13 ERA IB: Production of new bioactive compounds by plants and bacteria using new and improved halogenases

Natural products (complex molecules produced by plants and microorganisms) exhibit a wealth of agriculturally and medicinally significant activities. For example, auxin molecules, which are derived from the amino acid tryptophan, control how fast plants grow. Natural products also have tremendous medicinal value. Over the past three decades more than 60% of anticancer agents and over 70% of antibiotics entering clinical trials were based on natural products. Key examples include the antibiotic vancomycin (generated by a microbe) and the anticancer agent taxol (generated by a plant).

The introduction of a halogen- a chlorine or bromine atom- into a natural product has frequently been demonstrated to improve bioactivity and bioavailability. Furthermore, the incorporation of chlorine or bromine provides a reactive handle that may be utilised to further derivatise the product, and thereby modulate its bioactivity even further. The inherent complexity of many natural products renders them and their analogues hard to produce using chemical methods. Therefore, development of enzymatic syntheses for these compounds is highly attractive. Enzymes called halogenases that catalyse the installation of chlorine or bromine into natural products have been recently discovered.

In this proposal, we describe a consortium of five academic partners and one industrial partner to develop and implement these synthetic processes that utilise halogenases. The proposal describes a highly inter-disciplinary approach to:
a) discover new halogenases
b) engineer halogenases to change their specificity
c) develop chemical methods to further derivatise the halogen after it has been enzymatically installed
d) evaluate the structure and agrochemical bioactivity of any of the new compounds that are produced.

We envision that as a result of the work performed in this proposal we will:
a) have in hand a "suite" of halogenase enzymes that academic and industrial scientists can use to produce a wide range of halogenated compounds with a broad range of applications on a small or large scale
b) assemble a library of novel "unnatural natural products" with unusual halogenation patterns and chemical derivatisations from the halogen site
c) assay this library of compounds for biological activity for the agricultural industry (e.g. screen for new pesticides) in collaboration with Syngenta, the industrial partner

Natural products exhibit a wealth of agriculturally and medicinally significant activities. The introduction of a halogen into a natural product has frequently been demonstrated to improve bioactivity and bioavailability. Furthermore, the incorporation of chlorine or bromine into a natural product provides a reactive handle that may be utilised for further site-specific functionalization. The inherent complexity of many natural products renders them and their analogues hard to access synthetically. A more expeditious approach is to harness enzymatic processes that can generate these compounds. It has been shown recently that natural product biosynthetic pathways can be modified by introducing halogenase genes into natural product producing organisms (bacteria and plants). Tryptophan halogenases are especially suited for this purpose since they use free tryptophan as the substrate, and can be used to obtain novel tryptophan derived metabolites such as halogenated indole alkaloids or halogenated indole-3-acetic acid derivatives (auxins). The introduction of the halogen on these two classes of natural products may result in the development of molecules with new or improved biological and agricultural activity. The use of these halogenases, however, is limited because the enzymes will only recognise and react with a small number of starting materials. Notably, it was recently shown by members of this consortium that these enzymes can be engineered to display different substrate and regio-selectivities.

This proposal describes a highly collaborative, inter-disciplinary effort to discover new tryptophan halogenases with unprecedented regioselectivity, continue to engineer the selectivities of known halogenases, and to incorporate these halogenases into natural product producing organisms to generate new, highly active compounds. The new halogenated products that are generated will be tested for agrochemical activity in collaboration with an industrial partner

Impact Summary
WHO WILL BENEFIT FROM THE RESEARCH, AND HOW? The outputs of this research will shed light on how halogenation occurs enzymatically, how these enzymes can be manipulated to alter their specificity, and how these halogenases can be more broadly incorporated into bioprocess and metabolic engineering applications to generate potentially useful new-to-nature compounds. These products have a broad structural range, and have the potential to benefit human welfare and the UK economy across several sectors. Specifically, the indole based compounds that we will generate, given their chemical structure, are likely to act as auxin derivatives. We will, in collaboration with the industrial partnership, evaluate the widespread use and application of these compounds. We will also explore the potential of utilising the halogenase enzymes that emrge from this proposal in large scale biocatalytic processes to inexpensively produce high-value halogenated compounds.

WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH? Academic research at the John Innes Centre (where PI O'Connor is based) that has potential commercial application is patented through Plant Biosciences Ltd. (PBL), a technology transfer company based at JIC that is jointly owned by the BBSRC, the John Innes Centre, and the Sainsbury Laboratory. The purpose of Plant Biosciences Ltd. is to bring the results of research in plant and microbial sciences at the Centre into use for public benefit through commercial exploitation. The University of St. Andrews (where co-I Goss is based) also has a long history of vigorous IP development through their tech-transfer group, the Knowledge Transfer Centre. Moreover, the John Innes Centre, UEA and the University of St. Andrews have a long history of involvement with industry, particularly in the area of understanding and manipulating of metabolic processes. O'Connor and Goss also actively working to engage with industry. We both have ongoing interactions with industry, and will work to increase and improve these interactions over the course of the proposal.