Engineering Of The LysR Family Transcriptional Regulator FkbR1 And Its Target Gene To Improve Ascomycin Production

Ascomycin(FK520),a macrocyclic polyketide natural antibiotic,displays high antifungal and immunosuppressive activity.Ascomycin and its derivatives show diverse biological and pharmacological activities,containing antispasmodic,anti-malarial,anti-fungal,nerve regeneration and functional recovery.Especially as the effected immunosuppressive agents,ascomycin and its derivatives have attracted attention for potential treatment of autoimmune disease and organ transplant rejection.However,the ascomycin production still remains a lower level,and the regulatory factors and mechanism of ascomycin biosynthesis have been poorly elucidated,which limite the applied of ascomycin.In this study,to further understand complex regulatory mechanism of ascomycin biosynthesis and improve production of ascomycin,the role of the LysR family regulator FkbR1 in ascomycin biosynthesis was explored in S.hygroscopicus var.ascomyceticus FS35.Inactivation of fkbR1 led to about 67.5% reduction of ascomycin production,which was restored by complementation of fkbR1.Overexpression of fkbR1 resulted in a 33.5% increase in ascomycin production compared with the parent strain FS35.These findings indicated that FkbR1 was a positive regulator for ascomycin production.The potential genes regulated by FkbR1 were speculated based on the transcription level of the genes located in the ascomycin biosynthetic gene cluster using RT-PCR analysis.The result revealed that the expression of fkbE,fkbF,fkbS and fkbU were downregulated obviously in the fkbR1 deletion strain,but upregulated in the fkbR1 overexpression strain.The ?-glucuronidase reporter system was introduced indicated that FkbR1 negatively regulated the fkbR1 expression.Electrophoretic mobility shift assays(EMSAs)in vitro and chromatin immunoprecipitation(Ch IP)-qPCR assays in vivo indicated that FkbR1 particularly bound to the intergenic region of fkbR1-fkbE.The factor FkbR1 directly regulated the expression of target gene fkbE and fkbF.To investigate the role of the target genes fkbE and fkbF in ascomycin production,the deletion,complementation and overexpression of fkbE and fkbF were implemented,respectively.Overexpression of fkbE resulted in a 45.6% increase in ascomycin production.Deletion of fkbE led to about 75% reduction of ascomycin biosynthesis.However,little change was observed on fkbF overexpression strain and deletion of fkbF resulted in 14.5% decrease of ascomycin production.To further enhance ascomycin production,the fkbR1 and fkbE combinatorial overexpression strain OfkbRE was constructed with the ascomycin yield increased by 69.9% to 536.7 mg/L compared with that of the parent strain.Our research provides a helpful strategy to increase ascomycin production via rationally engineering FkbR1 and its target gene.