title: Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida.
abstract: BACKGROUND
  Pseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation.
  
  
  RESULTS
  A genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms.
  
  
  CONCLUSIONS
  Production of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Borrero-de Acuña
authors.forename: Agata
authors.initials: A
authors.lastname: Bielecka
authors.forename: Susanne
authors.initials: S
authors.lastname: Häussler
authors.forename: Max
authors.initials: M
authors.lastname: Schobert
authors.forename: Martina
authors.initials: M
authors.lastname: Jahn
authors.forename: Christoph
authors.initials: C
authors.lastname: Wittmann
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
authors.affiliation: Universidad Andrés Bello, Facultad de Ciencias Biológicas, Biosystems Engineering group, 8340176 Santiago, Chile. ignacio.pobletecastro@helmholtz-hzi.de.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Poblete-Castro
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D005947:Glucose / Q000378:metabolism; D005948:Glucose Dehydrogenases / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000254:growth & development / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D054813:Polyhydroxyalkanoates; D021122:Protein Subunits; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D009249:NADP; D005948:Glucose Dehydrogenases; D002265:Carboxylic Ester Hydrolases; C022363:6-phosphogluconolactonase; D005947:Glucose