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Summary In contrast to the current paradigm of using microbial mono‐cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed‐species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co‐culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4‐dinitrotoluene (2,4‐DNT). By encapsulating S. elongatus within a barium–alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4‐DNT, enhancing the performance of the co‐culture. The synthetic consortium was able to convert 2,4‐DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose‐dependent accumulation of polyhydroxyalkanoate, an added‐value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4‐DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs., This work describes the development of a synthetic microbial consortium composed of a cyanobacterium engineered to use light to fix inorganic carbon, and a heterotrophic bacterium engineered to degrade the environmental pollutant, 2,4‐dinitrotoluene. Together, the synthetic consortium displayed the capacity to remediate the industrial compound 2,4‐DNT while simultaneously synthesizing biopolymers using light and carbon dioxide as the primary inputs.

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 Prha, the anhydrotetracycline-inducible PL03, and the Co2+-inducible PcoaT. 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.

Research in the field of synthetic biology highly depends on efficient, well-characterized promoters. While great progress has been made with other model organisms such asEscherichia coli, photosynthetic cyanobacteria still lag behind. Commonly used promoters that have been tested in cyanobacteria show weaker dynamic range or no regulation at all. Alternatives such as native metal-inducible promoters pose the problem of inducer toxicity.Here, we evaluate four different inducible promoters, both previously published and new, using the modular plasmid pSHDY, in the model cyanobacteriumSynechocystis sp. PCC 6803 - namely the vanillate-inducible promoter PvanCC, the rhamnose-inducible Prha, and the aTc-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages, as well as dynamic range and strength of each promoter 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 order to facilitate the construction of more complex regulatory synthetic networks, as well as more complicated biotechnological pathways for cyanobacteria.

Inducible promoters are an important tool for synthetic biology. They enable temporal control of gene expression, as well as controlled expression of toxic genes. However, prior to working with an inducible promoter system, it is important to assess whether the inducer compount might positively or negatively affect growth or viability of the cell. This protocol describes the methodology for toxicity assays in the unicellular cyanobacterium Synechocystis sp. PCC 6803 to quantify its growth in the presence of different concentrations of inducer.

Inducible promoters are an important tool for synthetic biology. They enable temporal control of gene expression, as well as controlled expression of toxic genes. This protocol describes the methodology for fluorescence-based reporter assays in the unicellular cyanobacterium Synechocystis sp. PCC 6803 to quantify the dose-dependent response of an inducible promoter to its inducer.