Biology subjects

Fabris, M.

Publications and source records attributed to Fabris, M..

8 recordsLinked to original sources

bioRxiv2026

AUREOCHROME 1b is a transcriptional regulator of pigment biosynthesis in Phaeodactylum tricornutum

Diatoms have attracted attention for their potential to produce high-value metabolites, such as terpenoids. However, the regulatory mechanisms governing their isoprenoid metabolism remain poorly understood, which poses a major bottleneck in engineering them for greater production efficiency. By mining the Phaeodactylum tricornutum co-regulation network (PhaeoNet), we identified two candidate transcription factors (TFs) co-regulated with the methylerythritol 4-phosphate (MEP) pathway, suggesting a potential regulatory role in isoprenoid biosynthesis: AUREOCHROME 1b (AUREO1b) and sigma factor 70.4 ({sigma}70.4). To elucidate their mechanistic roles in isoprenoid metabolism, we generated episomal overexpression lines for both candidate TFs. These were characterized alongside lines overexpressing HSF1 and HSF3, previously identified regulators of carotenoid biosynthesis, to compare growth, pigment content, and global transcriptomic profiles. Phenotypically, overexpression of AUREO1b, PtHSF1, and PtHSF3 led to increased pigment accumulation, whereas {sigma}70.4 overexpression did not alter pigment levels. Transcriptomic analyses revealed that each TF upregulates distinct sets of genes involved in pigment biosynthesis, pigment binding, and photosynthesis. These results expand our functional understanding of the transcriptional regulation of isoprenoid and pigment pathways in diatoms and broaden the possibilities of engineering diatom microalgae for improved production of high-value compounds.

Molecular Biology↗

bioRxiv2025

Repurposing the diatom periplastidial compartment for heterologous terpenoid production

Diatoms are promising microorganisms to provide sustainable routes for photosynthetic terpenoid production from CO2, yet their potential for compartmentalized engineering remains largely unexplored. Here, we systematically profiled the biosynthetic capacity of Phaeodactylum tricornutum by targeting representative synthases for hemi, mono, sesqui, and tetraterpenoids to the cytosol, chloroplast, and periplastidial compartment (PPC). This comprehensive analysis revealed that all major prenyl phosphate precursors, DMAPP, GPP, FPP, and GGPP, are accessible in all compartments, including in the PPC and can sustain heterologous flux without major physiological penalties, although production efficiency varies across compartments and product classes. By determining precursor availability, we propose the diatom PPC as a minimal engineerable organelle directly interfaced with a eukaryotic chloroplast. Moreover, we highlight its utility as a unique interface to investigate metabolic exchange between the MVA and MEP pathways. These findings provide a systematic framework for compartment-specific terpenoid engineering in diatoms and open new opportunities for modular pathway assembly and synthetic biology in photosynthetic eukaryotes.

synthetic biology↗

bioRxiv2025

Iterative and modular expression of Botryococcus braunii genes enhances isoprenoid production in the diatom Phaeodactylum tricornutum

Isoprenoids are essential natural compounds with high structural and functional diversity. Among them, carotenoids and triterpenoids such as squalene have high biotechnological value but remain challenging to produce sustainably. The green microalga Botryococcus braunii synthesizes large amounts of triterpenoids through specialized methylerythritol phosphate (MEP) pathway configurations involving distinct 1-deoxy-D-xylulose-5-phosphate synthase (DXS) isoforms, a bifunctional squalene synthase (SQS) and squalene synthase-like (SSL) enzymes working together for the synthesis of the compound. However, its slow growth limits industrial application. In this work, we established the fast-growing diatom Phaeodactylum tricornutum as a heterologous chassis for terpenoid production using a synthetic biology Design-Build-Test-Learn (DBTL)-based approach. Episomal uLoop assembly enabled rapid expression and evaluation of B. braunii DXS and SQS variants, enhancing precursor flux and resulting in the co-accumulation of squalene and carotenoids. This work demonstrates the functional transfer of specialized algal terpenoid enzymes into a tractable diatom host and highlight P. tricornutums potential as a versatile platform for sustainable, high-value isoprenoid biosynthesis.

