title: Metabolic pathway engineering using the central signal processor PII.
abstract: BACKGROUND
  PII signal processor proteins are wide spread in prokaryotes and plants where they control a multitude of anabolic reactions. Efficient overproduction of metabolites requires relaxing the tight cellular control circuits. Here we demonstrate that a single point mutation in the PII signaling protein from the cyanobacterium Synechocystis sp. PCC 6803 is sufficient to unlock the arginine pathway causing over accumulation of the biopolymer cyanophycin (multi-L-arginyl-poly-L-aspartate). This product is of biotechnological interest as a source of amino acids and polyaspartic acid. This work exemplifies a novel approach of pathway engineering by designing custom-tailored PII signaling proteins. Here, the engineered Synechocystis sp. PCC6803 strain with a PII-I86N mutation over-accumulated arginine through constitutive activation of the key enzyme N-acetylglutamate kinase (NAGK).
  
  
  RESULTS
  In the engineered strain BW86, in vivo NAGK activity was strongly increased and led to a more than tenfold higher arginine content than in the wild-type. As a consequence, strain BW86 accumulated up to 57 % cyanophycin per cell dry mass under the tested conditions, which is the highest yield of cyanophycin reported to date. Strain BW86 produced cyanophycin in a molecular mass range of 25 to >100 kDa; the wild-type produced the polymer in a range of 30 to >100 kDa.
  
  
  CONCLUSIONS
  The high yield and high molecular mass of cyanophycin produced by strain BW86 along with the low nutrient requirements of cyanobacteria make it a promising means for the biotechnological production of cyanophycin. This study furthermore demonstrates the feasibility of metabolic pathway engineering using the PII signaling protein, which occurs in numerous bacterial species.
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany. bjoern.watzer@student.uni-tuebingen.de.
authors.forename: Björn
authors.initials: B
authors.lastname: Watzer
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany. alicia.engelbrecht@student.uni-tuebingen.de.
authors.forename: Alicia
authors.initials: A
authors.lastname: Engelbrecht
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany. waldemar.hauf@gmail.com.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Hauf
authors.affiliation: Central Facilities, Analytics, ZMBP, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 32, 72076, Tübingen, Germany. mark.stahl@zmbp.uni-tuebingen.de.
authors.forename: Mark
authors.initials: M
authors.lastname: Stahl
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany. iris.maldener@mikrobio.uni-tuebingen.de.
authors.forename: Iris
authors.initials: I
authors.lastname: Maldener
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076, Tübingen, Germany. karl.forchhammer@uni-tuebingen.de.
authors.forename: Karl
authors.initials: K
authors.lastname: Forchhammer
mesh_terms: D000641:Ammonia / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering; D046529:Microscopy, Electron, Transmission; D009566:Nitrates / Q000378:metabolism; D051922:PII Nitrogen Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000378:metabolism; D017354:Point Mutation; D046939:Synechocystis / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009566:Nitrates; D051922:PII Nitrogen Regulatory Proteins; C024250:cyanophycin; D000641:Ammonia; D001120:Arginine; D017851:Phosphotransferases (Carboxyl Group Acceptor); C018721:acetylglutamate kinase