Synthetic probes of natural product biosynthesis: implications for synthetic biology and drug discovery.

Natural products represent a major source of powerful agents for the treatment of human, animal and plant diseases. Indeed the most potent antibiotics, anticancer agents, antiparasitic agents and pesticides in use today are natural substances produced in microorganisms and plants by dedicated sets of enzymes. Natural products owe their extraordinary bioactivity to their highly complex chemical structures, which still constitute a challenge for chemical synthesis. Intriguingly microorganisms and plants have the ability to produce these products from very simple precursors (e.g. acetate, amino acids and sugars) for their survival. Advances in research have increased our knowledge of the bacterial, fungal and plant enzymes and the genes responsible for their existence; however little is known about the chemical details of their biosynthesis. This is because the biosynthetic processes comprise of multiple transformations that take place in rapid succession and for which we currently have no generic and straightforward method of investigation. The limited amount of information obtained so far in relation to the number and the complexity of the existing natural product pathways prevent us from fully understanding and utilizing these pathways for improving the production of known products (via the metabolic engineering of microorganisms) and the generation of new ones (via synthetic biology approaches aimed at new drug discovery). At the present time these are extremely desirable goals in view of the many global challenges in health, environment, sustainability and energy that the scientific and the nonscientific communities face.

This research project aims at developing a novel and general methodology for the study of natural product biosynthesis based of the use of synthetic probes. These are chemically prepared compounds that mimic the basic precursors utilized in natural product formation and interfere in the pathways leading to the natural product assembly, 'catching' and retrieving the intermediate species from complex biological mixtures and throughout their processing. The pathways targeted in this research project are those leading to polyketide and nonribosomal peptide products, among which we count many commercially and industrially relevant products such as the blockbuster cardiovascular statin drugs and the antibiotics of last resort vancomycin and teicoplanin.
To develop this 'chemical' methodology, which is of general applicability and is capable of providing immediate answers to complex mechanistic issues, we have identified specific research objectives concerning (1) the development of synthetic probes of improved bioavailability for the study of polyketide biosynthesis 'in vivo' (meaning directly in live microorganisms); (2) the design, the development and the validation of putative probes of nonribosomal peptide biosynthesis, and (3) the application of the polyketide and nonribosomal peptide probes to elucidate poorly understood pathways leading to products of commercial and industrial interest.

The main outcomes of this research will be (a) powerful yet very practical tools capable of providing unique mechanistic insights into biological processes, whose beneficiaries include researchers in the broad area of biosciences; (b) new understanding of natural product biosynthesis, which will constitute the basis of for the biosynthetic engineering of new products.
The major beneficiary of this research outside the academic community will therefore be the biotechnology industry, who is going to utilize the information gained through our methodology to develop new products for the nation's health and for the growth of the UK economic competitiveness.

Understanding the factors that control the programming of natural product biosynthetic enzymes is undeniably one of the greatest challenges and priorities in view of synthetic biology for drug discovery and production in microorganisms. One way to gain insights into the programming is to disclose the fine mechanistic details of the catalysis via the isolation of biosynthetic intermediates.

The overall aim of this project is to develop a chemical method for the investigation of polyketide and nonribosomal peptide biosyntheses based on the use of synthetic probes. These have been devised as nonhydrolysable analogues of the malonyl and the aminoacyl building blocks normally recruited in polyketide and nonribosomal peptide formation and compete with them for the natural product chain extension, capturing and off-loading premature biosynthetic species from the multifunctional enzymes.

In order to establish this chemical strategy as a leading method of investigation, we aim at: 1) developing the methodology for the investigation of polyketide biosynthesis in vivo, via second-generation probes of improved bioavailability and/or the aid of biochemical tools; 2) extending the methodology to other assembly lines, specifically to nonribosomal peptide synthetases (NRPSs), via nonhydrolysable aminoacyl cysteamine probes; 3) applying the methodology herein developed to elucidate poorly-understood biosynthetic pathways leading to commercially and industrially important natural products, such as thiolactomycin. These objectives will be accomplished through the combination of organic synthesis, in vivo feeding experiments, LC-HRMS and NMR analysis, molecular biology and protein chemistry tools. The outcome of this research will be powerful yet very practical probes that will provide unique insights into the sophisticated biosynthetic enzymes, laying the foundations for their novel exploitation in synthetic biology

Beside the academic beneficiaries, our research will benefit:

1) the biotechnology commercial private sector, who will be the main user of the research outputs, both immediately and in the longer term.
The data acquired through our methodology will be indeed used by biotechnology companies for the rational re-engineering of microorganisms to improve the production of pharmaceuticals and agrochemicals currently in use, as well as to generate new ones of improved or different bioactivity.
At the present time these are extremely desirable goals in view of the many global challenges in health, environment, sustainability and energy that the scientific and the non-scientific communities face worldwide. After the completion of this project, and in particular of objective 3 which concerns the methodology application for the elucidation of a pharmaceutically relevant product, we will already have in our hands relevant and significant data for the beginning of a research programme addressing the generation of novel anticancer and anti-obesity agents via synthetic biology, with the realistic possibility of generating a library of novel compounds for testing within the next 10 years. This is just the initial example of how our methodology will have long-term impact and lead to many benefits for both the industrial and the public sector.

2) Nation's health. The public health sector (NHS) is currently facing a growing demand for the treatment of conditions associated to ageing population and obesity. By leading to the generation of new products for human health that can tackle these challenges, our methodology will ultimately contribute to the enhancement of the quality of life for a larger number of people at reduced costs for the public sector.

3) the UK economic competitiveness. By introducing and enhancing a new creative output for synthetic biology, our basic research will ultimately contribute to maintaining and leading UK innovation in biotechnology for animal health, agriculture, biomaterials and biofuels. This is particularly relevant in face of climate change and a growing demand for clean energy, for which UK research and technology need to provide novel and prompt solutions.

4) the staff working on the project. The researcher working full-time on this project will benefit of an extensive cross-disciplinary training which will include organic synthesis, microbiology, molecular biology, protein chemistry and analytical chemistry. The practical skills acquired by PDRA will be useful for his/her employment in both the academic and the industrial sectors at the end of the project. In addition the researcher will have the opportunity to develop key transferable skills such as work planning, project management and development, teamwork and public outreach, which are invaluable tools also for non-research based careers. The collective skills acquired throughout this project will give the researcher a very competitive edge which is indispensable in the current global job market.