Browsing by Author "Cataldo, Vicente F."

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    Heterologous production of the epoxycarotenoid violaxanthin in Saccharomyces cerevisiae
    (2020) Cataldo, Vicente F.; Arenas, Natalia; Salgado, Valeria; Camilo, Conrado; Ibanez, Francisco; Agosin, Eduardo
    Microbial production of carotenoids has mainly focused towards a few products, such as beta-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and beta-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a beta-carotenogenic yeast strain, we first evaluated the performance of several beta-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/g(DCW) of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/g(DCW)). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/g(DCW) in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date.
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    p53 Related Protein Kinase is Required for Arp2/3-Dependent Actin Dynamics of Hemocytes in Drosophila melanogaster
    (2022) Molina, Emiliano; Cataldo, Vicente F.; Eggers, Cristian; Munoz-Madrid, Valentina; Glavic, Alvaro
    Cells extend membrane protrusions like lamellipodia and filopodia from the leading edge to sense, to move and to form new contacts. The Arp2/3 complex sustains lamellipodia formation, and in conjunction with the actomyosin contractile system, provides mechanical strength to the cell. Drosophila p53-related protein kinase (Prpk), a Tsc5p ortholog, has been described as essential for cell growth and proliferation. In addition, Prpk interacts with proteins associated to actin filament dynamics such as alpha-spectrin and the Arp2/3 complex subunit Arpc4. Here, we investigated the role of Prpk in cell shape changes, specifically regarding actin filament dynamics and membrane protrusion formation. We found that reductions in Prpk alter cell shape and the structure of lamellipodia, mimicking the phenotypes evoked by Arp2/3 complex deficiencies. Prpk co-localize and co-immunoprecipitates with the Arp2/3 complex subunit Arpc1 and with the small GTPase Rab35. Importantly, expression of Rab35, known by its ability to recruit upstream regulators of the Arp2/3 complex, could rescue the Prpk knockdown phenotypes. Finally, we evaluated the requirement of Prpk in different developmental contexts, where it was shown to be essential for correct Arp2/3 complex distribution and actin dynamics required for hemocytes migration, recruitment, and phagocytosis during immune response.