Projects / Programmes
Regulation of the FK506 (tacrolimus) biosynthetic process
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
immunosuppressants, FK506, polyketides, Streptomyces, genetics, biosynthesis process
Researchers (8)
Organisations (1)
Abstract
Over the last two decades the calcineurin inhibitors, cyclosporine and FK506 (tacrolimus) natural products, have been the mainstay of immunosuppressive therapy. Increased understanding of the cellular immune response and other complex signalling pathways, which are regulating biological processes in the cell, have enabled us to significantly improve immunosuppressive treatment of organ transplant recipients, and develop novel applications of current drugs or drug analogues such as the structurally related microbial products rapamycin, FK520 and FK506. Interestingly, these drugs have also recently shown great potential for therapeutic use in the treatment of cardiovascular, autoimmune and neurodegenerative diseases. Rapamycin and its derivatives are presently in clinical trials as anticancer agents. FK506 and a closely related compound, FK520, and their analogues have been licensed for treatment of inflammatory skin diseases. Considering their biosynthetic origin, rapamycin, FK506 and FK520 belong to polyketides, synthesised by so-called polyketide synthases (PKS), multi-enzyme complexes encoded by large gene clusters. Recent advances in the understanding of the PKS biosynthetic “machinery” have resulted in the development of an entirely novel approach to biosynthetic engineering aimed at generation of novel polyketide analogues of macrolide molecules such as erythromycin, epothilone, rapamycin and FK520; this could not have been achieved by a standard synthetic chemistry. Entire gene clusters of structurally related compounds rapamycin and FK520 have been completed and a number of their analogues generated. The FK506 gene cluster, on the other hand, has only been partially sequenced. There is an obvious need to complete the sequencing of this cluster, considering the medical and economical importance of this drug. The aim of our research proposal is to complete the DNA sequence of the FK506 gene cluster in Streptomyces sp. ATCC 55098. In particular, as a part of this proposal, our research will be focused on cloning the regulatory genes and studying their role in the regulation of FK506 biosynthesis. The understanding of the regulation of the FK506 gene cluster expression, as a longer term aim, will bring an important contribution to the knowledge needed for fermentation process improvements in the industrial environment.
Significance for science
Polyketides are a large group of natural products which includes antibacterials such as erythromycin and tetracyclin, antifungals (nystatin and amphotericin), anti-cancer compounds (doxorubycin, rapamycin and epothilon), anti-parasitic agents (avermectin), immunosuppressives such as FK506 and FK520 and many other clinically important metabolites with great economic significance. Due to the importance of these compounds the majority of their biosynthetic gene clusters have been cloned and sequenced. Nevertheless, many aspects of the biosynthesis of these secondary metabolites have remained obscure. Apart from few exceptions, the knowledge on the regulation of secondary metabolite biosynthesis including the regulation of expression of PKS genes has remained scarce, however, many processes in the production organism are known to be involved. Currently, literature data about the regulation of biosynthesis of structurally related macrolactones rapamycin, FK506 and FK520 is limited. Entire biosynthetic gene clusters of rapamycin and FK520 have been sequenced and several putative regulatory gene homologues have been identified. On the other hand, the published sequence of the FK506 gene cluster from Streptomyces sp. ATCC55089 does not contain putative gene homologues encoding regulatory proteins. In the scope of this project, the entire FK506 biosynthetic gene cluster has been sequenced and novel open reading frames (putative genes) have been identified which seem to be involved in the provision of unusual building blocks according to bioinformatic data. In addition, three regulatory genes have been identified and their role in the regulation of FK506 biosynthesis has been thoroughly studied. We have established that two of these genes are positive pathway-specific regulators which drastically affect the yield of the target product.
Significance for the country
Pharmaceutical industry, especially two companies, Krka d. d. and Lek Pharmaceuticals d. d. a member of the Sandoz group,, plays a vital role in the social and economic development of Slovenia. Bioprocess technology is an important part of activities of these two companies. Krka d. d. is an important producer of diverse secondary metabolites, such as antibiotics bacitracin, oxytetracyclin, salinomycin, lipstatin and the cholesterol lowering drug lovastatin. Likewise, Lek Pharmaceuticals produces ergot-alkaloids, vancomycin, gentamicin, clavulanic acid and pravastatin. In the near future, rapamycin, FK506 and FK520 as well as their novel analogues are sure to play an important role in medical practice as well as in the pharmaceutical industry, whereby Slovenia will not be exempted. Lek/Sandoz is producing FK506 (tacrolimus) using a biosynthetic procedure based on the microorganism Streptomyces sp. Deeper understanding of the regulation of FK506 biosynthesis will clearly be applicable in increasing the final yield of FK506 and reducing the concentration of undesired intermediates and side products. In addition to showing that manipulation of regulatory genes can improve the yield of the final metabolite, better understanding of regulatory systems can also accelerate the development of novel FK506 analogues. We believe that our results have significantly improved the understanding of the regulation of FK506 biosynthesis as well as secondary metabolites in general and moreover, new insights will be widely applicable in the field of strain improvement and bioprocess development in industrial environment.
Most important scientific results
Annual report
2008,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2008,
final report,
complete report on dLib.si
