Abstract: To address microbial contamination in industrial fermentation, we constructed an engineered Bacillus subtilis strain expressing the phosphite dehydrogenase gene (ptxD) as an antibiotic-free selection marker, enabling contamination-resistant fermentation under non-sterile conditions. Using “plug-and-play” prolonged overlap extension-polymerase chain reaction (POE-PCR), a ptxD expression vector was constructed; promoter optimization was used to enhance the efficiency of phosphite metabolism, resulting in the engineered strain growing 1.56 times faster than the original strain in the phosphite medium. After 30 generations of adaptive evolution, the strain exhibited further enhanced phosphite metabolism and a 1.34-fold increase in growth rate. After elimination of the kanamycin resistance gene, the resulting antibiotic-free strain stably grew on phosphite as the sole phosphorus source. The validation under non-sterile fermentation conditions showed uniform cellular morphology by microscopy and gradient plating, and a single band in 16S rRNA PCR amplification with > 99% homology. The results of high-throughput sequencing confirmed that after 72 h of fermentation in an unsterilized phosphite medium, the relative abundance of Bacillus reached up to 98.77%. This ptxD-based system enables non-sterile fermentation without the need for high-temperature sterilization or antibiotics, offering an environmentally friendly solution for industrial biomanufacturing.

Key words: Bacillus subtilis; phosphite dehydrogenase; antibiotic-free selection; non-sterile fermentation