TY - ABST

T1 - Development of a screening approach for exploring cell factory potential through metabolic flux analysis and physiology

A1 - Knudsen,Peter Boldsen

A1 - Nielsen,Kristian Fog

A1 - Thykær,Jette

AU - Knudsen,Peter Boldsen

AU - Nielsen,Kristian Fog

AU - Thykær,Jette

PB - Elsevier BV

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

U2 - 10.1016/j.nbt.2012.08.594

DO - 10.1016/j.nbt.2012.08.594

JO - New Biotechnology

JF - New Biotechnology

SN - 1871-6784

SP - S212

ER -