A Long-Range Acting Dehydratase Domain As The Missing Link For C17-Dehydration In Iso-Migrastatin Biosynthesis And Cross-over Regulation

Iso-migrastatin belongs to the glutarimide-containing polyketide family of natural products;members of this family include migrastatin,dorrigocin A,13-epi-DGN A,DGN B,lactimidomycin,cycloheximide,streptimidone,and 9-methylstreptimidone.The glutarimide-containing polyketides exhibit a multitude of biological activities.Migrastatin was found to inhibit tumor cell migration and has served as an anti-metastatic drug lead.The dorrigocins appear to inhibit a carboxylmethyltransferase involved in the processing of Ras-related proteins.Except for acting as potent inhibitors of tumor cell migration,LTM can block the trans-location step in eukaryotic protein translation initiation to display cytotoxicity.CHX can inhibite the transcription of gene to a protein in eukaryotic organisms.So LTM and CHX had been developed as biomedical research tools to study protein synthesis in eukaryotic cells in vitro.In a word,the compounds of this family are potent for drug development.But so far.there are still many unsolved problems,one of the most important is the identification of the gene related to dehydration of the C-17 hydroxy group of iso-migrastatin.Because the gene clusters of iso-migrastatin and lactimidomycin,which are very similar,could produce two diferent type compounds with or without the C17 hydroxy group.Moreover,this C17 hydroxy group is important for the bioactivity,which can increase the IC50 from ?M to nM.Hence,it is an urgent problem needed to be solved.To solve this problem,we use pBS11001,the BAC which contains the intact mgs gene cluster,as a research object and heterologous expressed the gene cluster to study the missing DH domain for dehydration of the C-17 hydroxyl group.Based on previous study of mgs gene cluster and biosynthesis,three domains,located in module 6,module 9 and module 11,were chosen as the candidates for the C17 dehydration and the active sites of these DH domains were mutated to abolish the function of these DHs.After the identification of expected mutant strains,it showed that only DH9 and DH11 mutant strains generated novel compounds and DH6 mutant strain completely abolished the production of iso-migrastatin.However,we still cannot correlate the DHs with the C17 dehydration.It suggested that biosynthesis of iso-migrastatin is not correspond to previous study.Thus,the DH domain in module 10 became our last candidate.In previous study of mgs gene cluster,this DH domain was identified as a non-functional domain,due to the loss of conserved HxxxGxxxxP motif.However,after much closer check of the sequence,we found this DH domain actually contain an extra loop,which separate the conserved HxxxGxxxxP into two part.After confirming that the DH10 domain is a functional domain,we carried out the site-directed mutagenesis to abolish the function of DH10 again.Fermentation of this DH10 mutant strain indicated three C17 hydroxyl iso-migrastatin analogous.Thus,the missing dehydratase domain was identified from mgs gene cluster.Moreover,we did the DH10 and DH11 double mutant to generate three new analogous for further experiment.In order to understand the timing of the DH10 domain for working on the C17 hydroxyl group,beyond the in vivo experiment,we carried out the in vitro experiment to answer the question.Thus,we overproduced and purified DH10 to homogeneity and demonstrated its ability to catalyze dehydration of two candidates in vitro.Two compounds were chosen as the potential substrates of DH10,they are the proposed intermediates that attached to module 4 and module 10 of mgs biosynthetic pathway.The compound for module 4 was obtained by chemical synthesis,while the compound for module 10 was isolated from both DH11 and DH10 mutant strain.The MGSDH10 can only catalyze dehydration of long chain compound and showed no activity using short chain compound as an alternative substrate.The kinetic study showed Km's for 21 at 7.9 ±0.75 mM and apparent Kat's at 10.35 ± 0.47 min-1.Finally,we test DH10 with a full-length substrate with C-3,17-OH moieties,to demonstrate whether DH10 can specifically catalyze the distant ?-dehydration or can also catalyze the normal ?-dehydration.Surprisingly,It showed that the DH10 is capable of catalyzing the dehydration of both C3 and Cl 7 hydroxyl group.But DH10 still prefers the distant Cl7 hydroxyl group.These findings provided direct evidence,further supporting the DH10 catalyzed the dehydration of C-17 hydroxyl group,and moreover,the dehydration was carried out on module 10,featuring an unusual DH domain.Furthermore,Comparison of iso-MGS,LTM,CHX gene clusters,two regulator mgsA and chxA were identified from iso-MGS gene cluster and CHX gene cluster respectively.However,the gene cluster of LTM do not contain any regulator.In previous experiment,the mgsA deletion mutant strain cannot yield MGS;Using RT-PCR,we also can not detect the transcription of mgsA in heterologous host.Thus,we think it is a chance for us to study the regulation of mgsA and chxA.So,based on high copy vector pKC1139 and pUWL201PW,we constructed several plasmids for overexpression of mgsA and chxA.After the fermentation and HPLC analysis,it showed that the overexpression of mgsA and chxA only improved the yield of iso-MGS about 10%.For CHX production,it is the same like the case of MGS,mgsA and chxA cannot significantly increase the titer of CHX.For LTM producer,specifically,overexpression of mgsA and chxA both significantly improved the production of ltm(about four-fold)compared with the wild-type strain.The titer was increased from 25 mg/L(WT)to 106 mg/L(overexpression of mgsA or chxA).