Non-sterile fermentation of butyl butyrate by Clostridium tyrobutyricum with an anti-microbial contamination system.

Microbial contamination is a major challenge in industrial fermentation, while conventional sterilization is energy-intensive and costly. Here, an anti-contamination system was developed in Clostridium tyrobutyricum for non-sterile production of butyl butyrate (BB). C. tyrobutyricum L319 grow on unconventional nitrogen sources, namely formamide (50 mM) and urea (20 mM), as well as phosphite (50 mM) as the sole phosphorus source. Heterologous expression of phosphite dehydrogenase (ptxD) further enhanced phosphite utilization and raised the intracellular level of reducing equivalents. The engineered strain maintained up to 88% population dominance under non-sterile conditions. In 3 L bioreactors, the engineered strain produced 0.5 g/L BB under non-sterile batch fermentation. This proof-of-concept strategy has the potential to reduce energy input and production costs by eliminating the need for sterilization and antibiotics. These findings represent an initial step toward low-cost, antibiotic-free biomanufacturing using C. tyrobutyricum as cell factory.