Biology subjects

Merindol, N.

Publications and source records attributed to Merindol, N..

11 recordsLinked to original sources

bioRxiv2025

Branch-Enabling N-Methyltransferase in Golgi Reconciles Divergent Models of Galanthamine Biosynthesis

Galanthamine, a therapeutic Amaryllidaceae alkaloid produced exclusively by species within the Amaryllidoideae subfamily, is a key treatment for early-stage symptoms of Alzheimers disease. Elucidating its biosynthetic pathway is essential for strategies aimed at enhancing production through metabolic engineering. Galantamine derives from the metabolic precursor 4'-O-methylnorbelladine, which undergoes cytochrome P450-mediated para-ortho C-C phenol coupling to yield nornarwedine. Two competing terminal routes have been proposed: (i) reduction of nornarwedine to norgalanthamine, followed by N-methylation, or (ii) N-methylation of nornarwedine to narwedine prior to reduction. Here, we identify three N-methyltransferase (NMT) candidates from Leucojum aestivum: LaNMT, related to coclaurine NMTs, and two {gamma}-tocopherol methyltransferases (TMT) homologs, LaTMT1 and LaTMT2. Subcellular localization studies revealed distinct compartmentalization, with LaNMT targeted to the ER-cytosol, LaTMT1 to plastids, and LaTMT2 to the Golgi apparatus. In vitro enzyme assays demonstrated that LaTMT2 methylates both nornarwedine and norgalanthamine, with a kinetic preference for nornarwedine. Agroinfiltration for transient expression in Nicotiana benthamiana further confirmed LaTMT2 as a catalytically efficient and substrate-promiscuous enzyme that supports both terminal routes. These findings identify LaTMT2 as a key branch-enabling N-methyltransferase that reconcile long-standing models of galanthamine biosynthesis and provides a strategic target for metabolic engineering strategies to enhance galanthamine production. Significant statementThis study identifies LaTMT2, a Golgi-localized {gamma}-tocopherol methyltransferase homolog, as a branch-enabling N-methyltransferase that resolves competing models of galanthamine biosynthesis. By revealing an unanticipated Golgi-associated step in Amaryllidaceae alkaloid metabolism, it redefines the subcellular organization of specialized metabolic pathways and provides a strategic enzymatic target for metabolic engineering of high-value therapeutic alkaloids.

plant biology↗

bioRxiv2025

Insights into cannabinoid biosynthesis in Chlamydomonas reinhardtii : successes with NphB and limitations of CBDAS expression

The growing legalization of Cannabis has increased demand for cannabinoids (CBs). Currently, pharmaceutically relevant CBs are primarily extracted from Cannabis, a process that presents challenges related to yield variability and purity. To overcome these limitations, microbial platforms are being explored for the sustainable production of specific CBs. Compared to conventional microbial hosts, the photosynthetic microalga Chlamydomonas reinhardtii offers plant-like post-translational modifications, low-cost cultivation, CO2 fixation, and low endotoxin contamination, making it a potential chassis for CB biosynthesis. Here, we expressed a codon-optimized, soluble aromatic prenyltransferase (NphBG286S/Y288A) from Streptomyces and Cannabis sativa cannabidiolic acid synthase (CBDAS) in the nuclear genome of C. reinhardtii; transformants were screened for integration, gene expression, protein accumulation, enzymatic activity, and CB production. Our results provide evidence of functional NphB expression in C. reinhardtii, with in vitro CBGA production reaching up to 633 {+/-} 58 {micro}g/L. Although CBDAS transcripts were detected under multiple construct designs, neither protein nor CBDA was accumulated, suggesting limitations in expression, localization, or post-translational processing in C. reinhardtii. Our study provides the first demonstration of in vitro CBGA biosynthesis in the photosynthetic model microalga C. reinhardtii, highlighting its potential as a future platform for CB production. It also highlights key challenges in nuclear expression of plant-derived enzymes, such as CBDAS, emphasizing that improved regulatory control, subcellular targeting, and mRNA processing will be required to achieve full pathway reconstruction in C. reinhardtii. HighlightsO_LIFirst report of active NphB expression in Chlamydomonas reinhardtii C_LIO_LINphB exhibits measurable in vitro activity, producing cannabigerolic acid in algal extracts C_LIO_LIEnzyme localization, construct design, and substrate availability critically influence functionality. C_LI

bioengineering↗

bioRxiv2025

AmarylOmicBase: A unified transcriptome database to accelerate gene discovery in Amaryllidoideae species

