Insights into guanidine assimilation, its regulation and utilization to drive synthetic processes in cyanobacteria

Cyanobacteria receive increasing attention in bioeconomy research as they could be used as whole-cell biocatalysts for a light-driven, CO₂-neutral and hence, sustainable production of chemicals and fuel components. The realization of this potential requires deep understanding of cyanobacterial metabolism and its regulation as well as the simultaneous development of molecular tools for metabolic engineering. Here, we provide insights into the assimilation of guanidine via a recently identified guanidine hydrolase (GdmH) [1]. The corresponding gdmH gene is widely distributed among cyanobacteria and allows them to grow on guanidine as sole nitrogen source. Consistently, strains lacking gdmH, either naturally or by gene deletion, do not grow on guanidine. In the model strain Synechocystis sp. PCC 6803, the expression of gdmH is controlled at the transcriptional level by NtcA that mediates transcriptional activation in response to nitrogen limitation. Moreover, a conserved RNA motif is present in the 5"UTR that forms the aptamer of a guanidine I riboswitch. Via in-line probing we demonstrate specific and high affinity binding of guanidine to the aptamer. In vivo, the riboswitch enabes guanidine-specific and titratable induction of gene expression. In addition, the gdmH gene is frequently co-localized with ABC transporter genes. Their mutation also interferes with guanidine-dependent growth of Synechocystis pointing towards a guanidine-specific substrate binding protein in the periplasm. However, this interference can only be observed in presence of rather low guanidine concentrations between 0.1 and 0.5 mM. At concentrations >1 mM the mutants grow similar to WT indicating the presence of additional, yet to be identified guanidine uptake systems. In addition, we made use of the guanidine riboswitch to achieve precise and dynamic regulation of heterologous gene expression in cyanobacteria. As guanidine is quite cheap and the required guanidine concentrations are rather low, guanidine riboswitches enable gene expression control at large scale without significant costs for inducers. Altogether, our findings not only contribute to the understanding of cyanobacterial metabolism, but also highlight the potential of guanidine riboswitches as valuable tool for synthetic biology applications in cyanobacteria.

[1] Funck, D. […], Hartig, J.S., 2022. Discovery of a Ni²⁺-dependent guanidine hydrolase in bacteria. Nature 603, 515–521.