| Myxobacteria are one group of excellent producers of a great deal of bioactive compounds with anticancer, antibacterial, fungicidal or immune-modulating activities. In the past three decades, over100basic structures and600structural variants have been discovered in myxobacteria, accounting for approximately3.5%of the presently known microbial secondary metabolites. Myxobacterial bioactive metabolites include aromatic, heterocyclic, polyene, alkaloids, macrocycle, polyether, peptide, etc, and most of which are completely novel. Of the myxobacterial bioactive secondary metabolites, nearly half were identified from Sorangium cultures, and almost100%of the Sorangium strains can produce secondary metabolites with biological activities. The production abilities of various chemical structures and functioning mechanisms by Sorangium can even compare with that of the famous drug-producer Streptomyces. Myxobacterial natural products are normally strain-specific, one compound is detectable in certain strains and one single strain is able to produce a variety of different secondary metabolites or various derivates of a basic structure. According to incomplete statistics, one-third of myxobacteria-sourced secondary metabolites are polyketides (PKs) or non-ribosomal peptides (NRPs). PK and NRP compounds are two important families of natural secondary metabolites, exhibiting extremely abundant structures and functions. Since the first report of myxobacterial modular PKS/NRPS assembly line in1995, almost all subsequent myxobacterial modular PKS/NRPS assembly lines disobey the classic assembly patterns and demonstrate novel genetic and biochemical characteristics. Based on the characteristics differences, the20presently identified myxobacterial modular PKS/NRPS assembly lines are classified into nine categories, including20subgroups. These unique genetic materials provide fresh vitality to the traditional genetic, biochemical, combinatorial biosynthesis and evolution research, and potentials in development of PKS/NRPS drugs.Epothilones are modular hybrid PKS-NRPS compounds with antitumor bioactivity produced by Sorangium cellulosum. As potential anticancer drugs, natural epothilone and their chemically modified analogs have inspired the enthusiasms of the researchers and pharmaceutical companies. Epothilones has become a hot cake in chemical, biological and clinical researches. Up to date, at least six epothilone analogs have been served into clinical assessment, one of which, ixabepilone (BMS) has been approved by FDA as a first-line drug against breast cancer. Epothilones were first discovered from the Sorangium cellulosum strain So ce90, and subsequent screening of thousands Sorangium cellulosum strain revealed approximately2.5%strains were able to produce epothilones. So0157-2, as a model Sorangium strain in our laboratory, has advantages in the production of epothilones. Comparison of the epothilone gene clusters in So ce90and So0157-2strains shows a98.67%similarity in sequences, and identical module and domain composition, but significant differences in the upstream and downstream sequences. So far,57epothilone analogs have been reported to be discovered from the two strains, including17epothiloneA derivatives,8epothilone B derivatives,16epothilone C derivatives, five epothilone D derivatives, eight18-member ring and three14-member ring epothilone analogs. The structures of epothilone analogs show diverse mutation sites and specificities in certain variable sites, suggesting particular organization and synthetic modes for the epothilone assembly line:special loading type, skipping type, stutter, non-specific substrate selection, module iterative type, and site selective cyclization. The open-loop structures and PKS modification characteristics, like modification of hydroxyl, oxygen, methylation, demethylation, glycosylation and trans, suggest that the upstream and downstream genes, together with strain-specific regulatory factors, play important roles in regulation and modification of epothilone synthesis. Epothilones are in low yield. However, so far, there is no report on the regulatory genes of epothilone biosynthesis. Comparative analysis of the four reported epothilones biosynthetic gene clusters showed that the main gene cluster sequences were almost identical, while the upstream and the downstream sequences varied significantly, presumed to be related to the potential regulatory elements.In this study, we performed sequence comparison of the epothilone biosynthesis gene clusters from So ce90and So0157-2, which suggested an existence of440-bp relatively conserved sequence in the upstream5’-UTR (untranslation region). Then, we designed the conserved primers to amplify the same region from eight epothilone-producing strains in our laboratory, and obtained five different sequences, while the other three were identical. The five sequences had94.27%identity, but showed50%variation in activity measurement. The transcription start site (TSS) position was located by primer extension experiments, and two TSSs were found in the conserved region located246bp and193bp upstream of translation start site (TLS). The corresponding-35and-10regions were identified and the spaces between each of the-35and-10region were17bp and22bp, respectively. Further activity tests showed that the two TSS were related to a strong promoter and a weak one, respectively. Meanwhile, a