Enhancement of Nitrogenase Activity in Recombinant <i>Escherichia coli</i> by Replacing Upstream Region of <i>nif</i> Operon from <i>Paenibacillus</i> Species

Recombinant Escherichia coli with nitrogen-fixation activity has been constructed using a simple approach of introducing a minimal gene set of nitrogen fixation (nif) genes along with its upstream region from Paenibacillus species; however, limitations may exist in nif expression. To understand the limitations of heterologous mass production of nitrogenase in E. coli, we evaluated this approach using various nif operons cloned from Paenibacillus species. Initial attempts revealed that, despite the high nitrogenase activity observed in the parental strains, the corresponding nif operons did not consistently function in E. coli. Further analysis revealed that certain upstream regions of the nif operons function as constitutive σ⁷⁰-dependent promoters in E. coli, and the host acquired nitrogenase activity only when the upstream region acted as a constitutive promoter. Notably, recombinant E. coli achieved high nitrogenase activity by linking a functional upstream region to the nif operon from a highly active parental strain. Additional improvements in nitrogenase activity were achieved by combining promoter replacement with synthetic biology approaches to enhance electron transfer from glucose metabolism to nitrogenase and metal cluster assembly for protein maturation. These findings provide new insights into heterologous nitrogenase production in E. coli for sustainable nitrogen fixation.