The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi.It is one of the important effective components in Agr...The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi.It is one of the important effective components in Agricultural Antibiotic120,which has been widely used as naturally-originated agents for treatment of crop decay in China.The chemical synthesis of anisomycin has recently been reported,but the complex process with low productivity made the biosynthesis still to be a vital mainstay in efforts.The biosynthetic gene cluster(BGC)of anisomycin in Streptomyces hygrospinosus var.beijingensis has been identified in our previous work,while poor understanding of the regulatory mechanism limited the yield enhancement via regulation engineering of S.hygrospinosus var.beijingensis.In this study here,we characterized AniF as an indispensable LuxR family transcriptional regulator for the activation of anisomycin biosynthesis.The genetic manipulations of aniF and the real-time quantitative PCR(RT-qPCR)revealed that it positively regulated the transcription of the anisomycin BGC.Moreover,the overexpression of aniF contributed to the improvement of the production of anisomycin and its derivatives.Dissection of the mechanism underlying the function of AniF revealed that it directly activated the transcription of the genes aniR-G involved in anisomycin biosynthesis.Especially,one AniF-binding site in the promoter region of aniR was identified by DNase I footprinting assay and an inverted repeat sequence(5′-GGGC-3′)composed of two 4-nt half sites in the protected region was found.Taken together,our systematic study confirmed the positive regulatory role of AniF and might facilitate the future construction of engineering strains with high productivity of anisomycin and its derivatives.展开更多
The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and con...The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.展开更多
基金Special Fund for Agro-scientific Research in the Public Interest(201003066)earmaked fund for Modem Agroindustry Technology Research System(CARS-26)Agricultural Science and Technology Innovation Program(ASTIP)
基金the National Natural Science Foundation of China(31630002,31700029,31770038,31470183,21661140002 and 31170085)the Ministry of Science and Technology,China+1 种基金Shanghai Pujiang Program from the Shanghai Municipal Council of Science and Technology(12PJD021)China Postdoctoral Science Foundation(2017M620151).
文摘The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi.It is one of the important effective components in Agricultural Antibiotic120,which has been widely used as naturally-originated agents for treatment of crop decay in China.The chemical synthesis of anisomycin has recently been reported,but the complex process with low productivity made the biosynthesis still to be a vital mainstay in efforts.The biosynthetic gene cluster(BGC)of anisomycin in Streptomyces hygrospinosus var.beijingensis has been identified in our previous work,while poor understanding of the regulatory mechanism limited the yield enhancement via regulation engineering of S.hygrospinosus var.beijingensis.In this study here,we characterized AniF as an indispensable LuxR family transcriptional regulator for the activation of anisomycin biosynthesis.The genetic manipulations of aniF and the real-time quantitative PCR(RT-qPCR)revealed that it positively regulated the transcription of the anisomycin BGC.Moreover,the overexpression of aniF contributed to the improvement of the production of anisomycin and its derivatives.Dissection of the mechanism underlying the function of AniF revealed that it directly activated the transcription of the genes aniR-G involved in anisomycin biosynthesis.Especially,one AniF-binding site in the promoter region of aniR was identified by DNase I footprinting assay and an inverted repeat sequence(5′-GGGC-3′)composed of two 4-nt half sites in the protected region was found.Taken together,our systematic study confirmed the positive regulatory role of AniF and might facilitate the future construction of engineering strains with high productivity of anisomycin and its derivatives.
基金supported by grants from the Ministry of Science and Technology of China (2012CB721005, 2012AA02A706, 2011ZX08009-001, 2012AA022107)the National Natural Science Foundation of China (31070070, 31121064)+1 种基金Ministry of Education of China (20110073110048)the SJTU-UM Collaborative Program
文摘The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.
