Projects / Programmes
Modulation of polyketid synthase complex involved in early and late stages of tetracycline biosynthesis
| Code |
Science |
Field |
Subfield |
| 4.06.04 |
Biotechnical sciences |
Biotechnology |
Microbe biotechnology |
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.09 |
Engineering and Technology |
Industrial biotechnology |
antibiotic, tetracycline, polyketide synthase type 2, Streptomyces, biosynthetic engineering
Researchers (17)
Organisations (3)
Abstract
In recent decades, the rise in bacterial resistance and the shortage of novel antibiotics has led to an urgent need for new antibacterial drug leads. Once highly-effective antibiotics, tetracyclines (TCs) are now unfortunately facing a widespread resistance phenomenon. Despite emerging resistance to TCs erupting during the 1980s, it was not until 2006, more than 4 decades later, that a third-generation TC, named tigecycline, was launched. Therefore, there is a critical need for novel “innovative” antibiotics, which are efficient against hospital multidrug resistant pathogens from the ESKAPE panel, particularly against Gram-negative pathogens such as Pseudomonas aeruginosa. Despite the significant efforts made in the past five decades into the study of TC biosynthesis, and despite very productive early work by McCormick and collaborators at the Lederle laboratories (USA) during the 1960s and 70s, which resulted in the isolation of a Streptomyces aureofaciens mutant strain that produced C6-demethyl-C7-chlorotetracycline, an important biosynthetically-produced intermediate in the production of second- and third-generation TCs, biosynthetic approaches in TC development have unfortunately not been productive for more than 50 years. Relatively slow and tedious molecular biology approaches for the genetic manipulation of TC-producing actinobacteria, as well as an insufficient understanding of the reiterative type of polyketide synthase (PKS) type II enzymatic mechanisms involved in TC biosynthesis, have significantly contributed to the low rate of success of such biosynthetic engineering efforts. The iterative nature of type-II PKS systems, which is much more difficult to genetically manipulate, compared to PKS type-I systems (such as erythromycin PKS), is one of the most likely reasons for the relatively low productivity in this area of research. Consequently, resulting in poor output in the biosynthetic engineering efforts made in TC drug discovery.
We have recently demonstrated in our laboratory that biosynthetic engineering of the type-II PKS can be productive. We believe that new opportunities in TC drug development have arisen thanks to significant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology approaches, and enormous and rapidly expanding genomics data available.
In the scope of this project proposal, we intend to take advantage of some relatively small, but essential differences in the substrate specificity of different parts of PKS complexes encoding type-II PKS systems, which are catalysing the biosynthesis of oxytetracycline, chlortetracycline and the atypical TC analogue chelocardin, produced by Streptomyces rimosus, Steptomyces aureofaciens and Amycolatopsis sulphurea, respectively. We intend to carry out in vivo and in vitro comparative studies into the priming and late stages in TC biosynthesis carried out by so-called minimal PKS, and additional enzymes that may be involved in starter unit selection. The structural features of these three TCs are ideally suited to study starter unit selection and later stages in the biosynthesis of these medically important drugs. We thus expect that novel approaches in the scope of this project proposal will result in the construction of unique TC analogues. This grant application is directed towards the study of the biosynthesis of clinically used TCs. However, the new findings in the scope of this proposal will likely be applicable to any other type-II PKS system, and will therefore likely have a direct impact on the drug discovery and industrial process development efforts.
