Co-expression of auxiliary genes enhances the activity of a heterologous O2-tolerant hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria bear great biotechnological potential as photosynthetic cell factories. In particular, hydrogenases are promising with respect to light-driven H₂ production as well as H₂-driven redox biocatalysis. Their utilization relies on effective strain design as well as a balanced synthesis and maturation of heterologous enzymes. In a previous study, the soluble O₂-tolerant hydrogenase complex from Cupriavidus necator (CnSH) could be introduced into the model cyanobacterium Synechocystis sp. PCC 6803. Due to its O₂-tolerance, it was indeed active under photoautotrophic growth conditions. However, the specific activity was rather low indicating that further engineering is required, for which we followed a two-step approach. First, we optimized the CnSH multigene expression in Synechocystis by applying different regulatory elements. Although corresponding protein levels and specific CnSH activity increased, the apparent rise in enzyme levels did not fully translate into activity increase. Second, the entire set of hyp genes, encoding CnSH maturases, was co-expressed in Synechocystis to investigate, if CnSH maturation was limiting. Indeed, the native CnSH maturation apparatus promoted functional CnSH synthesis, enabling a threefold higher H₂ oxidation activity compared to the parental strain. Our results suggest that a fine balance between heterologous hydrogenase and maturase expression is required to ensure high specific activity over an extended time period.