Amaryllidoideae plants produce structurally diverse and unique alkaloids with potent anti-cholinesterase, antiviral, and antitumor activities, making this subfamily a rich source of pharmaceutical leads. Despite the absence of reference genomes for any Amaryllidoideae species, many enzyme characterization and pathway reconstruction efforts to date have been made possible through transcriptome mining, often requiring bioinformatic expertise and data preprocessing. To facilitate new studies in this subfamily, here we present AmarylOmicBase, a unified transcriptomic dataset that integrates assemblies, annotations, and expression profiles from 39 studies, covering 27 species and four hybrid cultivars across 13 genera of Amaryllidoideae. The AmarylOmicBase includes both published and de novo assemblies generated from published raw data using Trinity or IsoSeq workflows and provides standardized functional annotation and quantitative expression datasets. AmarylOmicBase provides ready-to-use datasets that support gene discovery, comparative transcriptomics, and pathway-level investigations for specialized metabolism, including Amaryllidaceae alkaloid biosynthesis. By providing ready-to-use datasets and fully reproducible analysis scripts, this resource reduces computational barriers and expands access to transcriptomic information for researchers working on non-model plant species. AmarylOmicBase provides a centralized resource for transcriptomic data that can be reused in studies of enzyme function, pathway evolution, and regulatory processes in Amaryllidoideae.

bioinformatics↗

bioRxiv2025

Dissecting specialized metabolism in space: A MALDI-MSI atlas of Amaryllidaceae alkaloids in Hippeastrum papilio (Ravenna) Van Scheepen

Amaryllidoideae produce specific specialized metabolites known as Amaryllidaceae alkaloids (AAs), extensively studied for their significant pharmacological potential. AAs spatial distribution and biosynthesis within plant tissues remain poorly understood. This study investigates organ- and tissue-specific localization in Hippeastrum papilio, from precursors to galanthamine and haemanthamine, using matrix-assisted laser desorption/ionization mass spectrometry imaging. Consistent accumulation of AAs was observed in epidermal and vascular tissues, with leaves exhibiting a uniform distribution across all ages and positions. Bulbs exhibited higher concentrations in the outer-scales and basal-plates, while roots displayed compartmentalized patterns, with galanthamine being uniquely abundant in the vascular bundles. Haemanthamine and galanthamine were detected in high quantities in the leaves and bulbs mucilage, while precursors were scarce. Multivariate analyses revealed that precursors clustered separately from end-products and were specifically enriched in the middle-scales and apical-leaves of the bulbs. Nonetheless, biosynthetic intermediates were observed in all tissues, indicating widespread AA biosynthesis across all organs. These findings suggest a coordinated metabolic network in H. papilio, which challenges existing hypotheses on organ-specific AA biosynthesis and hints at the transport of end-products. This study refines current models of alkaloid biosynthesis and underscore the value of H. papilio as a promising resource for sustainable production of therapeutic AAs. HighlightsMALDI-MSI reveals widespread, tissue-specific alkaloid distribution in Hippeastrum papilio, challenging organ-specific biosynthesis and implicating epidermal and vascular tissues in Amaryllidaceae alkaloid production.

plant biology↗

bioRxiv2024

What is true for plants may not be true for Phaeodactylum tricornutum: The case of Vanilla planifolia vanillin synthase (VpVAN) targeted to four subcellular compartments of the diatom.

