Periodic Reporting for period 4 - ProgrES (Programmable Enzymatic Synthesis of Bioactive Compounds)
Periodo di rendicontazione: 2023-01-01 al 2024-06-30
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. As the enzyme toolbox for biocatalysis has expanded, so has the potential for the construction of powerful enzymatic cascades for efficient and selective synthesis of target molecules. Additionally, recent advances in computer-aided synthesis planning are revolutionizing synthesis design in both synthetic biology and organic chemistry. However, the potential for biocatalysis is not well captured by tools currently available in either field. In this project we have developed RetroBioCat, an intuitive and accessible tool for computer-aided design of biocatalytic cascades, freely available at retrobiocat.com. Our approach uses a set of expertly encoded reaction rules encompassing the enzyme toolbox for biocatalysis, and a system for identifying literature precedent for enzymes with the correct substrate specificity where this is available. Applying these rules for automated biocatalytic retrosynthesis, we show our tool to be capable of identifying promising biocatalytic pathways to target molecules, validated using a test set of recent cascades described in the literature.
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 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; ACS Catal. 2023, 13, 11771-11780 ). The software and database has been developed as part of this project with the retrobiocat tool freely available online (retrobiocat.com).
This database has been used to develop enzyme cascades towards amino polyols and iminosugars, which are highly sought-after pharmaceutical targets, but their chemical synthesis is lengthy and can suffer from poor scalability and purification. Using retrobiocat for design of the cascades, we have developed efficient routes to amino diols and amino polyols (Chem. Commun., 2020, 56, 7949-7952;JACS Au 2022, 2, 10, 2251–2258). We have developed cell-free protocols for isolation of highly polar iminosugar products from biotransformations in a single step through development of a gradient-elution cation exchange purification. The two-step pathway provides a short synthetic route that can be used as a cell-free platform for broader iminosugar synthesis (ACS Cent. Sci. 2023, 9, 1, 103–108).
A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof-of-principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross-reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O-methylnorbelladine (Angew. Chem. Int. Ed. 2021, 60, 18660-18665).
The retrobiocat database has also highlighted gaps in our toolbox of biocatalysts. During the project we have addressed two of these. Firstly, 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). This work was expanded to develop a family of biocatalysts for the regioselective mid- and end-chain C−H activation of fatty acids to generate hydroxy acids . Ancestral sequence reconstruction was successfully applied to the multidomain enzyme family of CYP116B cytochrome P450 monooxygenases to generate ancestor variants with distinct product and thermostability profiles.(Angew.Chem. IE, 2024, 63, e202314869). Secondly, biocatalytic amide bond formation was used effectively to selectively form commercially relevant surfactants (Angew.Chem. IE, 2022, 61, e202205054) in single steps with very high selectivity.
This database has been used to develop enzyme cascades towards amino polyols and iminosugars, which are highly sought-after pharmaceutical targets, but their chemical synthesis is lengthy and can suffer from poor scalability and purification. Using retrobiocat for design of the cascades, we have developed efficient routes to amino diols and amino polyols (Chem. Commun., 2020, 56, 7949-7952;JACS Au 2022, 2, 10, 2251–2258). We have developed cell-free protocols for isolation of highly polar iminosugar products from biotransformations in a single step through development of a gradient-elution cation exchange purification. The two-step pathway provides a short synthetic route that can be used as a cell-free platform for broader iminosugar synthesis (ACS Cent. Sci. 2023, 9, 1, 103–108).
A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof-of-principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross-reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O-methylnorbelladine (Angew. Chem. Int. Ed. 2021, 60, 18660-18665).
The retrobiocat database has also highlighted gaps in our toolbox of biocatalysts. During the project we have addressed two of these. Firstly, 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). This work was expanded to develop a family of biocatalysts for the regioselective mid- and end-chain C−H activation of fatty acids to generate hydroxy acids . Ancestral sequence reconstruction was successfully applied to the multidomain enzyme family of CYP116B cytochrome P450 monooxygenases to generate ancestor variants with distinct product and thermostability profiles.(Angew.Chem. IE, 2024, 63, e202314869). Secondly, biocatalytic amide bond formation was used effectively to selectively form commercially relevant surfactants (Angew.Chem. IE, 2022, 61, e202205054) in single steps with very high selectivity.