Biology subjects

Patwari, P.

Publications and source records attributed to Patwari, P..

3 recordsLinked to original sources

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

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

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↗