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Background   Penicillium chrysogenum , the main production strain for penicillin-G, has a high content of intracellular carbohydrates, especially reduced sugars such as mannitol, arabitol, erythritol, as well as trehalose and glycogen. In previous steady state  13 C wash-in experiments a delay of labeling enrichments in glycolytic intermediates was observed, which suggests turnover of storage carbohydrates. The turnover of storage pools consumes ATP which is expected to reduce the product yield for energy demanding production pathways like penicillin-G.    Results  In this study, a  13 C labeling wash-in experiment of 1 hour was performed to systematically quantify the intracellular flux distribution including eight substrate cycles. The experiments were performed using a mixed carbon source of 85% Cmol Glc /Cmol Glc+EtOH  labeled glucose (mixture of 90% [1- 13 C 1 ] and 10% [U- 13 C 6 ]) and 15% ethanol [U- 13 C 2 ]. It was found, that (1) also several extracellular pools are enriched with  13 C labeling rapidly (trehalose, mannitol, and others), (2) the intra- to extracellular metabolite concentration ratios were comparable for a large set of metabolites while for some carbohydrates (mannitol, trehalose, and glucose) the measured ratios were much higher.    Conclusions  The fast enrichment of several extracellular carbohydrates and a concentration ratio higher than the ratio expected from cell lysis (2%) indicate active (e.g. ATP consuming) transport cycles over the cellular membrane. The flux estimation indicates, that substrate cycles account for about 52% of the gap in the ATP balance based on metabolic flux analysis.

Substrate cycles in Penicillium chrysogenum quantified by isotopic non-stationary flux analysis