Isolation Of New Polyketide Synthase Gene Clusters/Fragments From East China Sea And Function Analysis Of A New Acyltransfrase

Polyketide synthase(PKS) catalyze certain substrates to produce structurally different polyketides and their vast analogues.Some of them have important pharmaceutical activities.In biotechnological drug research,it is important to isolate and express new PKSs which produce bioactive compound.Because of its structure characteristics,PKS has become an ideal material for research and development of combinatorial biosynthetic drugs.The already known PKSs are still not enough to further support these researches. Scientists need more new sequences of PKS genes to enlarge the PKS gene clusters data base to get more valuable information.In this study we isolated 26 new PKS gene fragments from metagenomic DNA of a marine sediment sample in the East China Sea.Phylogenetic relations of the protein sequences of all the new KS fragments were analyzed and the originating strains were predicted;a 7981 bp partial PKS gene cluster was isolated through construction of metagenomic DNA library.We analyzed the sequence of the partial PKS gene cluster and also predicted the substrate specificity of a new acyltransferase(AT,accession number EF080951) in it through substrate binding test in vitro;an AT domain AT2(3) in module 2 of the PKS in avermectin biosynthesis enzyme system was replaced with AT-EF080951 to testify the substrate specificity of AT-EF080951 in vivo.Through this study we preliminarily established a technical platform for gene isolation, structural and functional analysis of new PKS;through substrate binding test and domain replacement we validated the function of a new acyltransferase,thus made a ground for further study in polyketide combinatorial biosynthesis.1.Metagenomic DNA extraction from a marine sediment sampleMarine environment has the most enormous phylogenetic and metabolic diversity in the Earth but remain undersampled and essentially uncharted.In this study we choose the marine sediment as target and got marine sediment samples M1～5 approximately 10 ml of each from different sites about 10 m below the surface in the ECS in September 2005.Through microbiological isolation and culture we compared the population variety of each sample and choose M1(the one with the richest varieties of microbes among them) to extract its metagenomic.Because of the abundant components in marine sediment, traditional method(e.g.phenol/chloroform) is not suitable for the sample's DNA extraction. In this study we used the soil DNA isolation kit of Mo Bio Company to extract the metagenomic DNA from marine sediment sample M1 with modifications.The length of metagenomic DNA extracted was determined though pulsed-field gel electrophoresis;the concentration was determined through ultra-violet spectrometry;we used the metagenomic DNA as the template and designed 16S rDNA primers to run PCR to verify whether the DNA makes inhibition.2.Degenerative primers design and ketosynthase gene fragments isolationThe function and sequence of ketosynthase(KS) domain is conserved in PKS,so KS domain is appropriate for degenerative primer design.The method of degenerative primer design was referred to CODEHOP PCR invented by Rose and colleagues.For primer design,conserved and appropriate motifs DPQQR and HGTGT were selected through searching the Conserved Domain Database of NCBI.The nucleotide sequence distance between two motifs ranges from 600 to 900 bp.Several primers were designed targeting the two motifs and paired resulting totally 112 pairs of primers.The metagenomic DNA extracted in Part one was used as PCR template.Because of the high GC content of the target genes(mostly higher than 80%),GC buffer of Takara company was selected in PCR.The amplicons coincided with theoretical prediction were purified and ligated into vector pMD18-T for sequencing.The sequences were analyzed through GenBank database and software of NCBI.By degenerative PCR we isolated total 15 amplicons of correct molecular size,and 3 of them were new KS gene fragments.So far, our research team has isolated 26 new KS gene fragments from marine sediment samples.By construction of phylogenetic tree,all the new KS domains were classified into 2 PKS groups:typeⅠPKS and those from hybrid or mixed PKS/NRPS enzyme complexes primarily originating from marine actinobacteria,delta-proteobacteria,cyanobateria,and beta-proteobacteria.3.Construction of marine sediment sample metagenomic DNA library and isolation of PKS gene clusters.Although new PKS gene fragments can be isolated effectively by degenerative PCR, we can not acquire the whole sequences of the gene clusters,while by construction of metagenomic DNA library we can solve this problem.Since the molecular sizes of all the already known PKS genes are not above 50 