title: Substrate cycles in Penicillium chrysogenum quantified by isotopic non-stationary flux analysis.
abstract: 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 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 ¹³C 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% CmolGlc/CmolGlc+EtOH labeled glucose (mixture of 90% [1-¹³C₁] and 10% [U-¹³C₆]) and 15% ethanol [U-¹³C₂]. It was found, that (1) also several extracellular pools are enriched with ¹³C 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.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft 2628 BC, Netherlands.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhao
authors.forename: Angela
authors.initials: A
authors.lastname: Ten Pierick
authors.forename: Lodewijk
authors.initials: L
authors.lastname: de Jonge
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Heijnen
authors.forename: S Aljoscha
authors.initials: SA
authors.lastname: Wahl
mesh_terms: D057486:Carbon Cycle; D002247:Carbon Isotopes / Q000378:metabolism; D016000:Cluster Analysis; D000431:Ethanol / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007553:Isotope Labeling; D010408:Penicillium chrysogenum / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D000431:Ethanol; D005947:Glucose