(23f) Oxygen-Responsive Genetic Circuits Constructed in Synechocystis Sp. PCC 6803

Cyanobacteria exhibit promise as sustainable biotechnology platforms.  However, few synthetic regulatory circuits have been developed for these non-model organisms, and the simple circuits that have been created rely on the addition of expensive chemical inducers.  Environmental stimuli are often less costly, and can be physiologically relevant to production.  For instance, oxygen irreversibly inactivates molybdenum-dependent nitrogenase, the enzyme used by diverse prokaryotes to fix nitrogen.  Employing the fumarate and nitrate reduction system from Escherichia coli, we developed an oxygen-responsive circuit that can be used to control heterologous expression of nitrogenase in Synechocystis sp. PCC 6803.  As designed, transcripts for the genes encoding nitrogenase’s structural proteins, nifHDK, were produced only in the absence of oxygen.  In addition, we utilized the oxygen-responsive transcriptional regulator in a two-input AND gate, which allows Synechocystis to control heterologous gene expression in response to two signals, both low oxygen and high anhydrotetracycline.  To our knowledge, this two-input AND gate is the first complex genetic circuit built in a cyanobacterial strain, enabling more sophisticated regulatory control.  This work expands the synthetic biology toolbox for specific gene expression in cyanobacteria, furthering their utility in bio-manufacturing.