Engineering of genetic control tools in Synechocystis sp. PCC 6803 using rational design techniques

• Leveraged rational design strategies to manipulate promoters in Synechocystis.
• Developed promoters capable of a 78X range in expression in Synechocystis.
• We demonstrate near full repression of an inducible promoter in Synechocystis.
• Developed an inducible promoter capable of high levels of induction in Synechocystis.

Cyanobacteria show promise as photosynthetic microbial factories capable of harnessing sunlight and CO2 to produce valuable end products, but few genetic control tools have been characterized and utilized in these organisms. To develop a suite of control elements capable of gene control at a variety of expression strengths, a library of 10 promoter-constructs were developed and built via rational design techniques by adding individual nucleotides in a step-wise manner within the −10 and −35 cis-acting regions of the tac promoter. This suite produced a dynamic range of expression strength, exhibiting a 78 fold change between the lowest expressing promoter, Psca8- and the highest expressing promoter, Psca3-2 when tested within Synechocystis sp. PCC 6803. Additionally, this study details the construction of a chemically inducible construct for use in Synechocystis that is based on the tac repressor system most commonly used in Escherichia coli. This research demonstrates the construction of a highly expressed inducible promoter that is also capable of high levels of gene repression. Upon chemical induction with IPTG, this same mutant strain was capable of exhibiting an average 24X increase in GFP expression over that of the repressed state.