Understanding and Exploiting Tunicamycin (Bio)Synthesis to Enable Novel Antibiotics and Inhibitors

Streptomyces are bacteria that live in the soil and produce antibiotics to compete with other soil microorganisms. Tunicamycin is an antibiotic made by Streptomyces chartreusis with a very unusual chemical structure. It kills other bacteria by blocking the action of a protein (an enzyme) that performs an essential role in making the walls of bacterial cells. These cell walls are essential for bacteria to survive. The way in which tunicamycin kills bacteria is different to almost all other antibiotics that are used in medicine and so tunicamycin has the potential to become a new and very effective antibacterial treatment to counter, for example, MRSA infections.
The chemical structure of tunicamycin is actually similar to some of the building blocks used to make cell walls. This suggests that it may act my mimicking these building blocks, preventing the enzyme that makes cell walls from choosing the correct components.
We have discovered recently the genes that allow S. chartreusis to make tunicamycin, and we can now use these genes to produce the enzymes that make tunicamycin. We have also developed methods to make the building blocks, and slightly different versions of them, that are used to make tunicamycin. As a consequence, we are now in a very good position to understand not only how the very unusual tunicamycin structure is made by S. chartreusis, but also to use the enzymes and the variant building blocks to generate new tunicamycin-like compounds. Ultimately we aim to genetically manipulate S. chartreusis itself to produce such compounds.
Why is this important? Although tunicamycin is very good at killing bacteria, it also harms human cells, and so cannot be used as an antibiotic. By changing the structure of tunicamycin, we hope to remove the activity that is deleterious to humans, while retaining, or even improving, the activity against bacteria.

Tunicamycin was the first nucleoside antibiotic isolated and inhibits bacterial cell wall biosynthesis by blocking MraY, the enzyme that constructs the key biosynthetic precursor lipid I. This mode of action is orthogonal to antibiotics that are in clinical use. Tunicamycin's structure resembles that of substrates of MraY and suggests it behaves as an evolved scaffold for inhibition. Indeed, it is active against several enzymes, including TarO, the first enzyme in bacterial teichoic acid biosynthesis, and the GPT enzymes that create the lipid-linked precursors for all N-linked glycoprotein assembly. This latter property has seen its widespread use in cell biology (>8000 citations) yet creates toxicity that prevents its use in other applications (such as a new mode-of-action antibiotic based on its MraY activity). We have recently, through genome sequencing of the producer Streptomyces chartreusis followed by genome mining, discovered the gene cluster for tunicamycin biosynthesis. We have also developed methods for synthesizing key intermediates. This establishes a strong position to explore not only the mechanism of formation of the unique tunicamycin structure but to also probe the synthetic utility of associated enzymes (for chemical elaboration and diversification). The modular structure of tunicamycin suggests that the inhibitory scaffold may be altered in a systematic way to tune its activity towards and away from existing targets, and to create new ones. Early results have confirmed that core scaffold modules are active against other nucleotidyl-dependent enzymes. This suggests that synthetic analogues may be created that could uncouple existing activities, perhaps allowing eventual use as lead compounds for therapy. This collaborative project will explore tunicamycin biosynthesis, producer immunity, the enzymology (and structures) of key Tun enzymes, the synthesis of tunicamycin analogues, and their activity in screens against target organisms and enzymes.

Who will benefit from this research?
Longer term, the proposed work should benefit both society and the pharmaceutical industry through the generation of drugs to combat infectious diseases and other human ailments. There is an urgent need for new therapeutic agents for multi-drug resistant pathogens. Tunicamycin possesses a clinically unexploited mechanism of action and is therefore a highly attractive target. This proposal will provide the fundamental knowledge and technology to enable such an outcome. Shorter term, outputs of this research will be of value to fundamental scientists, to applied scientists working in the pharmaceutical industry, and to research clinicians.
Fundamental knowledge of the mechanism of action and properties of carbohydrate-processing proteins continues to underpin industrial applications in the detergent, paper pulp, fruit juice and food sectors. The use of biocatalysis combined with chemistry in the construction of potential therapeutics based on natural products is an area of current rapidly expanding growth and one where the UK has taken a lead not only through established companies e.g., Celltech (now UCB) but through new companies that place an even greater emphasis on biocatalysis and natural products pathways e.g. Biotica, Novacta (the latter founded on IP from the MJB laboratory).

How will they benefit from this research?
Streptomycetes account for ~80% of commercially important antibiotics, and are a rich source of other bioactive molecules, including anticancer agents and immunosuppressants (~$40 bn p.a. in the pharmaceutical industry worldwide). To fully exploit their biosynthetic potential for the production of compounds, we require a better understanding of natural product biosynthesis. Genetic engineering of tunicamycin biosynthesis has the potential to yield highly effective, clinically useful antibiotics directed at a thus far unexploited target (formation of Lipid I). Tunicamycin derivatives also have the potential for use in other human therapies, targeting specific carbohydrate processing enzymes. The fundamental studies proposed here will provide necessary underpinning knowledge to generate such compounds, and will therefore be of direct interest to pharmaceutical and biotechnology companies interested in anti-infectives and other human diseases.

BBSRC Strategic Priorities
Basic Bioscience Underpinning Health: It is a multi-disciplinary programme that combines microbiology, molecular genetics, biochemistry, protein purification and characterization, metabolite analysis, crystallography and chemical synthesis to understand and manipulate the biosynthesis of an unusual and clinically unexploited antibiotic.
Technology Development for the Biosciences: It will lead to the discovery and utilization of biologically-directed chemical tools (Chemical Biology tools) of both academic and commercial relevance that may lead to novel therapeutics.

What are the major aims that are likely to have significant impact?
1. The mutational analysis of the tun gene cluster (WP1) - this will prove or modify our proposed biosynthetic pathway.
2. Increased tunicamycin production from Streptomyces by manipulating gene expression and optimising culture media (WP2.2,2.3) - this will more readily allow production of compounds that will be useful for analogue synthesis.
3. Preparation of tunicamycin intermediates through degradation/relay methods (WP3.1) - this will also more readily allow production of compounds that will be useful for analogue synthesis.
4. In vitro reconstitution of synthetically useful enzyme activities combined with chemical methods to create tunicamycin analogues (WP3.2)
Aims 2. - 4. will more readily allow production of compounds that will be useful for analogue synthesis.
5. Screen analogue activities through zone inhibition, MICs, in vitro assay (MraY-type vs GPT-type) (WP4.1,4.2) - this will identify analogues with unique and enhanced activities.