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.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall (CW) layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of β-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counterdefensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.

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 CO 2 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 CO 2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrosedependent 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 CO 2 as the primary inputs.

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