Vanillin is the major organoleptic compound of the vanilla flavor. Since the extraction process from its natural source (Vanilla planifolia) is limited by a low yield, the market demand for vanillin is met by producing it through chemical synthesis or biotechnologically using microorganisms to convert ferulic acid, lignin, and guaiacol into vanillin. Our aim was to use the diatom Phaeodactylum tricornutum, a photosynthetic model organism already used to produce eukaryotic proteins and high-value compounds, to characterize V. planifolia vanillin synthase (VpVAN). This enzyme is the subject of conflicting results and was reported to be localized in the plastid of V. planifolia cells, which may play a role in its activity. For this purpose, we targeted VpVAN linked by an in vitro cleavable sequence (Xa) to the enhanced green fluorescent protein (eGFP) to the cytoplasm, peroxisomes, plastid, and Golgi apparatus. We used an episomal expression system transforming P. tricornutum with VpVAN:Xa:eGFP by bacterial conjugation. Positive cell lines were selected by fluorescence and characterized by sequencing the episomes, confirming stable replication in the diatom, as well as assessing the subcellular localization in the targeted cell compartments. Strategies such as enriching GFP-positive cells by cell sorting, protein purification, and organelle isolation did not yield detectable levels of the fusion protein. A better understanding of the diatom expression system is needed to complete the characterization of this enzyme and reveal whether the subcellular localization could be a missing aspect of VpVAN expression in microorganisms.

synthetic biology↗

bioRxiv2024

T7 RNA polymerase-independent expression of reporter genes from a T7 promoter-driven SARS-CoV-2 replicon-encoding DNA in human cells

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Surprisingly, however, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

microbiology↗

bioRxiv2022

Characterization of norbelladine synthase and noroxomaritidine/norcraugsodine reductase reveals a novel catalytic route for the biosynthesis of Amaryllidaceae alkaloids including the Alzheimer's drug galanthamine

Amaryllidaceae alkaloids (AAs) are a large group of pharmacological plant-specialized metabolites. Norbelladine is the entry compound in AAs biosynthesis. Two enzymes are capable of catalyzing this reaction in-vitro, both with low-yield; 1) norbelladine synthase (NBS) condenses tyramine and 3,4-dihydroxybenzaldehyde, while 2) noroxomaritidine/norcraugsodine reductase (NR), catalyzes a reduction reaction. To clarify the mechanisms involved in this controversial step, both NBS and NR were characterized from Narcissus papyraceus and Leucojum aestivum. Assays with each enzyme suggested that NBS and NR function together for norbelladine formation. Using molecular-homology modeling and docking studies, we predicted models for the binding of substrates to NBS and NR. Moreover, NBS and NR physically interact, localize to the cell cytoplasm and nucleus and are expressed at high levels in bulbs. Our study establishes that both NBS and NR participate in the biosynthesis of norbelladine, catalyzing the first key steps involved in the biosynthesis of the Alzheimers drug galanthamine.

biochemistry↗

bioRxiv2020

Editing of the TRIM5 gene decreases the permissiveness of human T lymphocytic cells to HIV-1

TRIM5 is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5 the capacity to target and strongly restrict HIV-1 upon over-expression of the mutated protein. Here, we used CRISPR-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (Clone 6) had both alleles containing R332G but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following IFN-{beta} treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5 knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5.

microbiology↗

bioRxiv2020

Optimization of SARS-CoV-2 detection by RT-QPCR without RNA extraction

Rapid and reliable screening of SARS-CoV-2 is fundamental to assess viral spread and limit the pandemic we are facing. In this study we evaluated the reliability and the efficiency of a direct RT-QPCR method (without RNA extraction) using SeeGene Allplex 2019-nCoV RT-QPCR and the influence of swab storage media composition on further viral detection. We show that SeeGenes assay provides similar efficiency as the RealStar(R) SARS-CoV-2 RT-PCR kit (Altona Diagnostics), and that RNA extraction is not necessary nor advantageous if samples are stored in UTM or molecular water but is recommended if samples are stored in saline solution and in Hanks medium.

