title: Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803.
abstract: The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P(trc) resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P(A1lacO-1), induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.
authors.affiliation: Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
authors.forename: Fernando
authors.initials: F
authors.lastname: Guerrero
authors.forename: Verónica
authors.initials: V
authors.lastname: Carbonell
authors.forename: Matteo
authors.initials: M
authors.lastname: Cossu
authors.forename: Danilo
authors.initials: D
authors.lastname: Correddu
authors.forename: Patrik R
authors.initials: PR
authors.lastname: Jones
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005030:Ethylenes / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial; D008190:Lyases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D044224:Pseudomonas syringae / Q000737:chemistry* / Q000201:enzymology; D053038:Quorum Sensing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D005030:Ethylenes; D011993:Recombinant Fusion Proteins; C036216:ethylene; D008190:Lyases; C034693:ethylene forming enzyme