Mechanism Of Chain Release In The Biosynthesis Of Polyther Antibiotic In Streptomyces Nanchangensis

Streptomyces nanchangensis NS3226 was a new Streptomycete that was isolated from the rhizosphere soil of a tea plant in the Meiling Mountain in Jiangxi Agriculture University in 1970s. Early research discovered that this strain at least produced two antibiotics, one is the polyether antibiotic nanchangmycin, and the other is the 16-membered macrolide antibiotic meilingmycin. Nanchangmycin (also known as dianemycin), has been found to inhibit gram-positive bacteria and to cure coccidiosis in chickens. More recently polyethers have been identified as agents with activity against drug resistant strains of malaria and HIV virus. Meilingmycin has the same core structure with the world famous antibiotic avermentin, but they have different side chain group, so they have similar bioactivity, and in some cases, meilingmycin even better than avermectin. The biosynthesis gene cluster of nanchangmycin has been cloned and fully sequenced, and the sequence gave us a lot of information to know how the polyether has been synthesized.By analyzing sequence of several known gene cluster of polyether, the deduced genetic organization indicated that the polyketide skeleton of nanchangmycin was assembled by large modular polyketide synthases. Interestingly, the modular polyether PKS appears to include a type I thioesterase domain of the type usually located at the C terminus of the last polyketide module. The two candidates CR domain and nanE in the nanchangmycin biosynthesis gene cluster have been deleted in frame and complemented, and used type I thioesterase AVER TE and DEBS TE to replace CR domain on the chromosome. In vivo experiments indicated that both the putative CR domain and the nanE appeared to be genetically relevant, and type I DEBS TE and AVER TE couldn't replace the CR domain. Among the three heterologously expressed soluble proteins (recombinant CR domain, the ACP-CR didomain, and NanE) tested, only NanE hydrolyzed the polyether-SNAC. By contrast, recombinant DEBS TE from the erythromycin pathway, and the recombinant MonAX, a type II TE associated with the polyether monensin biosynthesis for which a homolog has not been detected in the nanchangmycin cluster, hydrolyzed a diketide-SNAC but not the polyether-SNAC. We could thus conclude that NanE is a dedicated thioesterase mediating the specific release of the polyether chain during nanchangmycin biosynthesis.Site-Directed Mutagenesis has been performed to locate the active sites of this thioesterase, which proved that S96, D120 and H261 was the catalytic triad, and also identified W97 as a very important amino acid in helping this thioesterase choose its substrate and adjust its activity. The glycosyltransferase (nanG5) has been interrupted by an aparmycin resistance gene to accumulate the nanchangmycin aglycone. In theΔnanG5 mutant, the production of nanchangmycin aglycone is much lower than the production of nanchangmycin in the wild-type. Several polyether-SNACs of similar structure have been synthesized to test the substrate tolerance of NanE. It was found that NanE can hydrolyze the nanchangmycin-SNAC, nanchangmycin aglycone-SNAC and monensin-SNAC to generate the corresponding polyether, but it could not react with salinomycin-SNAC. From the results of the steady state kinetic analysis of NanE toward the SNAC substrates, nanchangmycin-SNAC was found to be the best substrate for NanE. Both in vivo and in vitro results indicated that the glycosyltransferase attachment of the sugar on nanchangmycin aglycone happens on the PKS prior to the Chain Release.Crystallization of NanE has been tried. The removal of His6-tag is good for crystal structure in the other cases of thioesterases. Thrombin, the first choice was used to remove the His6-tag from NanE, can cut the NanE into two pieces in the middle of NanE in this case. Finally protease HRV 3C were uesd to remove the His-tag from NanE very efficiently and specifically. The work for getting the crystal structure is in the process in our collaborated lab.The protein-protein interaction of NanE with ACP13 and ACP14- CR did not show that the obviously interaction between the NanE and ACPs, and further experiment need to be done to prove it.For understanding of mechanism of converting polyketide to polyether, soluble protein NanO has been got from E. coli, and try to use (±)-linalool to mimic the nature substrate of NanO, but only very low yield of product has been detected. Moreover, nanI gene has been interrupted by apramycin resistance gene, as the result, the production of nanchangmycin in the mutant is much lower than the wild type. A method of using degenerated primers to clone homologous genes of NanO to locate the novel polyether gene cluster has been developed. Compared with the traditional way that use heterogeneous PKS probe to hybridize the genomic library by southern, this method is very fast and convenient to clone the new polyether gene cluster.For understanding the confirmation of two double bonds of pre-nanchangmycin, NANS module3+TE and NANS module7+TE have been cloned into pET28a with N-terminal His6-tag, and these macroproteins (more than 200KDa) were successfully expressed as soluble protein, and showed the activity to substrate of di-ketide-SNAC. Therefore, for deeply understanding how the module work in the polyketide chain elongation, NANS Module 2 + TE has been successfully expressed as soluble protein. Modelu 2 + TE is a first full module, it included KS, AT, DH, ER, KR, ACP and DEBS TE domain. It will be a wonderful material to research DH, ER domain and stereochemistry of KR domain.The left side of existing Meilingmycin gene cluster has been deleted 20 kb to confirm that this region is involved with the biosynthesis of meilingmycin. As the result, meilingmycin still exist when this big fragment (including PKS gene) was deleted, so the left side genes didn't involve with the biosynthesis of meilingmycin. By constructing a 7-9 kb SacI fragment subclone library to chromosome walking to the right side, 4 positive subclones have been got, and one of them named 4H1 has been sequenced. The information from bioinformatics indicated these genes may be responsible for the biosynthesis of side chain of meilingmycin. Continuing to chromosome walking to the right side, the cosmid 9B8 has been got. This cosmid has been fully sequenced, though it completed the gene cluster of the biosynthesis of side chain of meilingmycin, the PKS part (including Loading module, module 1 and module 2) is still missing. According to the sequence of ER domain in module 2 in milbemycin gene cluster to design the primer, a highly homologous fragment has been got from genome DNA of S. nanchangensis. The cosmid 8B3 has been got by using the same primer from cosmid library S. nanchangensis. By deletion of 30 kb fragment covering by 8B3, the mutant lost the ability to produce any components of meilingmycin. It means that the meilingmycin biosynthesis gene cluster has been completely cloned.