Abstract
BackgroundCyanobacteria 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).ResultsWe 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.ConclusionsThe 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.
Highlights
Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking
Construction of a LacI-expressing and a Chloramphenicol resistance-only strain of Synechocystis In our previous study [25] we found that LacI expressed from pPMQAK1 using PrnpB and the BBa_B0034 [34] ribosome binding site was sufficient for repression of the original Ptrc2O promoter
Since the copy number of pPMQAK1 is expected to be similar to the copy number of the Synechocystis chromosome [25] or lower, taking newer data measuring the copy number of the chromosome to about 40 to 220 per cell depending on strain and growth phase into account [35], and assuming that the promoter activities are similar on the pPMQAK1 plasmid and the chromosome, we expected levels of LacI expression to be similar or higher than in our previous study
Summary
Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. The metabolic engineering efforts of even common biotechnological chassis such as Escherichia coli (E. coli), has illustrated the need for synthetic biology to provide new tools, e.g. minimal chassis, vectors and genetic control systems [3]. This is all the more true for cyanobacteria, and even though tools are available for cyanobacterial synthetic biology, more tools, especially promoters, are needed [4,5]. Most promoters previously used for engineering cyanobacteria are native to the chassis in question [8], so there is a specific lack of orthogonal promoters, constitutive as well as regulated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
