Final Report Summary - EXPLOGEN (Exploitation of actinomycetes genomics using synthetic and system biology approaches) The major aim of the project is the exploitation of the biosynthetic potential of actinobacteria in order to produce bioactive compounds. Using synthetic and system biology approaches we aim to access these so far unavailable natural products. The major components of our technology platform are: 1) library of synthetic controlling elements (promoters, RBSs, terminators etc); 2) native and reconstructed biosynthetic gene clusters for the production of natural products; 3) the chassis or host for the heterologous expression.1) The library of constitutive synthetic promoters, terminators and RBSs have been constructed for the fine-tuned expression of genes in Streptomyce albus, Streptomyces lividans and others non-streptomyces actinobacteria. Briefly, a synthetic constitutive and inducible promoter library to drive gene expression in actinomycetes has been constructed and comprehensively characterised using several reporter genes and RNA-seq. The library consists of approximately 60 synthetic promoter of different strength including the strongest promoter described for the gene expression in actinobacteria. These promoters have been used already in frame of the project to generate natural product overproducer and drive the expression of otherwise silent gene clusters. Several terminators have been generated and characterised to stop the transcription of genes in S. albus. We have found three very strong terminators, which block the 99 % of transcription and thus allow construction of the synthetic genes operons more efficiently. In addition, we have developed more than 100 synthetic RBSs and generated a design rules for the efficient RBSs in streptomycetes.The system for the optimal RBSs screening, so called RBS selector, have been developed for actinobacteria. Finally, we have combined all the elements mentioned above and constructed a dual control expression system for actinomycetes where the expression of genes is controlled on both transcriptional and translational levels.2) More than twenty different actinobacteria’s genomes have been sequenced, assembled and annotated. After bioinformatics analysis we have identified and mapped biosynthetic gene clusters putatively encoding active secondary metabolites to the genomes. Several dozens of biosynthetic gene clusters have been cloned into the BAC and cosmid vectors for heterologous expression. We have used a conventional BAC (cosmids) construction technology as well as yeast assembly methods to directly clone biosynthetic gene clusters. Five silent gene clusters have been modified using synthetic biobrick libraries and expressed in heterologous host. More than ten new antibiotically active compounds have been isolated after the cluster activation.3) The silent gene cluster activation and the optimisation of the natural product synthesis level of every Actinobacteria strain that is currently in use represents an insurmountable task, and the development of one (or several) improved strains, or a so-called chassis, is more appropriate. Two different issues should be considered for the development of a chassis strain: deletion of redundant genetic information, such as IS elements and secondary metabolite clusters, thus increasing genetic stability and reducing the metabolic burden and inactivation, silencing and activation of a number of host metabolic steps as well as introduction and balancing of foreign metabolic pathways, which together, should direct the flux to the production of higher yields of biosynthetic precursors. To tackle the first issue concerning genome engineering and deletion of the unnecessary DNA regions from the genome of the chassis strain we have developed iterative marker excision system (IMES). The system allows excision of DNA fragments without leaving active recognition sites and therefore can be used subsequently any number of times without risk of genetic instability. Using the system 14 different biosynthetic gene clusters have been excised from the genome of S. albus in order to improve its features as a heterologous host. Finally, we have expressed more than 100 native and reconstructed gene clusters in our final chassis strain. The extracts of the recombinant strains have beed analysed and more than 10 new antibiotically active compounds have been isolated, thus validating the synthetic biology approach for drug discovery.