synthetic biology↗

bioRxiv2025

Real-time tracking of intracellular prenyl phosphate pools in the marine diatom Phaeodactylum tricornutum with a metabolite protein-based biosensor

Metabolite-responsive, protein-based biosensors are a powerful tool for monitoring cellular metabolite dynamics in vivo and accelerating strain engineering workflows in microorganisms. In this study, we introduced a previously developed protein-based biosensor, computationally designed to detect farnesyl diphosphate (FPP), in the marine diatom Phaeodactylum tricornutum. We expressed two versions of the biosensor constitutively, under a strong promoter-terminator pair using extrachromosomal episomes, and we parameterized the capacity of both designs in detecting intracellular metabolite levels. Initial assays revealed that the two versions of the biosensor we investigated, S3-2D and S3-3A, had specificity not only for FPP but also for other exogenously supplied prenyl phosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) in a dose-dependent manner. We further demonstrated the capacity of S3-3A to track perturbations in the endogenous prenyl phosphate pools by testing it in the presence of pharmacological inhibition of the mevalonate pathway. Moreover, S3-3A generated signal "hot-spots" around the peroxisomes, suggesting their involvement in isoprenoid biosynthesis, which led us to characterize the subcellular localization of the key enzyme mevalonate kinase. These findings lay the groundwork for developing metabolite-responsive biosensors as robust tools for monitoring and investigating prenyl phosphate dynamics, providing a foundation for advanced metabolic engineering of microalgae.

synthetic biology↗

bioRxiv2024

DiatOmicBase, a gene-centered platform to mine functional omics data across diatom genomes

Diatoms are prominent microalgae found in all aquatic environments. Over the last 20 years, thanks to the availability of genomic and genetic resources, diatom species such as Phaeodactylum tricornutum have emerged as valuable experimental model systems for exploring topics ranging from evolution to cell biology, (eco)physiology and biotechnology. Since the first genome sequencing in 2008, numerous genome-enabled datasets have been generated, based on RNA-Seq and proteomics, epigenomes, and ecotype variant analysis. Unfortunately, these resources, generated by various laboratories, are often in disparate formats and challenging to access and analyze. Here we present DiatOmicBase, a genome portal gathering comprehensive omics resources from P. tricornutum and two other diatoms to facilitate the exploration of dispersed public datasets and the design of new experiments based on the prior-art. DiatOmicBase provides gene annotations, transcriptomic profiles and a genome browser with ecotype variants, histone and methylation marks, transposable elements, non-coding RNAs, and read densities from RNA-Seq experiments. We developed a semi-automatically updated transcriptomic module to explore both publicly available RNA-Seq experiments and users private datasets. Using gene-level expression data, users can perform exploratory data analysis, differential expression, pathway analysis, biclustering, and co-expression network analysis. Users can create heatmaps to visualize precomputed comparisons for selected gene subsets. Automatic access to other bioinformatic resources and tools for diatom comparative and functional genomics is also provided. Focusing on the resources currently centralized for P. tricornutum, we showcase several examples of how DiatOmicBase strengthens molecular research on diatoms, making these organisms accessible to a broad research community. Significance statementIn recent years, diatoms have become the subject of increasing interest because of their ecological importance and their biotechnological potential for natural products such as pigments and polyunsaturated fatty acids. Here, we present an interactive web-based server that integrates public diatom omics data (genomics, transcriptomics, epigenomics, proteomics, sequence variants) to connect individual diatom genes to broader-scale functional processes.

genomics↗

bioRxiv2024

Specific light-regime adaptations, terpenoid profiles and engineering potential in ecologically diverse Phaeodactylum tricornutum strains

