title: Mapping global effects of the anti-sigma factor MucA in Pseudomonas fluorescens SBW25 through genome-scale metabolic modeling.
abstract: BACKGROUND
  Alginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds. The polymer is also synthesized as an exo-polysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter, and these organisms may represent an alternative alginate source in the future. The current work describes an attempt to rationally develop a biological system tuned for very high levels of alginate production, based on a fundamental understanding of the system through metabolic modeling supported by transcriptomics studies and carefully controlled fermentations.
  
  
  RESULTS
  Alginate biosynthesis in Pseudomonas fluorescens was studied in a genomics perspective, using an alginate over-producing strain carrying a mutation in the anti-sigma factor gene mucA. Cells were cultivated in chemostats under nitrogen limitation on fructose or glycerol as carbon sources, and cell mass, growth rate, sugar uptake, alginate and CO(2) production were monitored. In addition a genome scale metabolic model was constructed and samples were collected for transcriptome analyses. The analyses show that polymer production operates in a close to optimal way with respect to stoichiometric utilization of the carbon source and that the cells increase the uptake of carbon source to compensate for the additional needs following from alginate synthesis. The transcriptome studies show that in the presence of the mucA mutation, the alg operon is upregulated together with genes involved in energy generation, genes on both sides of the succinate node of the TCA cycle and genes encoding ribosomal and other translation-related proteins. Strains expressing a functional MucA protein (no alginate production) synthesize cellular biomass in an inefficient way, apparently due to a cycle that involves oxidation of NADPH without ATP production. The results of this study indicate that the most efficient way of using a mucA mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation.
  
  
  CONCLUSIONS
  The insights gained in this study should be very useful for a future efficient production of microbial alginates.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, N 7491, Norway.
authors.forename: Sven E F
authors.initials: SE
authors.lastname: Borgos
authors.forename: Sergio
authors.initials: S
authors.lastname: Bordel
authors.forename: Håvard
authors.initials: H
authors.lastname: Sletta
authors.forename: Helga
authors.initials: H
authors.lastname: Ertesvåg
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Jakobsen
authors.forename: Per
authors.initials: P
authors.lastname: Bruheim
authors.forename: Trond E
authors.initials: TE
authors.lastname: Ellingsen
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
mesh_terms: D000464:Alginates; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D002478:Cells, Cultured; D005285:Fermentation; D020869:Gene Expression Profiling / Q000379:methods; D023281:Genomics / Q000379:methods; D020723:Glucuronic Acid / Q000096:biosynthesis; D006603:Hexuronic Acids; D046228:Microarray Analysis; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D025341:Principal Component Analysis; D011551:Pseudomonas fluorescens / Q000378:metabolism*
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; C107895:MucA protein, Pseudomonas; D020723:Glucuronic Acid