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

Ascomycin (FK520) is a 23-membered macrolide antibiotic primarily produced by the Streptomyces hygroscopicus subsp. ascomycetoides. Structurally similar to tacrolimus and rapamycin, it serves as an effective immunosuppressant widely used in the treatment of rejection reactions following 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 biosynthetic gene cluster that has not been studied, this paper focuses on the overexpression of the regulatory gene fkbR2 in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (WT). By constructing a strain with overexpressed fkbR2 gene, we initially obtained a high-yielding 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-yielding strain, this paper defines two indices: high-yield index and stability index. After two consecutive rounds of natural selection, strain R2-17 achieved a high-yield index of 100% and a stability index of 80%, ultimately leading to the selection of a stable high-yielding strain R2-17-3-10 with a 34% increase in yield compared to WT, reaching 686.47 mg/L. A comparative analysis between the high-yielding strain R2-17-3-10 and the original strain WT revealed differences in fermentation process parameters such as FK520 synthesis rate, pH, bacterial concentration, glycerol consumption rate, amino nitrogen level, and ammonium ion concentration. Additionally, the transcription levels of genes involved in the synthesis of precursors DHCHC (fkbO), ethylmalonyl-CoA (fkbE, fkbU, fkbS), and piperidine acid (fkbL), as well as pathway-specific regulatory genes (fkbN, fkbR1), were significantly increased at different time points in the high-yielding strain R2-17-3-10. This suggests that the gene fkbR2may enhance the supply of FK520 synthetic precursors by regulating the transcription 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 may be a regulatory gene that has not been fully studied, and it is worth further studying its regulatory mechanism.