presumed stem-loop structure was knocked out, which was proved a negative regulatory element. In addition, the remote non-conserved region was also verified to have an impact on promoter activity. These results revealed that the regulation of transcription of epothilone gene cluster is influenced by double promoters, the stem-loop structure and distal non-conserved upstream regions. These core elements are conserved in the five sequenced from different sources, but the variable bases and sites determined the corresponding activity of the different promoters. Here, we characterized the epothilone promoter and detected activities of the functional element using a promoter verification system in heterologous host.Next, we took a prediction of inner promoters presented between ORFs of epothilone gene cluster, which was generally considered as a huge operon because of the short space between each ORFs (15-147bp) or even overlap and no terminator structure was predicted to exist within the gene cluster. The space sequences between ORFs were amplified and programmed for promoter activity detection. The results showed that some of these sequences had promoter activity, and some were in rather low-level expressions. It was presumed that these additional inner promoters responded for transcriptional regulation of the relative ORFs during the transcription of the whole gene cluster. However, the specific mechanism remains to be further verified experimentally.Many research groups have focused on the heterologous expression attempts of epothilone gene cluster in the hosts with PKS/NRPS pathways, shorter generation time and more practical genetic manipulation system, such as Streptomyces colicolor, Streptomyces venezuelae, Streptomyces lividans, Myxococcus xanthus, Pseudomonas putida and recombinant Escherichia coli, etc. M. xanthus is a relative species to Sorangium cellulosum and is able to produce plenty of PKS/NRPS secondary metabolites, which provide the necessary substrates and enzymes for heterologous epothilone biosynthesis. In addition, the bacterium has a short generation time (4h), and dispersion growth characteristics in liquid. These characteristics suggest that M. xanthus is fermentation-friendly host, and is able to turn out with the higher yield of epothilone, compared with other heterologous hosts. The heterologous expression was based on two plasmids from the genomic library clones of So0157-2. Cosmid10contains complete epoA-epoC and partial epoD, while Fosmid3B11contains complete epoD-epoF and partial epoC. The two plasmids cover the entire epothilone gene cluster and share a6,538b overlap, containing partial region sequences upstream or downstream the cluster. We attempt to express epothilone gene cluster heterologously in Myxococcus xanthus. After many attempts in several recombinant hosts and technical manipulations, we selected an E. coli host with Red-ET recombination system for splicing and assembling the complete epothilone gene cluster. Subsequently, three different recombination elements for introduction of the cluster into M. xanthus chromosome were added:homology arms, Mx8site-specific recombination and transposon. Finally, we succeeded in integrating the gene cluster into Myxococcus chromosome by way of transposition, and obtained tens of mutants with significantly varied epothilone yields. The transposon insertion sites were scattered randomly throughout the whole chromosome, which indicated that the epothilone production may be affected by the different insertion sites in different mutants. Three mutants with different yields of epothilones were further performed for transcriptome sequencing, which showed that the expression of these foreign genes varied greatly in M. xanthus host. The results suggested that the corresponding genes with an up-regulated or down-regulated expression were presumably to influence the synthesis or product of epothilone by affecting transcription factors or global regulation factors.The mutants M. xanthus DZ2-9with the highest epothilone yield was used as a objective for the following genetic manipulations and modifications. The resistance gene was knocked-out to simplify the mutant background for subsequent genetic manipulation. Meanwhile, the autonomously replicating plasmid pZJY41was employed to express of individual ORFs. The results showed that overexpression of epoA or co-overexpression of epoP and epoA raised the epothilone production. Overexpression of other ORFs, knockout of epoF and genetic modifications are still under progress.This work proposed:1. The elements and functions of the promoter of epothilone gene cluster were analyzed by means of the E. coli heterologous system. The TSS was located by primer extension and two promoters with different transcription activity were identified in the5’-UTR of epothilone the cluster.2. The inner promoters within the epothilone gene cluster were predicted and functionally analyzed. These potential promoters may associate with the transcriptional regulation of the whole epothilone gene cluster with a low-level activity.3. Epothilone gene cluster was successfully introduced into the M. xanthus host by transposition. Effects of random integration sites were studied by transcriptome sequencing.4. The autonomously replicating plasmid pZJY41was applied in the genetic manipulations in M. xanthus host. |
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