A Library of Tunable, Portable, and Inducer-Free Promoters Derived from Cyanobacteria

Abstract

Cyanobacteria are emerging as hosts for various biotechnological applications. The ability to engineer these photosynthetic prokaryotes greatly depends on the availability of well-characterized promoters. Inducer-free promoters of a range of activities may be desirable for the eventual large-scale, outdoor cultivations. Further, several native promoters of cyanobacteria are repressed by high carbon dioxide or light, and it would be of interest to alter this property. We started with PrbcL and PcpcB, the well-characterized native promoters of the model cyanobacterium Synechococcus elongatus PCC 7942, found upstream of the two abundantly expressed genes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, and phycocyanin β-1 subunit, respectively. The library of 48 promoters created via error-prone PCR of these 300-bp-long native promoters showed 2 orders of magnitude dynamic range with activities that were both lower and higher than those of the wild-type promoters. A few mutants of the PrbcL showed greater strength than PcpcB, which is widely considered a superstrong promoter. A number of mutant promoters did not show repression by high CO2 or light, typically found for PrbcL and PcpcB, respectively. Further, the wild-type and mutant promoters showed comparable activities in the fast-growing and stress-tolerant strains S. elongatus PCC 11801 and PCC 11802, suggesting that the library can be used in different cyanobacteria. Interestingly, the majority of the promoters showed strong expression in E. coli, thus adding to the repertoire of inducer-free promoters for this heterotrophic workhorse. Our results have implications in the metabolic engineering of cyanobacteria and E. coli.

The Effect of Promoter and RBS Combination on the Growth and Glycogen Productivity of Sodium-Dependent Bicarbonate Transporter (SbtA) Overexpressing Synechococcus sp. PCC 7002 Cells

“…Among the promoters tested, P cpcB560 had the strongest effect, in agreement with its super-strong activity due to the presence of multiple TFBS in its sequence (Zhou et al, 2014). This is in spite of previous reports that P cpcB is inhibited under strong light conditions (Sengupta et al, 2020a). The strong and continuous illumination was chosen in our study because previous studies, including our own preliminary studies, have shown that it leads to higher biomass accumulation for the WT PCC 7002 cells.…”

“…P rbcL2A had a slightly lesser effect on SbtA expression, growth and glycogen accumulation compared to the P cpcB promoters. Our study shows that even under air bubbling and high illumination, very similar to the "optimal" conditions for high P rbcL activity (Sengupta et al, 2020a) P cpcB560 has higher activity compared to PrbcL2A. Promoter activity may also be affected by the length of the promoter chosen.…”

Current Status and Future Strategies to Increase Secondary Metabolite Production from Cyanobacteria

“…PCC 6803 [141,142]. Recently, a mutant promoter library from two popular promoters, P psbA and P cpc of S. elongatus PCC 7942, were generated to achieve promoters of varying strength [143]. A collection of 48 unique promoters were validated in three additional S. elongatus strains, expanding the cyanobacterial synthetic biology toolbox.…”

Construction of Fully Segregated Genomic Libraries in Polyploid Organisms Such as Synechocystis sp. PCC 6803

“…But clearly, integrating such a library on all the chromosomes of a polyploid microorganism is a major challenge, without going through the regular time-consuming segregation steps for a limited number of representatives. 7 How can one ensure that the genomic library integrated on the chromosomes of polyploid microorganisms is fully segregated? In other words, how can one ensure that all existing copies of the chromosome contain the intended fragment?…”

“…Our work marks the first report of construction of a fully segregated chromosomal genomic library in polyploid microorganisms, without going through the time-consuming segregation step of only a small number of representatives. 7 This library could in principle be exploited either for fundamental research ( e.g. , detailing complex phenotypes) or synthetic biology applications ( e.g.…”