Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.6/3211
Título: Resveratrol production strategies: their influence on cell physiology and plasmid stability
Autor: Afonso, Ana Margarida da Silva
Orientador: Silva, Filomena Augusta Almeida e
Domingues, Fernanda da Conceição
Palavras-chave: HPLC.DAD (High Performance Liquid Chromatograph-Diode Array Detector)
DOE (Design of Experiments)
Real-time qPCR
qPCR (Quantitative polymerase chair reaction)
Data de Defesa: 2013
Resumo: Resveratrol (3, 5, 4’–trans-hydroxystilbene) has been used since immemorial times in traditional medicine as an antimicrobial and antioxidant compound. Recent discoveries demonstrated other health benefits for this phytoalexin, such as anticancer and anti-ageing activities, making it desirable for the pharmaceutical, nutraceutical and cosmetic industries. This polyphenolic is a plant secondary metabolite mainly produced by peanuts and grapevines. Although plant cells were traditionally used as a biological alternative for resveratrol production, in recent years, recombinant microorganisms, such as yeast and bacteria, were proposed to improve resveratrol production. The present work describes resveratrol production in two recombinant microorganisms – Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) – its optimization of production in bioreactor using Design of Experiments (DoE) and the impact on cell physiology and plasmid stability, which was assessed by flow cytometry and real-time qPCR, respectively. For resveratrol quantification, a liquid-liquid extraction from culture media was performed using ethyl acetate and then a method for quantification in High Performance Liquid Chromatography – Diode Array Detector (HPLC-DAD) was validated. In order to assess which recombinant microorganism yielded higher resveratrol production, the influence of medium composition, pH, temperature, agitation, precursor concentration and optical density (OD600) at the addition of precursor were evaluated for resveratrol production in shake flask using E. coli and S. cerevisiae. The data obtained were used to create a DoE approach in order to optimize resveratrol production in bioreactor. The bioprocess was monitored using the HPLC-DAD method for resveratrol quantification, flow cytometry to assess cellular viability and real-time qPCR to evaluate plasmid segregational instability. Shake flasks screening assays revealed a 30 times higher resveratrol yield by E. coli (about 100 μg/mL) when compared to S. cerevisiae (3.17 μg/mL), which led to the choice of the first microorganism for the scale-up optimization studies. Only the factors that had the highest impact on resveratrol production were considered for the DoE approach: temperature, pH, precursor concentration and optical density (OD600) at the addition of precursor. A Central Composite Design, rotatable and full fractioned was used, which allowed the obtention of 159.96 μg/mL of resveratrol. The population of depolarized cells varied according to the conditions used, which sometimes resulted in a 10 % difference between higher and lower production assays. Plasmid segregational instability had also been observed and variations in the values of plasmid copy number (PCN) were noticed between 22 and 30 hours of fermentation, with the highest PCN values obtained at 30 hours, when also the highest amounts of resveratrol were obtained. It is possible to conclude that cellular viability and plasmid segregational instability affect significantly resveratrol production. In sum, this work outlines the optimization of resveratrol production in bioreactors using flow cytometry and real-time qPCR for bioprocess monitoring. It was demonstrated that using the appropriate tools to optimize and monitor resveratrol production process, solutions can be found for mass production of this compound, providing an effective alternative to chemical synthesis and avoiding the depletion of natural sources.
URI: http://hdl.handle.net/10400.6/3211
Designação: Dissertação apresentada à Universidade da Beira Interior para a obtenção do grau de Mestre em Biotecnologia
Aparece nas colecções:FC - DQ | Dissertações de Mestrado e Teses de Doutoramento

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