Development of a screening approach for exploring cell factory potential through metabolic flux analysis and physiology

Peter Boldsen Knudsen, Kristian Fog Nielsen, Jette Thykær

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    Abstract

    The recent developments within the field of metabolic engineering have significantly increased the speed by which fungal recombinant strains are being constructed, pushing focus towards physiological characterisation and analysis. This raises demand for a tool for diligent analysis of the recombinant strains in order to evaluate their potential as fungal cell factories and for guiding further metabolic engineering strategies. To meet the demand for a fast and reliable method for physiological characterisation of fungal strains, a screening approach using a micro titer format was developed on a Hamilton robotic system. This method aimed at characterising physiology at two levels: (1) An approach focusing on the traditional growth related parameters, i.e. growth rate, yield coefficients and extracellular metabolites. (2) 13C-labelling experiments, where metabolic fluxes are quantified in the strains of interest during exponential growth. The novelty of this screening approach, is that potential cell factories are selected based on their metabolic capacity for producing various products on interest and these cell factories may in turn be characterised based on their flux distributions. As part of the validation, already well described fungal strains were selected and tested using the described method and the developed method was subsequently used to test recombinant fungal strains producing the model polyketide 6-methylsalicylic acid. Diligent application of this strategy significantly reduces the cost and work-load connected with screening and selection of potential cell factories with attractive properties, compared with more “traditional” methodologies where metabolic flux analysis is applied at a much later state in the characterisation process.
    Original languageEnglish
    JournalNew Biotechnology
    Pages (from-to)S212
    ISSN1871-6784
    DOIs
    Publication statusPublished - 2012
    Event15th European Congress on Biotechnology - The Grand Cevahir Hotel & Convention Center, Istanbul, Turkey
    Duration: 23 Sep 201226 Sep 2012

    Conference

    Conference15th European Congress on Biotechnology
    LocationThe Grand Cevahir Hotel & Convention Center
    CountryTurkey
    CityIstanbul
    Period23/09/201226/09/2012

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