molecular biology↗

bioRxiv2017

Editing of the Human TRIM5 Gene to Introduce HIV-1 Restrictive Mutations Using CRISPR-Cas9

The type I interferon (IFN-I)-inducible human restriction factor TRIM5 inhibits the infection of human cells by specific nonhuman retroviruses, such as N-MLV and EIAV, but does not generally target HIV-1. However, the introduction of two aminoacid substitutions, R332G and R355G, in the human TRIM5 (huTRIM5) domain responsible for retroviral capsid recognition leads to efficient HIV-1 restriction. Using a DNA transfection-based CRISPR-Cas9 genome editing protocol, we successfully mutated TRIM5 to its HIV-1-restrictive version by homology-directed repair (HDR) in HEK293T cells. Nine clones bearing at least one HDR-edited TRIM5 allele containing both mutations were isolated (5.6% overall efficiency), whereas another one contained only the R332G mutation. Of concern, several of these HDR-edited clones contained on-target undesired mutations, and none had all the alleles corrected. We observed a lack of HIV-1 restriction in the cell clones generated, even when cells were stimulated with IFN-I prior to infection. This, however, was partly explained by the unexpectedly low potential for TRIM5-mediated restriction activity in this cell line. Our study demonstrates the feasibility of editing the TRIM5 gene to in human cells and identifies the main challenges to be addressed in order to use this approach to confer protection from HIV-1.

microbiology↗

bioRxiv2017

Gene knockout shows that PML (TRIM19) does not restrict the early stages of HIV-1 infection in human cell lines

The PML (promyelocytic leukemia) protein is a member of the TRIM family, a large group of proteins that show high diversity in functions but possess a common tripartite motif giving the family its name. We and others recently reported that both murine PML (mPML) and human PML (hPML) strongly restrict the early stages of infection by HIV-1 and other lentiviruses when expressed in mouse embryonic fibroblasts (MEFs). This restriction activity was found to contribute to the type I interferon (IFN-I)-mediated inhibition of HIV-1 in MEFs. Additionally, PML caused transcriptional repression of the HIV-1 promoter in MEFs. By contrast, the modulation of the early stages of HIV-1 infection of human cells by PML has been investigated by RNAi with unclear results. In order to conclusively determine whether PML restricts HIV-1 or not in human cells, we used CRISPR-Cas9 to knock out its gene in epithelial, lymphoid and monocytic human cell lines. Infection challenges showed that PML knockout had no effect on the permissiveness of these cells to HIV-1 infection. IFN-I treatments inhibited HIV-1 equally whether PML was expressed or not. Over-expression of individual hPML isoforms, or of mPML, in a human T cell line did not restrict HIV-1. The presence of PML was not required for the restriction of nonhuman retroviruses by TRIM5 was inhibited by arsenic trioxide through a PML-independent mechanism. We conclude that PML is not a restriction factor for HIV-1 in human cell lines representing diverse lineages.\n\nImportancePML is involved in innate immune mechanisms against both DNA and RNA viruses. Although the mechanism by which PML inhibits highly divergent viruses is unclear, it was recently found that it can increase the transcription of interferon-stimulated genes (ISGs). However, whether human PML inhibits HIV-1 has been debated. Here we provide unambiguous, knockout-based evidence that PML does not restrict the early post-entry stages of HIV-1 infection in a variety of human cell types and does not participate in the inhibition of HIV-1 by IFN-I. Although this study does not exclude the possibility of other mechanisms by which PML may interfere with HIV-1, we nonetheless demonstrate that PML does not generally act as an HIV-1 restriction factor in human cells and that its presence is not required for IFN-I to stimulate the expression of anti-HIV-1 genes. These results contribute to uncovering the landscape of HIV-1 inhibition by ISGs in human cells.

immunology↗