title: Fermentation of mixed substrates by Clostridium pasteurianum and its physiological, metabolic and proteomic characterizations.
abstract: BACKGROUND
  Clostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as n-butanol and 1,3-propanediol. Previously we have shown that dual substrate fermentation using glucose and glycerol enhanced the cell growth and butanol production significantly. Although C. pasteurianum can grow efficiently with either glucose or glycerol alone, under certain conditions, glucose limitation in the mixed substrate fermentation leads to growth cessation. To understand this phenomenon and for process optimization, fermentation experiments were performed in the presence of excess glycerol but with varied initial concentrations of glucose which were followed by physiological, metabolic and proteomic analyses.
  
  
  RESULTS
  Physiological characterization showed that the observed cease of growth is not due to the toxicity of n-butanol. Furthermore, the growth can be resumed by addition of glucose or the intermediate oxaloacetate. Proteomic analysis shed more light on the system-level regulation of many proteins directly or indirectly associated with this phenomenon. Surprisingly, it is found that the specific growth rate of C. pasteurianum in the different growth phases (e.g. before and after glucose limitation) correlated well with the expression level of the ATP dependent pyruvate carboxylase and with the expression level of biotin synthase which provides the cofactor biotin for the formation of oxaloacetate from pyruvate. Bioenergetic analysis based on the formation rates of metabolites further show that ATP supply is not a limiting factor for the pyruvate carboxylation to oxaloacetate.
  
  
  CONCLUSIONS
  The results of physiological and proteomic analyses clearly show that the anaplerotic synthesis of oxaloacetate plays a key role in determining the growth behaviour of C. pasteurianum in fermentations with mixed substrates of glucose and glycerol. This study provides interesting targets for metabolic engineering of this emerging industrial microorganism.
authors.affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, 21071, Hamburg, Germany.
authors.forename: Wael
authors.initials: W
authors.lastname: Sabra
authors.affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, 21071, Hamburg, Germany.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, 21071, Hamburg, Germany.
authors.forename: Sruthi
authors.initials: S
authors.lastname: Surandram
authors.affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, 21071, Hamburg, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Groeger
authors.affiliation: Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickestrasse 15, 21071, Hamburg, Germany. aze@tuhh.de.
authors.forename: An-Ping
authors.initials: AP
authors.lastname: Zeng
mesh_terms: D020001:1-Butanol / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003013:Clostridium / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D011409:Propylene Glycols / Q000378:metabolism*; D040901:Proteomics
chemical_list: D001426:Bacterial Proteins; D011409:Propylene Glycols; C041787:1,3-propanediol; D020001:1-Butanol; D005947:Glucose
keywords: 1,3-propanediol; Butanol; Mixed substrate fermentation; Oxaloacetate; Pyruvate decarboxylation