Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink.

Glycerol has become an attractive substrate for bio-based production processes. However, Escherichia coli, an established production organism in the biotech industry, is not able to grow on glycerol under strictly anaerobic conditions in defined minimal medium due to redox imbalance. Despite extensive research efforts aiming to overcome these limitations, anaerobic growth of wild-type E. coli on glycerol always required either the addition of electron acceptors for anaerobic respiration (e.g. fumarate) or the supplementation with complex and relatively expensive additives (tryptone or yeast extract). In the present work, driven by model-based calculations, we propose and validate a novel and simple strategy to enable fermentative growth of E. coli on glycerol in defined minimal medium. We show that redox balance could be achieved by uptake of small amounts of acetate with subsequent reduction to ethanol via acetyl-CoA. Using a directed laboratory evolution approach, we were able to confirm this hypothesis and to generate an E. coli strain that shows, under anaerobic conditions with glycerol as the main substrate and acetate as co-substrate, robust growth (μ = 0.06 h

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