Projects / Programmes
Study of hom(e)ologous recombination in the evolution of polyketide synthases
| Code |
Science |
Field |
Subfield |
| 4.06.01 |
Biotechnical sciences |
Biotechnology |
Recombinant DNA technology |
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.09 |
Engineering and Technology |
Industrial biotechnology |
Streptomyces, rekombination, polyketide synthases, hyper-recombination strains
Researchers (22)
Organisations (3)
Abstract
Soil-dwelling gram positive bacteria Actinomycetes are commercially very important microorganisms, which produce by far the highest number of industrially and clinically important bioactive natural products currently in use. One of the most important groups of such natural products are structurally and bioactively diverse polyketide compounds, which are found in wide areas of clinical applications with antibacterial (macrolides), antitumor (anthracyclines, epothilone, rapamycin) and immunosuppressant (FK506) activities, among others. Many of these polyketide compounds are synthesised by large and complex multi-enzyme systems called type I modular polyketide synthases (PKSs). In order to manipulate these PKS biosynthetic gene clusters, with the aim of generating novel drug analogues, different biosynthetic engineering approaches were applied, most of them relying on the process of hom(e)ologous recombination (HR). Although successful in some cases, this approach was frequently found to be very difficult and time consuming due to a generally very low frequency of HR events in Actinomyces (Streptomyces) species, which produce these secondary metabolites. Generally the mechanisms of recombination in these microorganisms are still poorly understood, thus providing only very limited possibilities for genetic manipulation of actinomycetes HR enzymatic machinery. Recent findings obtained trough comparative studies of ever increasing number of Actinomyces sp. genomic DNA sequences, indicated the existence of novel genes encoding for enzymes possibly involved in different recombination pathways. In particular identification of previously unknown Actinomyces sp. homologues of the eukaryotic recombination protein Ku, involved in the double strand DNA break-repair by non-homologous end-joining (NHEJ), suggested that the efficiency of HR might be suppressed by existence of this competing recombination pathway. Furthermore, the successful construction of Streptomyces strains carrying null mutations in the recA gene, so far considered to be essential for cell viability, opened another possibility for potential improvement of the HR mechanisms in these microorganisms.
Based on these findings, the main purpose of this project proposal is to investigate possible enhancement of the HR frequency either by inactivation of genes encoding for enzymatic activity which suppress the HR process or by replacement of existing HR enzymatic machinery with their orthologs from other organisms with known highly efficient HR mechanisms. This study could greatly enhance our recent knowledge of HR mechanisms in actinomycetes in general, and at the same time could lead to development of hyper-rec Actinomyces (Streptomyces) sp. strains, as prerequisite for more efficient end versatile manipulation of PKS encoding gene clusters. Thus, targeted genetic modification of selected PKS modules by employing recombineering-like techniques directly in the Actinomyces (Streptomyces) sp. hosts would be easier to implement. Such an approach would represent a major step forward in finding novel solutions for efficient manipulation of otherwise complex and difficult to engineer PKS gene clusters and to produce novel polyketide-related compounds. Considering all this, the expected results of this proposed project would significantly increase the efficiency of biosynthetic-engineering methods as well as other synthetic biology efforts in general. Thus, the project entails on one side a highly academic and in-depth study of the underlying and unusual recombination processes in actinomycetes, while on the other side the findings of this research may lead to specific development of new bioactive compounds and potential drug candidates, as well as contribute to a wider field of biosynthetic engineering and synthetic biology approaches used in drug discovery by academic groups and industry worldwide.
Significance for science
The main goal of this project was to study in detail genetic recombination systems in actinomycetes, which are poorly understood and unlike most other known bacterial recombination and repair systems. Considering the actinomycete life cycles and their natural environment, it is not so unexpected that these species evolved recombination and DNA repair systems that are different compared to the ones in proteobacteria. In this project we focused on S. rapamycinicus strain, which produces industrially important immunosuppressant rapamycin. In this strain we studied in detail minimal efficient processing segments (MEPS) and the role of selected recombination genes. Based on the obtained results we prepared specific plasmid constructs for the rapamycin gene cluster manipulation and genetically modified S. rapamycinicus strains were obtained producing novel analogs. Better understanding of recombination processes will also be of great importance for advancing the field of synthetic biology and biosynthetic engineering in actinomycetes and will allow a more efficient manipulation of industrially and medically important strains for generation of novel bioactive compounds. In this project an investigation of a very basic biological subject of unique recombination mechanisms was performed, which bring us new powerful knowledge and tools, to generate novel drugs through »synthetic biology« or combinatorial biosynthetic engineering approaches.
Significance for the country
Actinomycetes are industrially important source of biological active compounds used in medicine today. However, decade-long intensive drug discovery programs by pharmaceutical corporations have largely exhausted accessible natural resources of bioactive compounds, and also the chemistry used in semi-synthetic derivatization of these compounds to produce novel analogs has been greatly exploited. Approaches such as synthetic biology and biosynthetic engineering present possibilities for development of novel compounds. However, biosynthetic gene clusters containing polyketide synthases, that present the greatest potential, are at the same time extremely difficult to manipulate genetically. Improved understanding of the mechanisms of recombination of these biosynthetic gene clusters is of crucial importance for generation of novel drug candidates, which allowed us to generate new analogues of bioactive compounds already in the course of this project. The results obtained in this project are directly applicable to the applicant, biotechnology company Acies Bio. One of the important R&D activities of Acies Bio is development of novel bioactive compounds using biosynthetic and semi-synthetic approaches. The more widely applicable findings and mechanisms that were discovered in the course of this project will also be used in new approaches for development of improved production technologies for secondary metabolites, which are being developed by Acies Bio for Slovenian and foreign pharmaceutical companies. The execution of project was carried out in Acies Bio, Biotechnical faculty of the University of Ljubljana and Jožef Stefan Institute as well as in cooperation with international academic partners. Through such inter-sectorial collaboration with academia and industry we promoted the exchange of experiences, know-how and best practice flow, which is of utmost importance in order to increase the competitiveness of Slovenian science as well as the business sector. Collaborative work on this project engaged experts from local and foreign universities. Moreover, the knowledge gained by this project will also be transferred to the students of university programs of biotechnology and microbiology at the Biotechnical Faculty of the University of Ljubljana and Jožef Stefan Institute.
Most important scientific results
Annual report
2012,
2013,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2012,
2013,
final report,
complete report on dLib.si
