employing public databases, including The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression Project (GTEx), Human Protein Atlas (HPA), UALCAN and Tumor Immune Single-cell Hub (TISCH), this work investigated the PUS7 carcinogenesis in pan-cancer. Differential expression analysis, prognostic survival analysis and biological function were systematically performed. Furthermore, PUS7 potential as an osteosarcoma biomarker for diagnosis and prognosis was assessed in this study. RESULTS: The findings indicated that PUS7 was overexpressed in the majority of malignancies. High PUS7 expression contributed to the poor prognosis among 11 cancer types, including Adrenocortical Cancer (ACC), Bladder Cancer (BLCA), Liver Cancer (LIHC), Kidney Papillary Cell Carcinoma (KIRP), Mesothelioma (MESO), Lower Grade Glioma (LGG), Kidney Chromophobe (KICH), Sarcoma (SARC), osteosarcoma (OS), Pancreatic Cancer (PAAD), and Thyroid Cancer (THCA). In addition, elevated PUS7 expression was linked to advanced TNM across multiple malignancies, including ACC, BLCA, KIRP, LIHC and PAAD. The function enrichment analysis revealed that PUS7 participates in E2F targets, G2M checkpoint, ribosome biogenesis, and rRNA metabolic process. Moreover, PUS7 is also a reliable biomarker and a potential therapeutic target for osteosarcoma. CONCLUSIONS: In summary, PUS7 is a putative pan-cancer biomarker that reliably forecasts cancer patients' prognosis. In addition, this enzyme regulates the cell cycle, ribosome biogenesis, and rRNA metabolism. Most importantly, PUS7 possibly regulates osteosarcoma initiation and progression.

Genome-wide identification of the ICS family genes and its role in resistance to Plasmodiophora brassicae in Brassica napus L.

The isochorismate synthase (ICS) proteins are essential regulators of salicylic acid (SA) synthesis, which has been reported to regulate resistance to biotic and abiotic stresses in plants. Clubroot caused by Plasmodiophora brassicae is a common disease that threatens the yield and quality of Oilseed rape (Brassica napus L.). Exogenous application of salicylic acid reduced the incidence of clubroot in oilseed rape. However, the potential importance of the ICS genes family in B. napus and its diploid progenitors has been unclear. Here, we identified 16, 9, and 10 ICS genes in the allotetraploid B. napus, diploid ancestor Brassica rapa and Brassica oleracea, respectively. These ICS genes were classified into three subfamilies (I-III), and member of the same subfamilies showed relatively conserved gene structures, motifs, and protein domains. Furthermore, many hormone-response and stress-related promoter cis-acting elements were observed in the BnaICS genes. Exogenous application of SA delayed the growth of clubroot galls, and the expression of BnaICS genes was significantly different compared to the control groups. Protein-protein interaction analysis identified 58 proteins involved in the regulation of ICS in response to P. brassicae in B. napus. These results provide new clues for understanding the resistance mechanism to P. brassicae.

An omics-based characterization of Wolfiporia cocos reveals three CYP450 members involved in the biosynthetic pathway of pachymic acid.

Wolfiporia cocos is a medicinal mushroom used in China. It biosynthesizes pachymic acid (PA), a main therapeutic triterpene associated with therapies. Nowadays, the unknown PA biosynthesis leads to difficulties in increasing its content in W. cocos. Herein, we report sequencing, assembling, and characterization of the genome and several transcriptomes of W. cocos. Sequence mining determined candidate genes that encode lanosterol synthase, sterol O-acyltransferase, and sterol C-24 methyltransferase likely involved in the steps from lanosterol to PA. Gene cluster analysis identified four CYP450 cDNAs likely involved in the biosynthesis of PA, namely WcCYP64-1, WcCYP64-2, WcCYP52, and WcCYP_FUM15, which were subjected to both overexpression and silencing in mycelia. The overexpression of each of WcCYP64-1, WcCYP52 and WcCYP_FUM15 increased the content of PA, 16α-hydroxytrametenolic acid, eburicoic acid, and tumulosic acid, while the silencing of each gene either significantly or slightly decreased the contents of these four compounds, indicating their involvement in the PA biosynthesis. In addition, different temperatures affected the expression of these genes and the formation of PA. By contrast, the overexpression and silencing of WcCYP64-2 did not alter the formation of these compounds. Taken together, these findings determine more potential steps in the biosynthetic pathway of PA for metabolic engineering.

