Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input–output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli, the available variety of tunable promoter parts for phototrophic cyanobacteria is still limited. Commonly used synthetic and semisynthetic promoters show weak dynamic ranges or no regulation at all in cyanobacterial models. Well-controlled alternatives such as native metal-responsive promoters, however, pose the problems of inducer toxicity and lacking orthogonality. Here, we present the comparative assessment of dose–response functions of four different inducible promoter systems in the model cyanobacterium Synechocystis sp. PCC 6803. Using the novel bimodular reporter plasmid pSHDY, dose–response dynamics of the re-established vanillate-inducible promoter PvanCC was compared to the previously described rhamnose-inducible P rha , the anhydrotetracycline-inducible PL03, and the Co2+-inducible P coaT . We estimate individual advantages and disadvantages regarding dynamic range and strength of each promoter, also in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in cyanobacteria, facilitating the scalability and robustness of synthetic regulatory network designs and of complex metabolic pathway engineering strategies.

Cyanobacteria are extremely adaptable, fast-growing, solar-powered cell factories that, like plants, are able to convert carbon dioxide into sugar and oxygen and thereby produce a large number of important compounds. Due to their unique phototrophy-associated physiological properties, i.e. naturally occurring isoprenoid metabolic pathway, they represent a highly promising platform for terpenoid biosynthesis. Here, we implemented a carefully devised engineering strategy to boost the biosynthesis of commercially attractive plant sequiterpenes, in particular valencene. Sesquiterpenes are a diverse group of bioactive metabolites, mainly produced in higher plants, but with often low concentrations and expensive downstream extraction. In this work we successfully demonstrate a multi-component engineering approach towards the photosynthetic production of valencene in the cyanobacterium Synechocystis sp. PCC 6803. First, we improved the flux towards valencene by markerless genomic deletions of shc and sqs . Secondly, we downregulated the formation of carotenoids, which are essential for viability of the cell, using CRISPRi on crtE . Finally, we intended to increase the spatial proximity of the two enzymes, ispA and CnVS , involved in valencene formation by creating an operon construct, as well as a fusion protein. Combining the most successful strategies resulted in a valencene production of 19 mg/g DCW in Synechocystis . In this work, we have devised a useful platform for future engineering steps.

In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5’/3’ gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.

8Research in the field of synthetic biology highly depends on efficient, well-characterized 9 promoters. While great progress has been made with other model organisms such as 10 Escherichia coli, photosynthetic cyanobacteria still lag behind. Commonly used promoters 11 that have been tested in cyanobacteria show weaker dynamic range or no regulation at 12 all. Alternatives such as native metal-inducible promoters pose the problem of inducer 13 toxicity. 14 Here, we evaluate four different inducible promoters, both previously published and new, 15 using the modular plasmid pSHDY, in the model cyanobacterium Synechocystis sp. PCC 16 6803 -namely the vanillate-inducible promoter PvanCC, the rhamnose-inducible Prha, and 17 the aTc-inducible PL03, and the Co 2+ -inducible PcoaT. We estimate individual advantages 18 and disadvantages, as well as dynamic range and strength of each promoter in 19comparison with well-established constitutive systems. We observed a delicate balance 20 between transcription factor toxicity and sufficient expression to obtain a dose-dependent 21 response to the inducer. In summary, we expand the current understanding and 22 employability of inducible promoters in order to facilitate the construction of more complex 23 regulatory synthetic networks, as well as more complicated biotechnological pathways for 24 cyanobacteria. 25 26

Stanier RY, Deruelles J, Rippka R, Herdman M, Waterbury JB: Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology 1979, 111:1–61. Recipes for standard and alternative BG11 for culturing freshwater cyanobacteria, such as Synechocystis sp. PCC 6803, as described. Media is usually not suitable for marine cyanobacteria. Final Concentration of Medium. CaCl2*2 H2O 0.036 g/L Citric acid 0.006 g/L NaNO3 1.4958 g/L MgSO4* 7 H2O 0.0749 g/L 0.25M Na2EDTA (pH 8) 0.0056 mL/L Na2CO3 20 µg/ml Fe(III) Ammonium citrate 6 µg/ml K2HPO4 * 3H2O 30 µg/ml TES Buffer (pH 8) 10 mM H3BO3 2.86 mg/L MnCl2 * 4 H2O 1.81 mg/L ZnSO4 * 7 H2O 0.222 mg/L Na2MoO4 * 2 H2O 0.390 mg/L Co(NO3)2 *6 H2O 0.049 mg/L (CuSO4 * 5 H2O 0.079 mg/L if required)