Optimized Genetic Tools and Overexpression Strategies in the Development of Efficient Production Systems in Photosynthetic Cyanobacteria

Because of the gradually increasing need of fossil fuels and because of the inherent ecological and geopolitical concerns, the search for alternative/renewable energy sources is imminent. 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. Although cyanobacteria have many advantages over heterotrophic microorganisms like E. coli or S. cerevisiae, their biotechnological 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 pathways in the cyanobacterium Synechocystis sp. PCC6803 to be used for the production of a wide range of industrially valuable compounds. Since genetic alterations can cause perturbations and metabolic imbalance, in order to establish stable and efficient heterologous metabolic pathways, fine-tuned genetic elements and related synthetic biology tools needs to be evaluated at transcriptional and translational level. A library of 18 integration sites (targeting the chromosome and the native plasmids), a library of 13 ribosome binding sites (7 cyanobacterial and 6 from E. coli), 8 inducible promoters (5 native and 3 from E. coli), enhancer-, insulator- and transcriptional terminator elements have been designed, constructed and are being evaluated for an optimized metabolite production in this strain. The aim is also to explore the effect of the order of the coding genes on the translation efficiency; therefore we have chosen 3 different fluorescent reporter genes for evaluation. In the assembly process we use a set of plasmids and a specific gene-brick system; subsequently these constructs will be integrated in the genome of the cyanobacterium or alternatively maintained on an autonomously replicating artificial plasmid.

On metabolic level, our goal is to establish an efficient production of desired end-products directly from CO₂ and water using sunlight as energy. In practice this means targeted modification of the host to maximize the photon conversion efficiency and metabolic flux towards the production of pyruvate. 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 have been chosen to serve as markers for the pyruvate production efficiency: lactate and ethanol. Besides these, we are monitoring sucrose production to evaluate the effect of the alteration of the photosynthetic electron transport pathway. 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.

With this comprehensive and extensive study we aim to design an efficient cyanobacterial strain with an improved biotechnological potential.