title: Ethylene production with engineered Synechocystis sp PCC 6803 strains.
abstract: BACKGROUND
  Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.
  
  
  RESULTS
  This study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.
  
  
  CONCLUSIONS
  Making use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.
authors.affiliation: Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.|Reliance Technology Group, Reliance Industries Ltd., Navi Mumbai, India.
authors.forename: Vinod Puthan
authors.initials: VP
authors.lastname: Veetil
authors.affiliation: Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.|Institute of Science and Technology (IST) Austria, Klosterneuburg, Austria.
authors.forename: S Andreas
authors.initials: SA
authors.lastname: Angermayr
authors.affiliation: Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands. K.J.Hellingwerf@uva.nl.
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: Hellingwerf
mesh_terms: D001120:Arginine / Q000378:metabolism; D005030:Ethylenes / Q000096:biosynthesis* / Q000378:metabolism; D018628:Gene Dosage; D006003:Glycogen / Q000235:genetics / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008190:Lyases / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D010788:Photosynthesis; D011401:Promoter Regions, Genetic; D044224:Pseudomonas syringae / Q000201:enzymology / Q000235:genetics; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism
chemical_list: D005030:Ethylenes; D007656:Ketoglutaric Acids; D006003:Glycogen; C036216:ethylene; D001120:Arginine; D008190:Lyases; C034693:ethylene forming enzyme
keywords: Arginine; Cyanobacteria; Ethylene; Glycogen; Oxoglutarate; Sustainable; Synechocystis