Enhancement of FK520 production in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 by overexpressing the regulatory gene fkbR2.

Abstract

Ascomycin (FK520) is a 23-membered macrolide antibiotic primarily produced by the Streptomyces hygroscopicus var. ascomyceticus. Structurally similar to tacrolimus and rapamycin, it serves as an effective immunosuppressant widely used in the treatment of rejection reactions after organ transplantation and certain autoimmune diseases. Currently, FK520 is mainly produced through microbial fermentation, but its yield remains low. Since the gene fkbR2 is a regulatory gene within the FK520 biosynthesis gene cluster that has not been studied, this paper focuses on the overexpression of the gene fkbR2 in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (WT). By constructing a strain with overexpressed fkbR2 gene, we initially obtained a high-yield strain R2-17 through shake flask fermentation, with a 28% increase in yield compared to WT. In the process of further improving the stability of the high-yield strain, this paper defines two indices: high-yield index and stability index. After two consecutive rounds of natural breeding, strain R2-17 achieved a high-yield index of 100% and a stability index of 80%. Finally, the high-yield strain R2-17-3-10 was successfully screened, and the yield was increased by 34% compared with the strain WT, reaching 686.47 mg/L. A comparative analysis between the high-yield strain R2-17-3-10 and the original strain WT revealed differences in fermentation process parameters such as FK520 synthesis rate, pH, bacterial growth, glycerol consumption rate, ammonia nitrogen level, and ammonium ion concentration. In addition, the transcription levels of genes involved in the synthesis of precursors 4,5-dihydroxycyclohex-1-enecarboxylic acid (fkbO), ethylmalonyl-CoA (fkbE, fkbU, fkbS), and pipecolic acid (fkbL), as well as pathway-specific regulatory genes (fkbN, fkbR1), were significantly increased at different time points in the high-yield strain R2-17-3-10. EMSAs analysis showed that the FkbR2 protein could not bind to the promoter region of above genes. This suggests that the gene fkbR2 may enhance the supply of FK520 synthetic precursors by indirectly regulating the transcription levels of these genes, thereby promoting an increase in FK520 production. These results demonstrate that modifying genes within the biosynthetic gene clusters of natural products can be successfully applied to increase the yields of industrially and clinically important compounds. However, it is found that fkbR2 gene is a regulatory gene that has not been fully studied, and it is worth further studying its regulatory mechanism.