| Antibiotics are the compounds that play an important role in human health.Rapamycin,which is known as "magic medicine",is a class of macrolide compounds produced by microorganisms.It has a variety of biological and pharmaceutical activities,including anti-fungal activities,immunosuppressant activities,anti-tumor and resisting skins senility.However,some clinical researches reported that rapamycin could not completely inhibit mTORC1 banding to downstream targets.The negative feedback signal will active the PI3K-AKT signal pathway and increase the survival rate of the cancer cells when rapamycin binds to mTORC1.In recent years,although some researches have modified the carbon C37-C42 of six-member ring of the rapamycin to obtain different rapalogues,few rapalogue has been found to reduce negative feedback when binding to mTORC1.So,it is of great significance to develop novel rapalogues.Glucopeptide antibiotics(GPAs)as the last defence in treatment of varieties of bacterial infection diseases in the past have more prominent advantages in treatment of disease causing by drug-resistant bacteria or pathogenic diseases,and it can eliminate the bacterial drug resistance by its regulating mechanism,therefore exploring more sugar peptide compounds also has the very broad application prospects.At present,most modifications of rapamycin focus on the 4-hydroxy-3-methoxycyclohexyl group,while the piperidyl of rapamycin was rarely modified.In this research,rapamycin-producing bacteria(GIM4.111)was used as the starting strain to knock out the rapL gene that is the biosynthesis gene of piperidine acid which is required by synthesis of rapamycin.At the same time,feeding piperidine acid analogue(piperazic acid)to get rapalogue with the piperazic acid incorperated.At present,rapL knockout strain has been successfully obtained on the basis of GIM4.111,and piperazic acid has been added to rapL knockout strain for fermentation detection.Simultaneously,ktzI-ktzT has been integrated into rapL knockout strain,and fermentation detection is in progress.In addition,rapL knockout strain integrating rapP*has been obtained and piperazic acid was added to its fermentation for detection.It is expected to obtain novel rapalogue.In the aspect of genome mining about GPAs,We found that X-domain was a crucial conservative domain existing in all GPAs,and the potential novel GPA biosynthetic gene clusters were found in the actinomycetes library of our laboratory by means of using the X-domain as the probe.Among the library,LS2102 is a potential novel nine-peptide GPA-producing strains through establishing and analysising phylogenetic tree.Based on above researches,we intend to knock out the key gene cluster that encoding GPA in LS2102 and to find the novel GPA according to the differential peak orientation strategy,gene cluster cloning and heterologous expression.At present,the knockout strain of key gene cluster has been obtained,meanwhile this gene cluster also cloned successfully which can be used for heterologous expression and fermentation detection to find the novel GPA.In summary,based on genetic engineering and genome mining strategies,an environment suitable for the expression of novel rapalogue and GPA was established successfully by genetic manipulation of GIM4.111 and LS2102,which is expected to obtain rapalogue and novel nine-peptide GPA.In the future,these original compounds can be further developed and applied in clinical treatment,thus helping to solve the current serious drug-resistant bacteria problem. |
