Veetil, Vinod ; Angermayr, AndreasIST Austria ; Hellingwerf, Klaas
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 (glgC) 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. Conclusion: 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.
Microbial Cell Factories
Veetil V, Angermayr A, Hellingwerf K. Ethylene production with engineered Synechocystis sp PCC 6803 strains. Microbial Cell Factories. 2017;16(1). doi:10.1186/s12934-017-0645-5
Veetil, V., Angermayr, A., & Hellingwerf, K. (2017). Ethylene production with engineered Synechocystis sp PCC 6803 strains. Microbial Cell Factories, 16(1). https://doi.org/10.1186/s12934-017-0645-5
Veetil, Vinod, Andreas Angermayr, and Klaas Hellingwerf. “Ethylene Production with Engineered Synechocystis Sp PCC 6803 Strains.” Microbial Cell Factories 16, no. 1 (2017). https://doi.org/10.1186/s12934-017-0645-5.
V. Veetil, A. Angermayr, and K. Hellingwerf, “Ethylene production with engineered Synechocystis sp PCC 6803 strains,” Microbial Cell Factories, vol. 16, no. 1, 2017.
Veetil V, Angermayr A, Hellingwerf K. 2017. Ethylene production with engineered Synechocystis sp PCC 6803 strains. Microbial Cell Factories. 16(1).
Veetil, Vinod, et al. “Ethylene Production with Engineered Synechocystis Sp PCC 6803 Strains.” Microbial Cell Factories, vol. 16, no. 1, 34, BioMed Central, 2017, doi:10.1186/s12934-017-0645-5.