Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002

Abstract

The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system had a 48-fold dynamic range and was shown to out-perform Ptrc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.

Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain

“…However, for many application areas, it is instead preferable to exploit the natural abilities of nonmodel microbes as specialists at consuming or producing molecules or thriving within niche environments (2). Recent work has described adapting common E. coli synthetic biology tools to work across different bacterial phyla (3,4) and has produced genetic toolkits for new bacteria, where collections of DNA constructs and methods for precise control of heterologous gene expression have been developed for engineering strains naturally specialized for photosynthesis or survival within the gut microbiome (5,6). An important application area for biotechnology is the production of materials, and bacteria that naturally secrete high yields of cellulose have attracted significant attention not just from people in industry and research (7) but also from those in art, fashion, and citizen science (8).…”

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…Unfortunately, such tools often do not function as expected in cyanobacteria (15,16). This is due to differences in the transcription (17) and translation (18) machinery, cyanobacterial traits such as polyploidy (19), an efficient recombination system (20), and a circadian rhythm (21,22), and other features that distinguish cyanobacteria from their heterotrophic cousins.…”

“…In E. coli, extensive knowledge of methods for manipulating protein expression has led to the creation of complex synthetic systems such as analog computational circuits (24), logic gates (25), and oscillators (26). To facilitate advanced synthetic biology applications in cyanobacteria, broad-host-range vectors (16), promoter libraries (27,28), and ribosome binding sites (15) have been characterized.…”

Cyanobacterial Farming for Environment Friendly Sustainable Agriculture Practices: Innovations and Perspectives

“…Therefore, it is important to optimize the inherent biological chassis for enhancing the yield of biochemicals from cyanobacteria. In recent years, several groups have emphasized on construction, designing, and expression of the biosynthetic pathways along with development of the toolboxes for metabolic engineering in cyanobacteria which could lead to economic profitability by increasing the production of existing and novel chemicals and biofuels (Wang et al, 2012;Berla et al, 2013;Desai and Atsumi, 2013;Oliver and Atsumi, 2014;Gudmundsson and Nogales, 2015;Markley et al, 2015).…”