Mining Of Bioactive Natural Products From Actinomycetes And Development Of Chassis

Natural products are an important source of drug lead compounds.Actinomycetes,as the mainstay of high production of active natural products,have made great contributions to the development of the pharmaceutical field and still occupy an important position until now.In recent years,as sequencing technologies continue to mature and DNA databases continue to grow,more and more genome mining methods,web tools and databases are being developed,which greatly facilitate the discovery,characterization,and modification of natural products,greatly shorten the cycle of discovery of novel natural products.At the same time,thanks to the emergence and development of synthetic biology,a "top-down" strategy can be adopted to streamline the genomes of dominant strains with a clear genetic background,thereby transforming them into highly efficient chassis cells that can be used to activate"silent" biosynthetic gene clusters for the extraction of novel natural products or for the efficient biosynthesis of high value-added molecules.In this study,the main objective was to discover new bioactive natural products.Firstly genome mining of Streptomyces sp.sdu3101 was conducted.Secondly,Micromonospora echinospora CCTCC M 201 8898 was constructed into efficient chassis cells for active natural product discovery and yield improvement.The main studies and results of this thesis are as follows:(1)Mining of new active natural products from the termite intestinal symbiotic Streptomyces sp.sdu3101In this study,we sequenced the genome of Streptomyces sp.sdu3101,a symbiotic bacterium from the gut of the termite Odontolermes formosanus,and found that Streptomyces sp.sdu3101 is rich of BGCs.First,the novel type I PKS BGC6 was directly cloned using Red/ET recombineering,and the heterologous expression of BGC6 in the host S.albus J1074 was achieved;BGC15 responsible for the synthesis of the polycyclic tetramer macrolactone was cloned,and heterologous expression of BGC15 in S.coelicolor A3(2)was achieved by inserting the constitutive strong promoter SP44 in front of the core gene.(2)Construction of optimized chassis cells of Micromonospora echinospora CCTCC M 2018898In this study,to construct an optimized chassis cell that can be used for the biosynthesis of drugs,we proposed to replace the highly expressed BGC in M.echinospora with RMCE exchange cassette elements,using M.echinospora CCTCC M 2018898 as the original strain.Displace the native high expression BGCs to reduce the competing pathways and integrating the exogenous BGCs into the highly expressed positions by RMCE exchange cassette is beneficial for the mining of natural products as well as yield improvement.(i)Four different recombinase-mediated RMCE(recombinase-mediated cassette exchange)exchange cassettes were constructed.(ii)The BGC of gentamicin in M.echinospora CCTCC M 2018898 was successfully knocked out,and the RMCE exchange cassette element was placed at the position of the original BGC of gentamicin.The exchange cassette was further constructed to integrate the exogenous BGC.(iii)To find other endogenous high expression loci in M.echinospora CCTCC M 2018898,this part of work screened three endogenous high expression loci on the M.echinospora CCTCC M 2018898 genome by real-time fluorescence quantitative PCR,namely BGC3 with high transcriptional level and two moderate transcriptional levels of BGC 1 and BGC5.In summary,the above results not only show that there are still many secondary metabolic pathways to be explored in actinomycetes of special habitats,but also show the high efficiency of genome mining strategies and the superiority of synthetic biology techniques,laying the foundation for the subsequent discovery,modification and yield optimization of novel active natural products,as well as providing ideas for targeted mining of natural products.