Enhancement of tacrolimus production in Streptomyces tsukubaensis by genetic engineering and process optimization

Tacrolimus, an important immunosuppressive drug widely used in clinical practice, is produced by Streptomyces tsukubaensis. In order to enhance the strain productivity, a high-yield mutant strain, 2108N-1-4, was obtained through natural isolation, screening and physical mutagenesis form S. tsukubaensis NRRL 18488. Transcriptome analysis was conducted to identify the potential modification targets, among which the overexpression of regulatory gene fkbN was found to significantly improve tacrolimus production. To further optimize the expression of fkbN through altering promoter strength and increasing the gene copy number, a strain 2108N-1-4/DfkbN carried three copies of fkbN were constructed. The engineered strain achieved a tacrolimus yield of 1342 mg/L. Additionally, it was found that in the late fermentation phase, high concentration of tacrolimus inhibits both the growth of the strain and the expression of genes within the tacrolimus biosynthetic gene cluster. To address this issue, adsorbents such as LX-60 and β-cyclodextrin were added in the fermentation medium to reduce the tacrolimus concentration. This approach increased the tacrolimus yield to 3746 mg/L in flask shake culture and 3639 mg/L when up-scaled to a 5 L fermenter. This study significantly improves tacrolimus production through strain mutation breeding, genetic engineering and process optimization, achieving the highest production yield reported to date. These findings provide valuable insights for the industrial-scale production of tacrolimus.