Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium

Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library).

We first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis.

The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.