Boosting ascomycin production by n-butanol addition in fermentation of Streptomyces hygroscopicus var. ascomyceticus ATCC 14891

Ascomycin (FK520) is a macrolide antibiotic known for its immunosuppressive activities. In this study, we screened several short-chain alcohols for their addition in fermentation of Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 to enhance the FK520 titer, particularly focusing on n-butanol addition. Optimizing the addition of 0.8% n-butanol at 27 h led to a 1.72-fold increase in FK520 yield, reaching 569.4 mg/L. This addition also elevated reactive oxygen species levels by 3.02 times compared to the control group. Analysis of CoA-esters showed significant increases in FK520 biosynthesis precursors at multiple time points, including ethylmalonyl-CoA, methylmalonyl-CoA, and malonyl-CoA. Notably, the highest ethylmalonyl-CoA content marked a 5.3-fold increase at 48 h, while the highest methylmalonyl-CoA and malonyl-CoA levels increased 4.33-fold and 3.33-fold at 72 and 120 h, respectively. Gene expression analysis via quantitative real-time RT-PCR revealed that the addition of n-butanol significantly elevated genes expression associated with the biosynthesis precursors of FK520 (fkbO and pcc), the positive regulatory gene fkbN, and the structural genes involved in FK520 biosynthesis (fkbA and fkbB). This indicates that the addition of n-butanol not only facilitates the accumulation of FK520 biosynthesis precursors but also enhances its utilization of related precursors to synthesize the secondary metabolite FK520, thereby resulting in an increased yield of FK520.