Engineering the Cyanobacterium Synechocystis Sp. PCC 6803 Towards Optimized Solutions for Future Biotechnological Applications

Nagy, C. I., University of Turku
Aro, E. M., University of Turku
Thiel, K., University of Turku
Dandapani, H., University of Turku
Mulaku, E., University of Turku
Kakko, L., University of Turku
Cyanobacteria can serve as cell factories for the production of fuels or other commodity chemicals because of the many benefits that their physiology and genetics offer: photosynthesis, CO2 fixation, fast growth rate, relatively simple genetic manipulation, and feasible culture conditions. With the emerging and sophistication of synthetic biology tools the genetic and metabolic engineering of the innate cyanobacterial metabolism is progressively advancing. Although cyanobacteria have many advantages over heterotrophic species like E. coli or S. cerevisiae, their potential is less explored because the relative scarce information about their genetic and metabolic processes. However by means of synthetic biology techniques cyanobacterial strains can be effectively modified to produce valuable compounds.
Our research focuses on the construction of synthetic metabolic pathways in the cyanobacterium Synechocystis sp. PCC6803 for the production of a range of industrially valuable compounds. In order to implement new heterologous metabolic pathways in Synechocystis fine-tuned genetic elements needs to be evaluated at transcriptional and translational level. A library of ribosome binding sites (cyanobacterial and from E. coli), a library of integration sites (targeting the chromosome and the native plasmids), inducible promoters (native and E. coli-derived), enhancer-, insulator- and transcriptional terminator elements have been designed, constructed and systematically evaluated for an optimized metabolite production in this strain. In order to explore the effect of the order of the coding genes on the translation efficiency and to simulate and analyze the integrity and robustness of multigene operons, we have chosen different fluorescent reporter genes for evaluation. To efficiently combine most genetic and metabolic elements, we adapted a novel set of plasmids and a specific gene-brick system. All constructs can be integrated in the host genome or alternatively expressed from an autonomously replicating artificial plasmid.
On metabolic level, our goal is to establish an efficient production of industrially valuable end-products directly from CO2 and water using sunlight as energy. Since pyruvate is one of the central metabolites, several metabolic modifications have been accomplished to direct and enhance the metabolic flux towards this chemical. Two native reporter compounds - lactate and ethanol - have been chosen to serve as markers for the pyruvate production efficiency. In parallel, an engineered sucrose pathway is used to evaluate the effects of upstream modifications targeting the photosynthetic machinery. In order to divert the metabolic flux from non-essential photosynthetic and biochemical processes towards target products, several genes and subsequently pathways have been altered that are related to alternative photosynthetic electron transport, glucose metabolism and Calvin cycle.
Our recent findings regarding the ribosome binding sites, genomic integrations, promoters and photosynthetic modifications for an improved metabolite production will have a major contribution to the field of the molecular biology and genetics of cyanobacteria. Expanded scope of validated genetic elements and quantitative comparative information on the functions will add valuable tools to the biotechnological applications of these significant microorganisms.