17-ERACoBioTech Methyl Transferases for the Functional Diversification of Bioactives: BioDiMet

BioDiMet aims to implement Nature's strategy to selectively methylate target compounds as a robust enzymatic platform ready for use at an early industrial scale. In Nature methylation is performed by enzymes called SAM-dependent methyl transferases, and this is a key step to accomplish and enhance the bioactivity of compounds. The reactions can occur in a selective manner and this is very difficult to achieve using established chemical synthetic methods where mainly toxic methylation agents such as methyl iodide and dimethyl sulfate are used. Enzymatic approaches have the advantages of sustainability, the avoidance of such toxic reagents, and reaction selectivity. This is of great relevance to the need for the development of novel bioactives in multi-billion pound markets of the pharma, agrochemical and fragrance/flavour industries.

Despite the importance in Nature, SAM-dependent methyl transferases are completely underexploited in industrial synthesis. This is mainly because an additional compound, a co-factor, is required known as S-adenosyl methionine (SAM). This is extremely expensive to purchase. To overcome this problem SAM co-factor supply/recycling systems can be used but these have not been available. In addition, a robust methyl transferase enzyme toolbox is needed. In recent years members of the BioDiMet consortium demonstrated that these limitations do not hold true anymore by establishing smart enzymatic methylation cascades with integrated SAM co-factor supply or recycling. BioDiMet is now the next step towards industrialisation of these enzymes in industry. The project will produce a toolbox of methyl transferases that are active towards a range of chemical structures and use SAM co-factor supply or recycling systems. They will be used with other enzymes to produce sustainable reaction cascades to important classes of compounds. In addition we will improve the robustness of the methyl transferases so they can be used by industry. Development of scaled-up reactions and down-stream processing methods within the project will also facilitate the industrial feasibility of the platform technology.

BioDiMet will therefore provide tools to synthesize new bioactives through powerful selective biocatalytic methylation and holds significant potential to develop novel active pharmaceutical ingredients via the methylation of compound libraries. Development of a spectrum of methyl transferases potentially allows access to a plethora of previously unobtainable diverse bioactives with desirable properties to deliver new products for industry.

BioDiMet aims to implement Nature's strategy to selectively methylate target compounds as a robust enzymatic platform ready for use at an early industrial scale. In Nature enzymatic methylation by SAM-dependent methyl transferases is a key step to accomplish and enhance bioactivity. The reactions can occur in a regio- or stereoselective manner and will be a valuable tool for the synthesis of novel bioactives in the pharma, agrochemical and fragrance/flavour industries.

Despite the importance in Nature, SAM-dependent methyl transferases are completely underexploited for use in industrial synthesis. This is mainly because SAM cofactor supply/recycling as well as a robust methyl transferase toolbox were not available. In recent years members of the BioDiMet consortium have established smart enzymatic methylation cascades with integrated SAM supply or recycling.

BioDiMet will now develop the next step towards industrialisation of the enzymes. The activities will be carried out in technology platforms that cover the synthesis of target molecules for methyl derivatisation, the discovery of novel enzymes for methylation and alkylation, and improved enzymes via enzyme engineering. It also involves the development of methylation cascades with cofactor supply and optimization together with applications in other enzyme cascades. The development of scaled-up reactions and down-stream processing methods will facilitate the industrial feasibility of the platform.

BioDiMet unites leading partners from academia, and industry in the areas of i) synthetic biology for methyl transfer cascade design, ii) enzymatic and chemoenzymatic synthesis to produce added value chemicals starting mainly from natural resources or bio-based molecules, iii) bioinformatics, and iv) available data from systems biology for the discovery and optimisation of enzymes in order to shift the emerging and highly promising enzymatic methylation technology into an industrial reality.

The sustainable methyl transferase toolbox in BioDiMet for the selective and straightforward synthesis of diverse products will target the interests and markets of pharma, agrochemicals, flavour & fragrances and cosmetics industry in both discovery and manufacturing. It will also have impact in academia. In many European countries there is a demographic change, and the ageing population has a continuing need for novel drugs to treat unmatched pathologic problems (www.euro.who.int). By contrast however, in pharmaceutical R&D there is a tension between the rapid availability of innovative drugs and the needs of patients. BioDiMet provides pivotal solutions with significant socio-economic impact. It will provide tools to synthesize new bioactives through powerful selective biocatalytic methylation and holds significant potential to develop novel APIs via the methylation of compound libraries. Development of a spectrum of methyl transferases potentially allows access to a plethora of previously unobtainable diverse bioactives with desirable properties to deliver new products in these major industries. For example, the global pharma market is estimated to be a 1.12 trillion US dollar market in 2022 (Pharma Market, 2022).

BioDiMet enables otherwise difficult selective methylation reactions to be achieved in an environmentally friendly way with non-toxic reagents. Catalytic cascade reactions remove the need for isolation of intermediates which will reduce manufacturing costs: e.g. removal of a single stage in an average pharma manufacturing process saves 4 M EU per annum. Similarly single step chemoselective methylation can remove upstream and downstream stages by avoiding protecting group manipulations that may be required for chemical methylation. The catalytic reactions that can be achieved via supply/recycling cascades are low cost in comparison to otherwise necessary stoichiometric supply of SAM, which would only be affordable for the highest value products. Co-factor recycling starting materials drastically decreases the costs or input of materials and requires development of the efficient recycling enzymes that BioDiMet will deliver.

BioDiMet will provide an efficient low cost method for O- and N-methylation that can be applied to many compounds of interest. The enzymatic methylation substrate mapping will demonstrate which methyl transferases can be used with each substrate type (a broad chemical space of methyl acceptors is represented in this consortium). Enzymes will then be available for others in the area of biotechnology and for those constructing synthetic biology pathways to bioactives. New insights into biosynthetic pathways (existing and artificial) will also be enabled. BioDiMet's added value will be evident also with regard to planned scientific publications and patents. At the same time well trained students, PhD students and post docs are the most valuable deliverable of the entire project and the largest addition that can be made to UK and European research and innovation.

BioDiMet will also have the potential to shorten timelines in drug development, resulting in cost savings in drug lead discovery. The methylation platform technology has the potential to bring new APIs on the market, and the technology can be transferred to the flavour & fragrance and agrochemical industries and others. Thus, the technology will further add new jobs in these fields.

In summary BioDiMet will have impact in each of the areas of economy, society, knowledge and people. Impact of this work extends beyond the immediate participants and project. It will benefit the UK economy by sustaining high-level research and the successfully developed methyl transferase cascades and pathways will be promoted widely within the pharmaceutical and fine chemical industries. Furthermore, with the bio-based processes suggested in BioDiMet matches key aspects of the European Bioeconomy Strategy (EU Bioeconomy Strategy, 2012).