title: Screening and genetic characterization of thermo-tolerant Synechocystis sp. PCC6803 strains created by adaptive evolution.
abstract: BACKGROUND
  Temperature tolerance is an important aspect for commercial scale outdoor cultivation of microalgae and cyanobacteria. While various genes are known to be related to Synechocystis sp. PCC6803's heat shock response, there is very limited published data concerning the specific genes involved in long term thermal tolerance. We have previously used random mutagenesis and adaptive evolution to generate a mixture of strains of Synechocystis sp. PCC6803 with significantly increased thermal tolerance. The genetic modifications leading to the phenotypes of the newly generated strains are the focus of this work.
  
  
  RESULTS
  We used a custom screening platform, based on 96-deepwell microplate culturing in an in house designed cultivation chamber integrated in a liquid handling robot for screening and selection; in addition we also used a more conventional system. The increased thermal tolerances of the isolated monoclonal strains were validated in larger bioreactors and their whole genomes sequenced. Comparison of the sequence information to the parental wild type identified various mutations responsible for the enhanced phenotypes. Among the affected genes identified are clpC, pnp, pyk2, sigF, nlpD, pyrR, pilJ and cya1.
  
  
  CONCLUSIONS
  The applied methods (random mutagenesis, in vivo selection, screening, validation, whole genome sequencing) were successfully applied to identify various mutations, some of which are very unlikely to have been identified by other approaches. Several of the identified mutations are found in various strains and (due to their distribution) are likely to have occurred independently. This, coupled with the relatively low number of affected genes underscores the significance of these specific mutations to convey thermal tolerance in Synechocystis.
authors.forename: Ulrich M
authors.initials: UM
authors.lastname: Tillich
authors.forename: Nick
authors.initials: N
authors.lastname: Wolter
authors.forename: Philipp
authors.initials: P
authors.lastname: Franke
authors.forename: Ulf
authors.initials: U
authors.lastname: Dühring
authors.affiliation: Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Bahnhofstraße 1, 16-2001, D-15745 Wildau, Germany. mfrohme@th-wildau.de.
authors.forename: Marcus
authors.initials: M
authors.lastname: Frohme
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D005075:Biological Evolution; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D010641:Phenotype; D017422:Sequence Analysis, DNA; D046939:Synechocystis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins