(773e) Developing a Tunable and Tightly Controlled Gene Expression System in Synechocystis sp. PCC6803

Cyanobacteria are photosynthetic microbes that can derive energy from sunlight and carbon from atmospheric carbon dioxide, as well as other sources. In the past two decades, there were numerous efforts geared towards developing cyanobacteria as bioproduction platforms. Several of these organisms are amenable to genetic manipulation, thus facilitating synthetic biology/metabolic engineering applications. The unique demands of living on light as a primary nutrient make the use of many established gene expression control systems difficult or impossible to apply. Among other approaches, researchers have used endogenous promoters such as PpsbA2 and PcpcB (promoters that drive expression of major photosynthetic components), and synthetic riboswitches with limited/mixed success. Hence, developing an effective genetic toolkit to regulate gene expression in cyanobacteria is of utmost importance. In this work, as a first case study, we combined a previously developed theophylline-responsive riboswitch with the T7 control system to drive expression of a yellow fluorescence protein (YFP) reporter in Synechocystis sp. PCC 6803. We then demonstrated, as a second case study, the application of this system by using PnblA to drive expression of this same reporter. Note that, under environmental stresses such as nitrogen or sulfur starvation, cyanobacteria utilize phycobilisomes (that account for 30-40% of total cellular protein) as a nutrient source through a degradation process that leads to yellowing or bleaching of the culture. The nbl (nonbleaching) genes are involved in this process, with nblA interacting directly with the phycobilisome, possibly causing rod instability and the beginning of the disassembly process. Overall, the utility of the designed gene expression control system was clearly shown via reduced baseline expression and higher ratio of induction. Thus, our work shows the utility of a tightly controlled and tunable gene expression control system that can exploit the differences in light/dark life cycle and expedite biotechnological applications in cyanobacteria.