Studies On Biosynthesis Of 2'-Chloropentostatin,2'-Amino-2'-Deoxyadenosine And 5-Methy Luridine

2'-chloropento statin(2 '-Cl PTN,2 '-chloro-2 '-deoxycoformycin)and 2 '-amino-2'-deoxyadenosine(2'-amino dA)are two adenosine-derived nucleoside antibiotics coproduced by Actinomadura sp.ATCC 39365.2'-Cl PTN is a potent adenosine deaminase(ADA)inhibitor,featuring an intriguing 1,3-diazepine ring,as well as a chlorination at C-2' of ribose.It plays a key role in the protection of 2'-amino dA from deamination to form 2'-amino dl and was also clinically used in the treatment of blood cancers.2'-amino dA shows a distinctive bioactivity against RNA-type virus infection such as measles virus.But the biosynthetic logic of both two nucleosides has been pending.As suggested by in silico analysis,using Pentostatin biosynthesis genes PenA,PenB,PenC as probes conducting BLASTP analysis against the genome of Actinomadura sp.ATCC 39365,leads to the location of the candidate 2'-Cl PTN and 2'-amino dA biosynthetic gene cluster(ada),and deeper investigation of relating genes results in the unveiled of the genetic organization of the ada gene cluster.For further examination,a regin covering adaJKLM were deleted to give LG1,the results demonstrated the mutant lost the ability to generate 2'-Cl PTN,PTN or '-amino dA,consequently identified the target gene cluster is simultaneously resoponsible for the biosynthesis of 2'-Cl PTN,PTN and 2'-amino dA.Later,a postitive Cosmid 3G12 housing the ada gene cluster was successfully screened from the genomic library of Actinomadura sp.ATCC 39365 and subsequent engineered production of 2'-Cl PTN,PTN and 2'-amino dA in heterologous host S.aureochromogenes CXR14 was also realized.In addition,minimal 13-gene cluster for the biosynthesis of 2'-Cl PTN and 2'-amino dA was determined before conducting in-frame deletions of all the related genes on 3G12 via PCR-targeting strategy,which revealed 2'-Cl PTN and 2'-amino dA arise from independent biosynthetic pathways.We provide biochemical proofs that the biosynthesis of 2'-amino dA is initiated by Nudix hydrolase AdaJ,which catalyzes the hydrolysis of ATP to AMP with release of a pyrophosphate.And we speculate AdaE(a proposed cation/H+ antiporter)might be related to chlorination of 2'-Cl PTN.We also carried out in vitro studies on AdaF,AdaG,AdaM in order to elucidate their function.Furthermore,we confirmed that the adenosine deaminase ADA1 is capable of catalyzing the deamination of 2'-amino dA but relatively insensitive to the inhibition of PTN.And followed by combining the biosynthetic pathway of Pentostatin,we proposed and correct the biosynthetic pathways of 2'-Cl PTN and 2'-amino dA.Beginning from the end of last century,when 5-methyluridine was successfully used in the synthesis of anti-HIV drugs like AZT and d4T for the first time,much more attention has been paid to it all over the world,and it has become an important precursor for the treatment of AIDS.In addition,5-methyluridine also used as a raw material for the synthesis of thymidine,but it does not exist in nature.At present,the synthesis of 5-methyluridine in industry is still dominated by chemical synthesis,polluting the environment with relatively low production and little room for improvement.Under laboratory conditions,the application of enzymatic strategy to obtain 5-methyluridine has been realized,but the comparatively high production costs with quite tough reaction conditions,not only affects the 5-methylurdine final yield enhancement,but large-scale industrial application is therefore limited.Here according to bioinformatics analysis,the alkaline phosphatase gene(phoA)was cloned from E.coli BL21(DE3),and examed its activity in vitro.With the help of pRSFDuet-1,combine phoA and UMP C-5 methylation gene(polB),a 5-methyluridine produce vector pYJ023 was constructed successfully,wish to deliver exogenous 5-methyluridine biosynthetic pathway into E.coli.But initially when introduce it into BL21(DE3)we failed to detect the 5-methyluridine.After that three nucleoside hydrolase RihC,RihB and RihA,thymidine phosphorylase DeoA,uridine phosphorylase Udp,thymidine kinase Tdk and uridine kinase Udk,which might be associated with the metabolic of 5-methyluridine,were studied respectively in vitro,and we found that in addition to Udk cannot catalyze corresponding reaction of 5-methyluridine,rest of the proteins are all shown to be probably involved in the metabolism of 5-methyluridine in vivo.Based on these in vitro biochemical results and in silico analysis,GYJ1 was obtained by frame interrupting rihC at the chromosome level of E.coli.After delivering pYJ023 into the mutant GYJ1,the engineering product of 5-meththyluridine was successfully achieved.Inspired by this and expect to improve the final yield of 5-methyluridine,based on GYJ1,rihA interrupted mutant GYJ2 was then obtained,next based on GYJ2,deoA interrupted mutant GYJ3 was gained.Subsequently udp and tdk was interrupted sequentially to get correspondingly GYJ4 and GYJ5.Finally,we obtained maxium 5-methyluridine in GYJ5.In short,we combined metabolic engineering with synthetic biology,for the first time successfully developed a rational optimized model strain of E.coli to metabolically produce 5-methyluridine.