Metabolic Engineering, ethanol, biofuel, decoupling production
The model cell factory Synechocystis PCC6803 is utilized as a model strain for biofuel production. Engineered strains of the photoautotrophic Cyanobacterium Synechocystis PCC6803 must balance the metabolic flux towards growth and biomass production against conversion of metabolic intermediates to ethanol. In addition ethanol has been shown to induce a stress response in this non-native producer such that decoupling may have significant advantage. In the classical yeast system in Saccharomyces cerevisiae, growth occurs first under aerobic conditions and the switch to anaerobic conditions results in switch off of Krebs cycle with the consequent conversion of pyruvate to ethanol. The key issue is that significant biomass has been produced to now produce ethanol.
To mimic such a system in Synechocystis PCC6803 we engineered a riboswitch into the control region of an ethanol cassette and introduced this into the genome of Synechocystis PCC6803. This allowed growth to occur without ethanol production since the riboswitch prevented expression of pyruvate decarboxylase and alcohol dehydrogenase, the enzymes responsible for conversion of pyruvate to ethanol.
Growth rate analysis indicated that biomass levels were higher compared to non-riboswitch constructs and no ethanol was produced during this phase. The cassette was then induced via theophylline once biomass had reach mid to late exponential levels.
Our data provides a proof of concept to decoupling ethanol production from growth. Results comparing production levels to non-riboswitch strains are presented.