Design and characterisation of Novel synthetic biology tools in cyanobacterial model species

Abstract

Cyanobacteria are a diverse phylum of prokaryotes capable of conducting oxygenic photosynthesis. They can efficiently harvest CO2 as a carbon source and transform it into sugars and complex molecules using sunlight, water, and some trace minerals. Compared to other model heterotrophs (e.g. Escherichia coli), cyanobacteria has a more complex metabolism that allows them to produce a wide variety of complex molecules. Although cyanobacteria are promising microorganisms for sustainable biotechnology applications, yet unlocking their potential requires radical re-engineering and application of cutting-edge synthetic biology techniques and molecular tools which are currently quite limited.

To overcome the lack of synthetic biology tools to engineer cyanobacteria, we developed the CyanoGate toolkit, a molecular cloning system that unifies cyanobacteria, plants, and algae. The toolkit builds on the stablished Golden Gate MoClo syntax and assembly library for plants that has been adopted by the OpenPlant consortium. CyanoGate provides a wide range of tools including, well characterised promoters for gene expression, flanking regions and antibiotic resistance cassettes for marked and unmarked genome engineering, and CRISPR interference and sRNA tools for gene repression studies.

Building on the CyanoGate platform, I developed a strategy to adapt the Golden Gate MoClo syntax for the expression of operons. In prokaryotes, operons are an important element in genomic DNA organisation that clusters functionally related genes for simultaneous expression. Here I developed an easy-to-use system comprised of 14 new acceptor vectors called CyanOperon that allows the expression of up to 7 genes in a single operon construct. With this toolkit, a library of ribosome binding sites was characterised and compared between Escherichia coli, Synechocystis sp. PCC 6803 and Synechococcus elongatus UTEX 2973. Our findings showed that RBS activity was not only different between E. coli and cyanobacteria, but the library also performed differently between cyanobacterial species. Using the CyanOperon system, I reconstructed the violacein biosynthetic pathway in E. coli to validate the system for the expression of multigene constructs.

Another limitation to engineer cyanobacteria is the few available well-characterised inducible promoters. To address the lack of reliable inducible systems, I explored the field of optogenetics as an alternative to small molecule inducer systems. I designed and characterised a suite of 14 promoters responsive to blue-light. The system is based on the EL222 transcription factor from the marine specie Erythrobacter litoralis HTCC2594. Our results suggest that several promoters are responsive to blue light in both E. coli and Synechocystis sp. PCC 6803.