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Programmable Enzymatic Synthesis of Bioactive Compounds

Periodic Reporting for period 2 - ProgrES (Programmable Enzymatic Synthesis of Bioactive Compounds)

Reporting period: 2020-01-01 to 2021-06-30

The overall aim and objective of the programme is to design new biocatalytic routes to high value compounds using an integrated design, test, build system. This involves curating a database of enzyme activity and incorporating it in an automated retrosynthesis software to facilitate sustainable synthesis of high-value compounds for industrial application.
The global effort towards low-carbon and sustainable living is driving the chemical industry to develop novel manufacturing technology which embraces green sustainable production and mitigating the use of toxic chemicals such as heavy metals or fossil-derived chemicals. While it is difficult to completely phase-out oil-derived compounds, biocatalysis has in many ways provided an alternative route to minimizing the use of such compounds. Instead of using heavy metal catalysis, bio-derived enzymes are employed which drastically reduces solvent usage and temperature which in overall reduces energy consumption.
This drive towards integrating biotechnology in chemical manufacture will support the European bioeconomy creating jobs, diversity and support the European green deal.
The team has developed a novel computer-aided synthesis software called retrobiocat powered by a comprehensive biocatalyst database (Nature Catalysis, 2021, 4, 98-104). The software and database has been developed as part of this project with the retrobiocat tool freely available online (retrobiocat.com). In addition, a novel selection of biocatalysts has been added to the system to predict new biosynthetic pathways to pharmaceutically relevant imino sugars.
For industrial application, further development in screening enzyme activity, stability and tolerance to solvent/heat is required. The team has demonstrated the application of high throughput screening and in the scalability of biocatalytic transformation. By a technique called immobilization, an enzyme which selectively catalyses the oxidation of C-6 hydroxy group in lactose was performed with operational stability under process conditions greatly enhanced producing gram-scale quantities of products (ACS Catal. 2019, 9, 12, 11658–11662).

The activation of C-H bonds in synthetic chemistry is not trivial and requires harsh conditions and often heavy metal catalysis. Here, the team employed a mild pathway to formal C-H bind activation by employing a P450 enzyme. Subsequent lactonization yielded lactone derivatives in high selectivity which have a range of application in flavours, fragrance and in pharmaceuticals (Angew. Chem. 2019, 131, 5724 –5727).
The first enzymatic cascade developed in this project resulting in the synthesis of amino-piperidine and amino-azepanes has been completed and published (Chem. Commun., 2020, 56, 7949-7952). A second cascade to polyhydroxylated iminosugars including a novel use of imin reductases has been completed (manuscript in preparation).

Several new C-H activating oxygenases have been developed for early-stage functionalization (using ancestral sequence reconstruction and protein engineering, 2 manuscripts in preparation). Foe late-stage functionalization, oxygenases working on naked piperidines and pyrrolidines are being investigated.
The overall aim of the research programme is to develop a set of tools which can be employed by the chemical and pharmaceutical industry to manufacture a range of high-value chemicals (either drop-in replacements or novel compounds). This research will seek to integrate AI driven retrosynthetic analysis for rapid cascade design and new analytical and protein engineering techniques. Overall, a panel of imino sugars will be synthesised by the end of the project to demonstrate the bioinformatics platform being developed. An additional aspect of the workflow will be the application of immobilized enzymes in flow, which would enable ultimately fully programmable and automated synthetic strategies.