Enzyme Cascades and Synthetic Biology Routes to Non-Natural Alkaloids

Plants and medicinal compounds from plants have been used for centuries to treat pain, infections, asthma, cancer and many of these plant natural products are used in modern medicine, for example: the analgesics morphine and codeine;
anti-malarial quinine for malaria; vinblastine and homoharringtonine for cancer treatment, and ephedrine for asthma. There are over 14,000 natural compounds of the alkaloid family and many possess biological activities. Recently, there has been intensive interest in producing some of these natural compounds using microbes, rather than isolating them from plants in parts of the world that are difficult to access. Synthetic biology approaches to achieve this are proving to be extremely powerful and recent articles have highlighted the possibility of producing morphine from yeast.

In previous work we have investigated some of the early steps and enzymes that use amino acids and perform transformations towards a key intermediate in the pathways that plants use to synthesise the benzylisoquinoline alkaloids, a family of over 2500 alkaloids. We now aim to use these enzymes, together with other enzymes not used in the plant pathway, and new enzymes that have been little explored in biocatalytic applications. The enzymes will be used in a laboratory bacterium to generate a wide range of structurally diverse compounds that are not found in nature. Compounds will screened for antibacterial properties.

Enzymes will be used in in vitro cascades to generate three, four, five, or more multiple enzyme pathways, and also by establishing the pathways in bacteria we will be able to generate new compounds in a reproducible way using a renewable resource.

This project aims to design and assemble de novo pathways in vitro and in vivo for the synthesis of novel complex alkaloids with new functionalities. Notably, while existing approaches using plant enzymes have targeted naturally occuring alkaloids, here the focus is distinctive with the use of non-natural substrates as starting materials to generate diverse non-natural alkaloids.

We will use tyrosine hydroxylases, tyrosine decarboxylases and DOPA decarboxylases previously developed in our laboratory, and new enzymes identified via metagenomics to generate dopamine analogues from non-natural amino acids for use with the enzyme norcoclaurine synthase (NCS). We will also use enzymes including our available transaminases, alcohol dehydrogenases and ene-reductases to generate aldehydes in situ for use in coupling reactions with NCS. The amines and aldehydes will then be used in assays with our library of native and mutant NCSs to generate novel tetrahydroisoquinoline alkaloids (THIAs) that are structurally diverse and have potential for subsequent chemical cyclisation. From this selected reactions will be optimised, and compounds generated will be screened for antibacterial properties.

In preliminary work we have established that some NCSs can accept ketones (non-ketoacids) which is exciting as there are very few routes to 1,1'-substituted THIAs. This route to generate novel THIAs will be explored using a range of ketones together with amines produced using hydroxylases and decarboxylases. Further structural diversity will be incorporated using methyltransferases and a recently developed S-adenosyl methionine cascade. Furthermore we will use C-C phenolic coupling enzymes to mediate further ring forming reactions to produce for example novel aporphine alkaloids.

Using this toolbox of enzymes we will then assemble in vitro reaction cascades to and construct an in vivo pathway to build routes to structurally diverse novel alkaloids.

The project will have impact in each of the areas defined by the BBSRC and RCUK: economy, society, knowledge and people.

In terms of economic impact the PI and CoI have experience of working with industry, and the commercialisation of research findings and creation of a spin-out company. The enzymes and data resulting from this project will be made available (after IP protection) to the wider academic and industrial community via publication, and dissemination at conferences and meetings for example the BBSRC NIBB Networks. Furthermore, the novel alkaloids will be screened for antibacterial properties, which may identify new bioactive compounds. Beneficiaries of this work therefore extends beyond the immediate participants. It will benefit the UK economy by sustaining high-level research and the successfully developed biocatalytic cascades and pathways will be promoted widely within the pharmaceutical and fine chemical industries.

The impact of knowledge generation from the research will arise from the communication and publication of new scientific advances and public outreach activities to schools, detailed in the pathways to impact.

The societal impact will arise from the development of available and new enzymes in multienzyme systems to generate new alkaloids. The use of biocatalysis in the chemical and pharmaceutical industry is growing, but is limited by the types of enzymes used and the compounds generated. By identifying new enzymes and methods for assembling these to achieve sustainable synthesis, these resources will assist the UK government meet its targets on greenhouse gas emissions and help mitigate the negative environmental effects of global warming. The use of synthetic biology for pathway construction is a key part of the cross council research strategy and synthetic biology is one of the BBSRC strategic priority areas. In addition, if new biologically active compounds are identified that can be made sustainably and cheaply this could impact positively on the general public.

The impact on people will arise from training and career development of the PDRA and provision of skilled scientist in the industrial biotechnology sector.

The findings will also influence future research directions, since the successful completion of this proposal will provide the data to embed biocatalytic strategies to new alkaloids that has to date been little unexplored. In addition, a wide spectrum of potential end-users, which include commercial companies, charities and the health care systems may profit from the outputs of this project in the longer term. While the impact of the funded research will be at an international level, the intellectual property and thus the commercial profits will be likely to enhance the competiveness of the UK. UCL who will hold the intellectual property rights to the enzymes and compounds generated in this proposal, will benefit from licensing and industry collaboration.