Browsing by Author "Salgado, Valeria"

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    Genomic integration of unclonable gene expression cassettes in Saccharomyces cerevisiae using rapid cloning-free workflows
    (2020) Cataldo von Bohlen, Vicente Francisco; Salgado, Valeria; Saa Higuera, Pedro Andrés E.; Agosin T., Eduardo
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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.