Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

Automated summary

Biological nitrogen fixation is a crucial process that converts atmospheric nitrogen into bioavailable ammonia, but its engineering requires a comprehensive understanding of the gene regulatory dynamics and sequence-function space of nitrogenase, the central catalytic metalloenzyme. In this study, we analyzed the transcriptome of Azotobacter vinelandii engineered with a genetically inferred ancestral nitrogenase protein variant to investigate this relationship. Our results showed resilient expression of genes within the nitrogen fixation network despite nitrogenase sequence-level perturbations, while reduced expression of genes supporting trace metal and electron resource allocation to nitrogenase accompanied the sustained physiological compatibility with the ancestral nitrogenase variant. These findings provide important insights for the engineering of nitrogenase proteins and host diazotrophs to improve compatibility.