Significance for science
Secondary metabolites from the group of polyketides currently represent one of the most important groups of natural products, with extremely diverse activities used in all areas of medicine and agriculture. Therefore, the great interest shown by research institutions and industry in this field is not surprising. For example, Lek/Sandoz, partner in this project proposal, is industrially producing poliketide metabolites such as the anticancer rapamycin and the important immunosuppressant FK506. Not only Lek/Sandoz, but also other companies in Slovenia, such as Krka, market polyketide natural products or they develop the technologies for their costumers (Acies Bio Ltd). Efforts in the scope of this project proposal will be directed towards the study of one of the most complex enzymatic machineries; the type-II polyketide synthase (PKS) enzyme complex, which is catalyzing the biosynthesis of important secondary metabolites such as tetracycline antibiotics or anticancer drugs from the class of anthracyclines. Compared to the type-I PKS encoding the biosynthesis of a number of important drugs such as the antibacterial erythromycin or the anticancer drug rapamycin, which have been extensively studied, the programming of PKS complexes of type-II is still poorly understood. Due to the reiterative nature of the so-called minimal PKS enzymes, involved in the early stages of biosynthesis, we do not yet sufficiently understand their biosynthetic mechanism. This is likely also one of the reasons why we cannot efficiently generate novel analogues by applying biosynthetic engineering efforts.
We have recently demonstrated that biosynthetic engineering of type-II PKS can be productive (Lešnik, Lukežič et al., 2015, Angew Chem Int Ed Engl. 2015 54(13):3937-40). We believe that new opportunities in TC drug development have arisen thanks to a significant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology methods, and enormous and rapidly expanding genomics data available in the DNA databases. Therefore, by applying a unique approach for biosynthetic engineering in the scope of this project proposal, where in vivo experiments with chimeric PKS will be carried out, it will be possible to gain very valuable data on the programming of these complex enzyme systems. In addition, we will attempt in vitro evaluation of the catalytic activity and enzyme specificity involved in OTC biosynthesis, which has not been achieved to date. Finally, our work will not only be contributing towards high-quality science, but also rapidly translating this knowledge towards drug and process development efforts.
Significance for the country
Polyketide secondary metabolites represent today one of the largest groups of medically and agriculturally important drugs, with an estimated annual turnover reaching over 50 billion USD. It is thus not surprising that huge efforts are being invested in studying the biosynthesis of these important metabolites, which do display a broad spectrum of activities such as antiinfective, anticancer, antiparasitic, cholesterol-lowering, immunosuppressive and many others. In general, the use of secondary metabolites carries an enormous importance for modern society, as well as the biotechnology, chemical and pharmaceutical industry. To ensure steady progress in the development of novel drugs, advanced and environmentally friendlier bioprocesses, there is a critical need to study biosynthesis of these natural products. In the scope of this project proposal, we are collaborating directly with Sandoz scientists, and we will attempt to develop novel bioprocesses by applying advanced technologies of biosynthetic engineering, chemo-biosynthesis and biotransformation. In collaboration with Lek/Sandoz and their R&D efforts, we will particularly focus on halogenation reactions, towards the development of novel technologies for an environmentally friendly and economically improved production of the important generic products already marketed by Lek/Sandoz. Very productive collaborations of the Biotechnical faculty (Prof. Petković) and the Institute Josef Stefan (Prof. Stavber) with the Slovene industry (Sandoz, Krka, Acies Bio, Medis, TKI Hrastnik etc.) was already demonstrated in the past (US 8980585B2, US8361777B, EP2019869B1). It is also important to mention that our efforts in the past resulted in the formation of one spin-off company, Acies Bio Ltd (Ljubljana), which is currently employing over 35 researchers and successfully developing novel bioprocesses for already 10 years. In another aspect of this project proposal, we work on the particularly emerging problem of rapidly rising resistance of the most dangerous pathogens against clinically used antibiotics, causing diverse infections, particularly hospital infections caused by the multidrug-resistant pathogens from the ESKAPE panel. We do expect, in addition to our scientific aims, that our efforts will also result in the generation of truly innovative tetracycline antibiotics, which will complement future semi-synthesis efforts to produce novel TC antibiotics and other biosynthetically similar polyketide metabolites of medical importance. To summaries, in addition to the high quality science, the results of this project will most likely have a profound influence on the development of new technological expertise immediately transferrable to the industry.
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