kb,fosmid vector(range of inserts,20～50 kb) is appropriate for cloning of PKS gene clusters.Therefore we choose the fosmid library kit of Epicentre Company for metagenomic DNA library construction.The template for library construction was the metagenomic DNA extracted in Part 1.The protocol was also modified in our study.By tests and formula we calculated the titer of the library and plated library clones according to desired density;the library clones were transferred to nitrocellulose(NC) membrane;DNA fragment DQ924531 isolated from Part 2 was used as a probe and labeled with DIG;positive clones were screened out by Southern blotting and chemical staining;a 7981 bp gene cluster was obtained by sequencing the fosmid plasmids extracted from positive clones,but the whole sequence of the cluster could not obtained because of the high frequent super structure and the limitation of sequencing techniques;by genome walking basing on the known sequence we did not get longer sequence of the cluster either.We analyzed the structure of gene EF568935 by using GenBank database and bioinformatics software;typical domains like AT,KS and ACP were identified;locations of the domains and a promotor in the EF568935 sequence were also analyzed.4.The functional analysis of new acyltransferase AT-EF080951 in vitro through substrate binding testThe function of AT domain in a PKS is to select and incorporate extender unit in polyketide biosynthetic pathway.The substrate specificity has become a hot research point. The acyltransferase AT-EF080951 in the partial PKS gene cluster EF568935 was found to have the ability to incorporate malonyl-CoA by bioinformatic softwares.To analyze its substrate specificity,substrates CoA,acetyl-CoA,malonyl-CoA and methylmalonyl-CoA were used to carry the substrate binding test of AT-EF080951.MBP-AT-EF080951 fusion protein were expressed and purified,and single AT-EF080951 was separated through factor Xa cleavage reaction.Using MBP,BSA protein as controls we tested the binding ability of the 4 proteins to 4 substrates through ELISA method and calculated the binding constants of them.Through comparison to the binding constants of control proteins,we found the binding ability of AT-EF080951 to the substrates is Kα_(acetyl-CoA)> Kα_{(malonyl-CoA)}≈Kα_{(Methylmalonyl coenzyme A)}≈Kα_(CoA),(the later three belong to non-specific absorption).The result of binding test indicate that both MBP-AT-EF080951 and AT-EF080951 bind acetyl-CoA specifically and Kα_(acetyl-CoA)> Kα _{(malonyl-CoA)},which conflicts with the result by bioinformatics software.Soluble and specific substrate-binding active fusion protein MBP-AT-EF080951 and single AT-EF080951 acyltransferase were obtained through heterologous expression in pMAL-c2x system,which indicated the function of an acyltransferase selectively binding to a specific substrate still remains when the N-terminal KS domain,the C-terminal ACP domain and some intracellular factors are not present.The joining regions at both sides of AT-EF080951 remained no more than 14 amino acids,which also indicated that its function does not depend on the space structure of the joining regions flanking the AT domain.5.The functional analysis of new acyltransferase AT-EF080951 in vivo through domain replacementThe sequence and domains' functions of the avermectin biosynthetic gene cluster have already known.To further testify the result by substrate binding test in vitro,the acyltransferase AT2(3) gene(malonyl-CoA specific,within module 2) in aveA1 of avermectin PKS gene of S.avermitilis ATCC31271 was replaced with AT-EF080951.The products of avermectin PKS in S.avermitilis ATCC31271 were analyzed by HPLC.If AT-EF080951 only binds malonyl-CoA the component and content of avermectin will remain the same;if AT-EF080951 only binds acetyl-CoA the polyketide chain will terminate,and avermectin will not be detected;if AT-EF080951 binds to both of them the component of avermectin will remain the same while the content of it will decrease.We prepared protoplast of ATCC31271 and constructed targeting vector which was electrotransformated into the protoplast;through double crossover the module 2 AT2(3) domain gene in aveA1 was replaced by AT-EF080951;we used PCR to verify the mutants; the correct mutant was cultivated with fermentation medium and the broth was extracted with methanol;we applied the supernatant to liquid chromatography mass spectrometry; the components of avermectin were the same while the content decreased.The results showed that AT-EF080951 can incorporate malonyl-CoA to extend the polyketide chain.Based on results in this study and others',we concluded that the specificity of starter AT domain in vivo might be interfered or regulated by other factors and the substrate binding test of starter AT domain in vitro may lead to inaccurateconclusions.