Abstract
Microbially mediated biological nitrogen fixation is pivotal to sustainable agricultural development. However, optimizing nitrogenase activity in native biological nitrogen-fixing bacteria has been hindered by the complexities of genetic manipulation. Heterologous expression has served as a foundational strategy for engineering next-generation nitrogen-fixing microbial agents. In this study, genomic analysis of Paenibacillus polymyxa CR1 revealed an 11 kb nitrogen-fixing (nif) gene cluster. The nif cluster was first synthesized and then assembled using ExoCET technology and finally integrated into the genome of Bacillus subtilis 168 via double-exchange recombination. RT-PCR confirmed the transcription of the nif cluster; however, no nitrogenase activity was detected in the acetylene reduction assay. A promoter replacement strategy (replacing the native promoter with Pveg) enabled B. subtilis to produce active nitrogenase. However, stronger promoters-namely, P43 and Ptp2-did not further enhance nitrogenase activity. This demonstrates that promoter selection requires balancing transcriptional strength with systemic compatibility, particularly for metalloenzymes demanding precise cofactor assembly. This is the first report describing the heterologous expression of the nif gene cluster in B. subtilis, establishing a foundation for engineering high-efficiency nitrogen-fixing biofertilizers.