The Study On The Polyene Antibiotics FR-008 Biosynthesis Cascade Regulatory Network In Streptomyces Sp.FR-008

Streptomyces is a common type of microorganisms in nature that can synthesize some secondary metabolites with clinical application significance,such as anti-infective drugs,antibiotics,antiviral agents,etc.Most of the drugs with antifungal activity are polyene antibiotics,such as type I polyketone macrolides.Polyene antibiotics can specifically combine with ergosterol(the main component of fungal cell membranes)to form channels,leading to the leakage of intracellular bacteria,thereby achieving a bactericidal effect.With the rapid development of DNA sequencing technology,the whole genome sequence of many common Streptomyces has been reported.According to bioinformatics analysis,it can be known that the genes responsible for antibiotic biosynthesis usually come together to form a biosynthetic gene cluster(BGC),including structural genes and regulatory genes.BGC controls secondary metabolism at different levels,and the most common control method is the regulatory cascade formed by the regulatory genes inside and outside the cluster.Streptomyces secondary metabolic regulation system is very precise and complicated for regulation of antibiotic biosynthesis,especially for BGC with multiple regulatory genes.Streptomyces sp.FR-008 can produce polyene antifungins FR-008,and its BGC contains four regulatory genes fscRl,fscR2,fscR3 and fscR4,these genes encode for regulatory proteins from different families,and form subclusters.The synonymous arrangement of these regulatory genes is widely distributed in different polyene antifungal gene clusters,indicating that the regulatory mechanism of these antibiotics biosynthesis is highly conservative.However,the relationship between these regulatory genes is still unknown.Previously,relevant literature has reported that the regulatory genes fscRl and fscR4 are essential for the production of the polyene antibiotic FR-008.This study further characterized the roles of the other two regulatory genes fscR2 and fscR3,and focused on analyzing the cascade regulatory network between these four regulatory genes.Firstly,gene deletion was performed on Streptomyces sp.FR-008 by means of homologous recombination technology in this study.Four deletion mutant strains of? fscR2,?fscR3,?fscR2-3 and ?fscRl--1 were constructed.Using transcriptional analysis,bioassay,and HPLC analysis,it was confirmed that the knockout of single or multiple regulatory genes does not affect the growth of Streptomyces.However,the expression level of genes in FR-008 biosynthetic gene cluster decreased,and FR-008 synthesis was blocked,which indicated that all four regulatory genes were indispensable in FR-008 biosynthesis.In this study,the Realtime-PCR of Streptomyces sp.FR-008 revealed that fscRl-fscR4were differentially expressed throughout the growth phase,but it showed a similar temporal expression pattern,that is the relative expression suddenly increased to the highest level in the early exponential phase.This conclusion is consistent with the experimental results of biological activity determination.In addition,the results of Realtime-PCR of six deletion strains(fscR1??fscR2??fscR3??fscR4??fscR2-3 and ?fscR1-4)showed that the four regulatory genes fscRl-fscR4 have different degrees of control over the structural genes in BGC,and they are superimposed.The results of comprehensive cross-complementation experiments show that functional complementation occurs only between certain regulatory genes and is unidirectional.That is,fscRl can functionally compensate for the loss of fscR2 or fscR3,fscR3 can functionally compensate for the loss of fscR2,but the contrary is not true.Finally,based on the above cross-complementation and transcription analysis results,a clear cascaded regulatory network that exists between regulatory genes and controls the biosynthesis of FR-008 and other potential polyene antibiotics is analyzed.This study provides new perspectives and insights into the role of multiple regulatory genes in other BGC and their cascade regulatory networks.In summary,it can be seen that the four regulatory genes fscR1-fscR4 of Streptomyces sp.FR-008 are pathway-specific regulatory genes that regulate the synthesis of polyene antibiotics FR-008 and are necessary for the synthesis of polyene antibiotics FR-008.But they do not affect the morphology,growth and maturation of Streptomyces sp.FR-008.The four regulatory genes are differentially expressed,differentially control the expression of structural genes,cross-regulate each other,functionally complement some genes,forming a complex cascade of regulatory networks during the growth of Streptomyces sp.FR-008.