title: Integrated analysis of gene expression and metabolic fluxes in PHA-producing Pseudomonas putida grown on glycerol.
abstract: BACKGROUND
  Given its high surplus and low cost, glycerol has emerged as interesting carbon substrate for the synthesis of value-added chemicals. The soil bacterium Pseudomonas putida KT2440 can use glycerol to synthesize medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA), a class of biopolymers of industrial interest. Here, glycerol metabolism in P. putida KT2440 was studied on the level of gene expression (transcriptome) and metabolic fluxes (fluxome), using precisely adjusted chemostat cultures, growth kinetics and stoichiometry, to gain a systematic understanding of the underlying metabolic and regulatory network.
  
  
  RESULTS
  Glycerol-grown P. putida KT2440 has a maintenance energy requirement [0.039 (mmolglycerol (gCDW h)(-1))] that is about sixteen times lower than that of other bacteria, such as Escherichia coli, which provides a great advantage to use this substrate commercially. The shift from carbon (glycerol) to nitrogen (ammonium) limitation drives the modulation of specific genes involved in glycerol metabolism, transport electron chain, sensors to assess the energy level of the cell, and PHA synthesis, as well as changes in flux distribution to increase the precursor availability for PHA synthesis (Entner-Doudoroff pathway and pyruvate metabolism) and to reduce respiration (glyoxylate shunt). Under PHA-producing conditions (N-limitation), a higher PHA yield was achieved at low dilution rate (29.7 wt% of CDW) as compared to a high rate (12.8 wt% of CDW). By-product formation (succinate, malate) was specifically modulated under these regimes. On top of experimental data, elementary flux mode analysis revealed the metabolic potential of P. putida KT2440 to synthesize PHA and identified metabolic engineering targets towards improved production performance on glycerol.
  
  
  CONCLUSIONS
  This study revealed the complex interplay of gene expression levels and metabolic fluxes under PHA- and non-PHA producing conditions using the attractive raw material glycerol as carbon substrate. This knowledge will form the basis for the development of future metabolically engineered hyper-PHA-producing strains derived from the versatile bacterium P. putida KT2440.
authors.affiliation: Institute of Systems Biotechnology, Saarland University, Campus A1.5, 66123, Saarbrücken, Germany.
authors.forename: Veronique
authors.initials: V
authors.lastname: Beckers
authors.affiliation: Center for Bioinformatics and Integrative Biology, Biosystems Engineering Laboratory, Faculty of Biological Sciences, Universidad Andrés Bello, 8340176, Santiago, Chile.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Poblete-Castro
authors.affiliation: Research Group Microbial Communication, Helmholtz Centre for Infection Research, Brunswick, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Tomasch
authors.affiliation: Institute of Systems Biotechnology, Saarland University, Campus A1.5, 66123, Saarbrücken, Germany. christoph.wittmann@uni-saarland.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Wittmann
mesh_terms: D018533:Biomass; D002244:Carbon / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005990:Glycerol / Q000378:metabolism*; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways; D009584:Nitrogen / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D054813:Polyhydroxyalkanoates; D002244:Carbon; D007521:Isocitrate Dehydrogenase; D009584:Nitrogen; D005990:Glycerol
keywords: Elementary flux modes; Flux balance analysis; Glycerol metabolism; Metabolic flux analysis; Nitrogen and carbon limitation; Polyhydroxyalkanoates; Pseudomonas putida KT2440; Transcriptome