SA and NHP glucosyltransferase UGT76B1 affects plant defense in both SID2- and NPR1-dependent and independent manner.

KEY MESSAGE: The small-molecule glucosyltransferase loss-of-function mutant ugt76b1 exhibits both SID2- or NPR1-dependent and independent facets of enhanced plant immunity, whereupon FMO1 is required for the SID2 and NPR1 independence. The small-molecule glucosyltransferase UGT76B1 inactivates salicylic acid (SA), isoleucic acid (ILA), and N-hydroxypipecolic acid (NHP). ugt76b1 loss-of-function plants manifest an enhanced defense status. Thus, we were interested how UGT76B1 genetically integrates in defense pathways and whether all impacts depend on SA and NHP. We study the integration of UGT76B1 by transcriptome analyses of ugt76b1. The comparison of transcripts altered by the loss of UGT76B1 with public transcriptome data reveals both SA-responsive, ISOCHORISMATE SYNTHASE 1/SALICYLIC ACID INDUCTION DEFICIENT 2 (ICS1/SID2)- and NON EXPRESSOR OF PR GENES 1 (NPR1)-dependent, consistent with the role of UGT76B1 in glucosylating SA, and SA-non-responsive, SID2/NPR1-independent genes. We also discovered that UGT76B1 impacts on a group of genes showing non-SA-responsiveness and regulation by infections independent from SID2/NPR1. Enhanced resistance of ugt76b1 against Pseudomonas syringae is partially independent from SID2 and NPR1. In contrast, the ugt76b1-activated resistance is completely dependent on FMO1 encoding the NHP-synthesizing FLAVIN-DEPENDENT MONOOXYGENASE 1). Moreover, FMO1 ranks top among the ugt76b1-induced SID2- and NPR1-independent pathogen responsive genes, suggesting that FMO1 determines the SID2- and NPR1-independent effect of ugt76b1. Furthermore, the genetic study revealed that FMO1, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), SID2, and NPR1 are required for the SA-JA crosstalk and senescence development of ugt76b1, indicating that EDS1 and FMO1 have a similar effect like stress-induced SA biosynthesis (SID2) or the key SA signaling regulator NPR1. Thus, UGT76B1 influences both SID2/NPR1-dependent and independent plant immunity, and the SID2/NPR1 independence is relying on FMO1 and its product NHP, another substrate of UGT76B1.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a ß-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

Minor Hydroxylated Triterpenoids Produced in Engineered Yeast by the Enzymes OSC and CYP716s from the Plant Enkianthus chinensis and Their Anti-Inflammatory and Hepatoprotective Activities.

Triterpenoids are a type of specialized metabolites that exhibit a wide range of biological activities. However, the availability of some minor triterpenoids in nature is limited, which has hindered our understanding of their pharmacological potential. To overcome this limitation, heterologous biosynthesis of triterpenoids in yeast has emerged as a promising and time-efficient production platform for obtaining these minor compounds. In this study, we analyzed the transcriptomic data of Enkianthus chinensis to identify one oxidosqualene cyclase (EcOSC) gene and four CYP716s. Through heterologous expression of these genes in yeast, nine natural pentacyclic triterpenoids, including three skeleton products (1-3) produced by one multifunctional OSC and six minor oxidation products (4-9) catalyzed by CYP716s, were obtained. Of note, we discovered that CYP716E60 could oxidize ursane-type and oleanane-type triterpenoids to produce 6ß-OH derivatives, marking the first confirmed C-6ß hydroxylation in an ursuane-type triterpenoid. Compound 9 showed moderate inhibitory activity against NO production and dose-dependently reduced IL-1ß and IL-6 production at the transcriptional and protein levels. Compounds 1, 2, 8, and 9 exhibited moderate hepatoprotective activity with the survival rates of HepG2 cells from 61% to 68% at 10 µM.