Development of robust cyanobacterial strains for biotechnological applications : Stress tolerance and cell-specific expression in heterocyst-forming cyanobacteria

Abstract: Synthetic biology tools can be used to exploit potential applications of cyanobacteria, promising photosynthetic hosts for production of fuels and chemicals. Specific genetic tools are needed for the development of robust cyanobacterial strains for bioengineering. The key work presented in this thesis is a characterization and design of bioengineering tools for the heterocyst-forming cyanobacterium Nostoc punctiforme strain ATCC 29133. This multicellular cyanobacterium may express several oxidative stress-managing systems, including five Dps proteins. Two of these Dps proteins, NpDps2 and NpDps5, are involved in the tolerance against oxidative stress that induced by H2O2 or high light intensities. The capacity of NpDps2 and NpDps5 to further enhance oxidative stress tolerance, was confirmed by homologous overexpression and a constitutive strong promoter in N. punctiforme. The results show the potential of Dps proteins as tools to create robust cyanobacterial cells with improved stress tolerance. This work also establishes a Dps-mediated link among light tolerance, H2O2 scavenging, and iron homeostasis, and provides evidence on the non-redundant role of multiple Dps in multicellular cyanobacteria. To address the lack of well-defined promoters in cyanobacteria, a minimal synthetic promoter, SynDIF, was designed for heterocyst-specific expression. Promoters with 5’TCCGGA, the DIF motif, at the -35 region, have been identified to give heterocyst-specific transcription. To identify promoter elements, critical for cell-specificity, DIF promoter sequences from Anabaena PCC 7120 were used in a consensus sequence approach. The importance of the consensus regions for cell-specificity was investigated with promoter-eyfp reporters. This result provides new insights to the details of DIF promoters, which suggest that the DIF-motif, only together with the consensus or a native DIF promoter -10 region, are sufficient for heterocyst-specificity. Therefore, the DIF-35 region was (i) not independent of other promoter elements, and (ii) not sufficient for heterocyst-specific expression. Besides, the strength of the synthetic promoter was improved by including the upstream element from the native heterocyst specific promoter PNsiR1. Moreover, the SynDIF promoter is the shortest promoter ever reported to provide heterocyst-specific expression, indicating the potential of introducing this promoter in future biotechnological applications.