Microalgae, and among them, the diatom Phaeodactylum tricornutum stand out with their remarkable versatility and metabolic engineering potential. Diatoms exhibit substantial variability in metabolism, photosynthetic physiology and environmental adaptation, even across the same species. These factors can affect the design and outcome of metabolic engineering strategies. In this study, we profiled the diversity of biotechnologically relevant traits of three P. tricornutum strains (Pt1, Pt6, and Pt9) under different light regimes to identify the most suitable chassis to be employed as bio-factory to produce high-value terpenoids. We conducted detailed assessments of these strains, using pulse amplitude modulated (PAM) fluorometry to measure photosynthetic efficiency and we analyzed the composition of pigments and triterpenoids, as main terpenoid metabolic sinks. Parameters such as the maximum quantum yield of PSII (Fv/Fm), the efficiency of excitation energy capture (Fv/Fm), and OJIP kinetics were used to estimate photosynthetic performance in different light regimes. Additionally, we evaluated their transformation efficiency and their capacity to produce heterologous monoterpenoids, using geraniol as a model product. Our findings revealed that Pt1, widely used in laboratories, exhibits robust growth and photosynthetic performance under standard laboratory conditions. Pt6, adapted to intertidal environments, shows unique resilience in fluctuating conditions, while Pt9, with its high-temperature tolerance, excels under continuous high irradiance. Additionally, this variability across strains and light conditions influenced the metabolic output of each strain. We concluded that understanding the physiological responses of different P. tricornutum strains to light is crucial for optimizing their use in metabolic engineering. The insights gained from this research will facilitate the strategic selection and exploitation of these strains in algae biotechnology, enhancing the production of commercially valuable compounds such as high-value terpenoids and derivatives. This comprehensive characterization of strains under varying light conditions offers a pathway to more efficient and targeted metabolic engineering applications. HighlightsO_LIPt1, Pt6, and Pt9 exhibit distinct physiology under different light regimes. C_LIO_LIPt9 is photosynthetically more performant in continuous light, Pt6 in photoperiod. C_LIO_LILight regimes affect pigments and triterpenoid content in all three strains. C_LIO_LIEach strain exhibits a specific carotenoid and triterpenoid composition. C_LIO_LIBacterial conjugation of episomes varies across strains, it is more efficient in Pt1. C_LIO_LIPt1 is suited for the heterologous synthesis of monoterpenes (geraniol). C_LI

plant biology↗

bioRxiv2023

Real time mobilization of a novel diatom Mutator-Like Element (MULE) transposon to inactivate the uridine monophosphate synthase (UMPS) locus in Phaeodactylum tricornutum

Diatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic diversity, which often result in peculiar biochemical and biological traits. Transposable elements (TE) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic diversity and centrally contribute to genome evolution. Here, through long-read whole genome sequencing, we identified a novel Mutator-Like Element (MULE) in the model diatom Phaeodactylum tricornutum, and we report the direct observation of its mobilization within the course of one single laboratory experiment. Under selective conditions, this novel TE inactivated the uridine monophosphate synthase (UMPS) gene of P. tricornutum, one of the two only endogenous genetic loci currently targeted for selectable auxotrophy in functional genetics and genome editing applications. We report the first, real-time observation of the mobilization of a transposon in diatoms that possesses novel peculiar features. These include the combined presence of a MULE transposase domain with Zinc finger, SWIM-type domains, and of a diatom-specific E3 ubiquitin ligase of the zinc finger UBR type, which indicate a novel mobilization mechanism. Our findings provide new elements for the understanding of the role of TEs in diatom genome evolution and in the enrichment of intraspecific genetic variability. Ultimately, this raises relevant concerns on the targeting of loci such as UMPS as selectable markers for functional genetics and biotechnological applications in diatoms. Significance StatementWe identified a novel DNA transposon in the diatom Phaeodactylum tricornutum. This new Mutator-Like Element encodes a transposase and a diatom-specific E3 ubiquitin ligase, which suggest a novel mobilization mechanism. We documented independent insertions in real-time, which spontaneously inactivated the uridine monophosphate synthase (UMPS) locus, a common selectable marker. We provide new insights on the role of transposons in diatom genome dynamics and evolution and on the unsuitability of UMPS as selection locus in diatoms.

molecular biology↗

bioRxiv2020

Overexpression of key sterol pathway enzymes in two model marine diatoms alters sterol profiles in Phaeodactylum tricornutum

Sterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes; however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the native HMGR and a conventional, heterologous SQE gene in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates in P. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24)-dien-3{beta}-ol was observed in all P. tricornutum overexpression lines, and campesterol increased 3-fold in P. tricornutum lines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found in T. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols in P. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.

molecular biology↗